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Sample records for human neutrophil chemotaxis

  1. α-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8.

    LENUS (Irish Health Repository)

    Bergin, David A

    2010-12-01

    Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

  2. The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

    Science.gov (United States)

    Lomas, D A; Stone, S R; Llewellyn-Jones, C; Keogan, M T; Wang, Z M; Rubin, H; Carrell, R W; Stockley, R A

    1995-10-06

    Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

  3. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells.

    Science.gov (United States)

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  4. Suppression of blood monocyte and neutrophil chemotaxis in acute human malaria

    DEFF Research Database (Denmark)

    Nielsen, H; Kharazmi, A; Theander, T G

    1986-01-01

    tested monocyte chemotactic responsiveness in 19 patients with acute primary attack malaria. In addition, the neutrophil chemotaxis was measured in 12 patients. Before the initiation of antimalarial treatment a significant depression of monocyte chemotaxis was observed in approximately half...... of the patients when compared with healthy control subjects. The depression was found in Plasmodium falciparum malaria as well as in P. vivax or P. ovale malaria patients. The defective responsiveness was not receptor specific, since the responses towards casein and zymosan activated serum proved to be equally...... of treatment, and nearly normalized after 7 days (87% of controls). Furthermore, monocyte phagocytic and candidacidal activities were assessed in the same patients on admission and during the follow-up. In contrast to chemotaxis, these functions were normal in all of the patients whenever measured...

  5. The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

    Directory of Open Access Journals (Sweden)

    Tamding Wangdi

    2014-08-01

    Full Text Available Salmonella enterica serovar Typhi (S. Typhi causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a and C5a receptor (C5aR. Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

  6. Comparison of the effect of timegadine, levamisole, and D-penicillamine on human neutrophil metabolism of endogenous arachidonic acid and chemotaxis

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, O.H.; Ahnfelt-Roenne, I. Department of Pharmacology, Leo Pharmaceutical Products, Ballerup; Elmgreen, J.

    1988-01-01

    The effect of timegadine, a novel experimental antirheumatic drug, on human neutrophil (PMN) 5-lipoxygenase activity and leukotriene B/sub 4/ (LTB/sub 4/) chemotaxis was compared with that of two second-line antiinflammatory drugs, D-penicillamine and levamisole. 1-/sup 14/C-Arachidonic acid (AA) was incorporated into the purified cells until steady state conditions were obtained. After preincubation with serial dilutions of the three drugs, AA release and metabolism was stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thinlayer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxi of PMNs towards LTB/sub 4/ was measured in a modified Boyden chamber. Timegardine showed dose-dependent inhibition of both the 5-lipoxygenase pathway (IC50 3.4 x 10/sup -5/ M), and of chemotaxis (IC50 3 x 10/sup -4/ M). Inhibition of the release of AA from phospholipids, however, occurred only at therapeutically irrelevant doses (millimolar concentrations). Levamisole and D-penicillamine did not inhibit any of the cell functions investigated. Inhibition of both neutrophil motility and cellular synthesis of pro-inflammatory eicosanoids, may thus contribute to the clinical effects of timegadine in rheumatoid arthritis.

  7. ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis.

    Science.gov (United States)

    Mazaki, Yuichi; Onodera, Yasuhito; Higashi, Tsunehito; Horinouchi, Takahiro; Oikawa, Tsukasa; Sabe, Hisataka

    2017-10-02

    The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex. We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges. Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.

  8. Chemotactic Activity on Human Neutrophils to Streptococcus mutans

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    Tetiana Haniastuti

    2013-07-01

    Full Text Available Objective: The aim of this study was to evaluate chemotactic activity o neutrophil to S. mutans. Chemotaxis assay was performed in blind well chambers. Materials and Methods: Hanks balanced salt solution (HBSS containing 106 S. mutans,  108 S. mutans, 10-8 M fMLP, or HBSS alone were placed in the lower wells of the chamber and covered with polycorbonate membrane filter. Neutrophils suspension (2x105 cells was then placed in the upper compartment. After incubation for 60 mins at 37ºC in a humidified atmosphere with 5% CO2, the filters were removed and stained with Giemsa. Result: ANOVA revealed statistically significant differences among groups (p<0.05, indicating that S. mutans induced neutrophils chemotaxis. The number of neutrophils migration in response to 108 S. mutans and 106 S. mutans were signifiantly greater compared to fMLP (p<0.05. Conclusion: S. mutans may activate human neutrophils, resulting in the chemotaxis of the neutrophils.DOI: 10.14693/jdi.v16i2.99

  9. A dual-docking microfluidic cell migration assay (D2-Chip) for testing neutrophil chemotaxis and the memory effect.

    Science.gov (United States)

    Yang, Ke; Wu, Jiandong; Xu, Guoqing; Xie, Dongxue; Peretz-Soroka, Hagit; Santos, Susy; Alexander, Murray; Zhu, Ling; Zhang, Michael; Liu, Yong; Lin, Francis

    2017-04-18

    Chemotaxis is a classic mechanism for guiding cell migration and an important topic in both fundamental cell biology and health sciences. Neutrophils are a widely used model to study eukaryotic cell migration and neutrophil chemotaxis itself can lead to protective or harmful immune actions to the body. While much has been learnt from past research about how neutrophils effectively navigate through a chemoattractant gradient, many interesting questions remain unclear. For example, while it is tempting to model neutrophil chemotaxis using the well-established biased random walk theory, the experimental proof was challenged by the cell's highly persistent migrating nature. A special experimental design is required to test the key predictions from the random walk model. Another question that has interested the cell migration community for decades concerns the existence of chemotactic memory and its underlying mechanism. Although chemotactic memory has been suggested in various studies, a clear quantitative experimental demonstration will improve our understanding of the migratory memory effect. Motivated by these questions, we developed a microfluidic cell migration assay (so-called dual-docking chip or D 2 -Chip) that can test both the biased random walk model and the memory effect for neutrophil chemotaxis on a single chip enabled by multi-region gradient generation and dual-region cell alignment. Our results provide experimental support for the biased random walk model and chemotactic memory for neutrophil chemotaxis. Quantitative data analyses provide new insights into neutrophil chemotaxis and memory by making connections to entropic disorder, cell morphology and oscillating migratory response.

  10. Decreased numbers of chemotactic factor receptors in chronic neutropenia with defective chemotaxis: spontaneous recovery from the neutrophil abnormalities during early childhood

    International Nuclear Information System (INIS)

    Yasui, K.; Yamazaki, M.; Miyagawa, Y.; Komiyama, A.; Akabane, T.

    1987-01-01

    Childhood chronic neutropenia with decreased numbers of chemotactic factor receptors as well as defective chemotaxis was first demonstrated in an 8-month-old girl. Chemotactic factor receptors on neutrophils were assayed using tritiated N-formyl-methionyl-leucyl-phenylalanine ( 3 H-FMLP). The patient's neutrophils had decreased numbers of the receptors: numbers of the receptors were 20,000 (less than 3 SD) as compared with those of control cells of 52,000 +/- 6000 (mean +/- SD) (n = 10). The neutropenia disappeared spontaneously by 28 months of age parallel with the improvement of chemotaxis and increase in numbers of chemotactic factor receptors. These results demonstrate a transient decrease of neutrophil chemotactic factor receptors as one of the pathophysiological bases of a transient defect of neutrophil chemotaxis in this disorder

  11. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    International Nuclear Information System (INIS)

    Jannat, Risat A; Hammer, Daniel A; Robbins, Gregory P; Ricart, Brendon G; Dembo, Micah

    2010-01-01

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K D of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β 2 -integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

  12. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    Energy Technology Data Exchange (ETDEWEB)

    Jannat, Risat A; Hammer, Daniel A [Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 South 33rd Street, Philadelphia, PA 19104 (United States); Robbins, Gregory P; Ricart, Brendon G [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 311A Towne Building, 220 South 33rd Street, Philadelphia, PA 19104 (United States); Dembo, Micah, E-mail: hammer@seas.upenn.ed [Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215 (United States)

    2010-05-19

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K{sub D} of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against {beta}{sub 2}-integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

  13. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... towards the chemotactic peptide f-met-leu-phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan-activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63...... in a concentration-dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished...

  14. Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro

    DEFF Research Database (Denmark)

    Jensen, P; Kharazmi, A

    1991-01-01

    We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity were....... Another advantage of the assay is that it can be used to show the migration pattern of different populations of neutrophils from both healthy individuals and patients....

  15. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

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    Cheng-Yuk Lee

    Full Text Available Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis as well as upon contact (by serum-dependent adhesion and phagocytosis. This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

  16. CYP4F18-Deficient Neutrophils Exhibit Increased Chemotaxis to Complement Component C5a

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    Rachel Vaivoda

    2015-01-01

    Full Text Available CYP4Fs were first identified as enzymes that catalyze hydroxylation of leukotriene B4 (LTB4. CYP4F18 has an unusual expression in neutrophils and was predicted to play a role in regulating LTB4-dependent inflammation. We compared chemotaxis of wild-type and Cyp4f18 knockout neutrophils using an in vitro assay. There was no significant difference in the chemotactic response to LTB4, but the response to complement component C5a increased 1.9–2.25-fold in knockout cells compared to wild-type (P < 0.01. This increase was still observed when neutrophils were treated with inhibitors of eicosanoid synthesis. There were no changes in expression of other CYP4 enzymes in knockout neutrophils that might compensate for loss of CYP4F18 or lead to differences in activity. A mouse model of dextran sodium sulfate colitis was used to investigate the consequences of increased C5a-dependent chemotaxis in vivo, but there was no significant difference in weight loss, disease activity, or colonic tissue myeloperoxidase between wild-type and Cyp4f18 knockout mice. This study demonstrates the limitations of inferring CYP4F function based on an ability to use LTB4 as a substrate, points to expanding roles for CYP4F enzymes in immune regulation, and underscores the in vivo challenges of CYP knockout studies.

  17. Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

    DEFF Research Database (Denmark)

    Jaeger, K E; Kharazmi, A; Høiby, N

    1991-01-01

    concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect...... on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused...

  18. Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.

    Science.gov (United States)

    Bhuiyan, Md Saruar; Ellett, Felix; Murray, Gerald L; Kostoulias, Xenia; Cerqueira, Gustavo M; Schulze, Keith E; Mahamad Maifiah, Mohd Hafidz; Li, Jian; Creek, Darren J; Lieschke, Graham J; Peleg, Anton Y

    2016-08-23

    Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease.

  19. Influence of standard and novel LTB4 analogs on human neutrophil chemotaxis measured by the multiwell cap assay.

    Science.gov (United States)

    Psychoyos, S; Uziel-Fusi, S; Bhagwat, S; Morrissey, M M

    1989-11-30

    Standard and novel LTB4 analogs were tested for neutrophil chemoattractant activity using the multiwell cap assay (Evans et al. (1986) Biosc. Rep. 6, 1041). The assay uses disposable equipment and measures chemotaxis by the number of cells able to migrate across the full thickness of cellulose nitrate filters. Under standard conditions (90 min incubation at 37 degrees C in buffer containing 2% bovine albumin), LTB4 and 6-cis-LTB1 had EC50 values of 3.5 and 15,000 nM, respectively. 20-hydroxy-LTB4 was equipotent with LTB4 and exhibited a similar biphasic chemotactic response, however, only one third of the number of cells migrated through the filter. 20-carboxy-LTB4 was inactive up to 1,000 nM. 5-desoxy-((6,7)-cis-cyclopropyl)-LTB2, (6,7)-benzo-LTB2 and 5-desoxy-(8,10)-LTB2 had EC50 values of 11,300, 50,000 and 84,000 nM, respectively. Checkerboard analysis indicated a chemokinetic component of 42% for LTB4 at a concentration causing peak chemotaxis. Reduction of albumin in the buffer to 0.5% increased the apparent potencies of LTB4 and 6-cis-LTB1 five-fold. Since LTB4 is a mediator of inflammation, various anti-inflammatory agents were tested at peak concentrations observed in vivo for in vitro inhibition of LTB4-stimulated chemotaxis in the presence of 0.5% albumin. Under the conditions of the assay, chloroquine diphosphate, dexamethasone, indomethacin, penicillamine, piroxicam and diclofenac sodium were inactive; gold sodium thiomalate was inhibitory (IC50 = 20 microM).

  20. A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils.

    Science.gov (United States)

    Ohira, Taisuke; Bannenberg, Gerard; Arita, Makoto; Takahashi, Minoru; Ge, Qingyuan; Van Dyke, Thomas E; Stahl, Gregory L; Serhan, Charles N; Badwey, John A

    2004-08-01

    Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

  1. Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis.

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    Orion D Weiner

    2006-02-01

    Full Text Available Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes, scaffolded by hematopoietic protein 1 (Hem-1, that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b substantially weakens Rac activation and phosphatidylinositol-(3,4,5-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5-tris-phosphate/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

  2. Prostaglandin F2α–F-Prostanoid Receptor Signalling Promotes Neutrophil Chemotaxis via Chemokine (CXC motif) Ligand-1 in Endometrial Adenocarcinoma

    Science.gov (United States)

    Wallace, Alison E; Sales, Kurt J; Catalano, Roberto D; Anderson, Richard A; Williams, Alistair RW; Wilson, Martin R; Schwarze, Jurgen; Wang, Hongwei; Rossi, Adriano G; Jabbour, Henry N

    2009-01-01

    The prostaglandin F2α (PGF2α) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF2α signalling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue as compared to normal endometrium and localised to glandular epithelium, endothelium and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100nM PGF2α increased CXCL1 promoter activity, mRNA and protein expression, and these effects were abolished by co-treatment of cells with FP antagonist or chemical inhibitors of Gq, EGFR and ERK. Similarly, CXCL1 was elevated in response to 100 nM PGF2α in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalised to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF2α-treated FPS cells stimulated neutrophil chemotaxis which could be abolished by CXCL1 protein immunoneutralisation of the conditioned media or antagonism of CXCR2. Finally, xenograft tumours in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared to tumours arising from wild-type cells or following treatment of mice bearing FPS tumours with CXCL1-neutralising antibody. In conclusion, our results demonstrate a novel PGF2α-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis. PMID:19549892

  3. p21-Activated kinase (PAK regulates cytoskeletal reorganization and directional migration in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Asako Itakura

    Full Text Available Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP, and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

  4. Combined activity of post-exercise concentrations of NA and eHsp72 on human neutrophil function: role of cAMP.

    Science.gov (United States)

    Giraldo, Esther; Hinchado, María D; Ortega, Eduardo

    2013-09-01

    Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as "danger signals" during exercise-induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post-exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise-induced stress. Given this context, the aim of the present investigation was to study the combined activity of post-exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects. Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post-exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP. These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise-induced stress. Copyright © 2013 Wiley Periodicals, Inc.

  5. Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide: effect on human neutrophil chemotaxis and oxidative burst

    DEFF Research Database (Denmark)

    Kharazmi, A; Fomsgaard, A; Conrad, R S

    1991-01-01

    There are conflicting data on the effect of bacterial lipopolysaccharides (LPS) on the function of human neutrophils. The present study was designed to examine the relationship between chemical composition and the modulatory effect of LPS on human neutrophil function. LPS was extracted from five...

  6. Burn injury reduces neutrophil directional migration speed in microfluidic devices.

    Directory of Open Access Journals (Sweden)

    Kathryn L Butler

    2010-07-01

    Full Text Available Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72-120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.

  7. Imaging neutrophil migration dynamics using micro-optical coherence tomography (Conference Presentation)

    Science.gov (United States)

    Chu, Kengyeh K.; Yonker, Lael; Som, Avira; Pazos, Michael; Kusek, Mark E.; Hurley, Bryan P.; Tearney, Guillermo J.

    2016-03-01

    Neutrophils are immune cells that undergo chemotaxis, detecting and migrating towards a chemical signal gradient. Neutrophils actively migrate across epithelial boundaries, interacting with the epithelium to selectively permit passage without compromising the epithelial barrier. In many inflammatory disorders, excessive neutrophil migration can cause damage to the epithelium itself. The signaling pathways and mechanisms that facilitate trans-epithelial migration are not fully characterized. Our laboratory has developed micro-optical coherence tomography (μOCT), which has 2 μm lateral resolution and 1 μm axial resolution. As a high-resolution native contrast modality, μOCT can directly visualize individual neutrophils as they interact with a cell layer cultured on a transwell filter. A chemoattractant can be applied to the apical side of inverted monolayer, and human neutrophils placed in the basolateral compartment, while μOCT captures 3D images of the chemotaxis. μOCT images can also generate quantitative metrics of migration volume to study the dependence of chemotaxis on monolayer cell type, chemoattractant type, and disease state of the neutrophils. For example, a disease known as leukocyte adhesion deficiency (LAD) can be simulated by treating neutrophils with antibodies that interfere with the CD18 receptor, a facilitator of trans-epithelial migration. We conducted a migration study of anti-CD18 treated and control neutrophils using T84 intestinal epithelium as a barrier. After one hour, μOCT time-lapse imaging indicated a strong difference in the fraction of neutrophils that remain attached to the epithelium after migration (0.67 +/- 0.12 attached anti-CD18 neutrophils, 0.23 +/- 0.08 attached control neutrophils, n = 6, p < 0.05), as well as a modest but non-significant decrease in total migration volume for treated neutrophils. We can now integrate μOCT-derived migration metrics with simultaneously acquired measurements of transepithelial electrical

  8. SiMA: A simplified migration assay for analyzing neutrophil migration.

    Science.gov (United States)

    Weckmann, Markus; Becker, Tim; Nissen, Gyde; Pech, Martin; Kopp, Matthias V

    2017-07-01

    In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB 4 )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS ® FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB 4 , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and

  9. Growth factors G-CSF and GM-CSF differentially preserve chemotaxis of neutrophils aging in vitro

    NARCIS (Netherlands)

    Wolach, Baruch; van der Laan, Luc J. W.; Maianski, Nikolai A.; Tool, Anton T. J.; van Bruggen, Robin; Roos, Dirk; Kuijpers, Taco W.

    2007-01-01

    OBJECTIVE: The ability of human neutrophils to migrate was studied during culture in vitro. METHODS: Neutrophils were isolated from human blood and cultured at 37 degrees C. Apoptosis was determined by Annexin-V fluorescein isothiocyanate binding. Receptor expression was measured by fluorescence in

  10. CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

    Directory of Open Access Journals (Sweden)

    Rebecca A Drummond

    2015-12-01

    Full Text Available Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS. However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

  11. Inhalation of activated protein C inhibits endotoxin-induced pulmonary inflammation in mice independent of neutrophil recruitment

    NARCIS (Netherlands)

    Slofstra, S. H.; Groot, A. P.; Maris, N. A.; Reitsma, P. H.; Cate, H. Ten; Spek, C. A.

    2006-01-01

    BACKGROUND AND PURPOSE: Intravenous administration of recombinant human activated protein C (rhAPC) is known to reduce lipopolysaccharide (LPS)-induced pulmonary inflammation by attenuating neutrophil chemotaxis towards the alveolar compartment. Ideally, one would administer rhAPC in pulmonary

  12. Impaired neutrophil function in intestinal lymphangiectasia.

    OpenAIRE

    Bolton, R P; Cotter, K L; Losowsky, M S

    1986-01-01

    Impaired neutrophil chemotaxis and phagocytosis were shown in three patients with intestinal lymphangiectasia. Abnormalities in cell associated and serum derived activity occurred, and possible mechanisms are suggested.

  13. Distinct cellular sources of hepoxilin A3 and leukotriene B4 are used to coordinate bacterial-induced neutrophil transepithelial migration.

    Science.gov (United States)

    Pazos, Michael A; Pirzai, Waheed; Yonker, Lael M; Morisseau, Christophe; Gronert, Karsten; Hurley, Bryan P

    2015-02-01

    Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response. Copyright © 2015 by The American Association of Immunologists, Inc.

  14. Neutrophil chemotactic activity in bronchoalveolar lavage fluid of patients with AIDS-associated Pneumocystis carinii pneumonia

    DEFF Research Database (Denmark)

    Benfield, T L; Kharazmi, A; Larsen, C G

    1997-01-01

    been shown to confer a poor prognosis in PCP. We therefore investigated the potential of BAL fluid from 17 patients with PCP to induce neutrophil chemotaxis. BAL fluid from patients induced considerable neutrophil chemotactic activity compared to normal controls. Elevated levels of IL-8 were detected...... in patient samples as compared to controls. A specific anti-IL-8 antibody significantly reduced chemotactic activity of patient samples by more than 50%. In conclusion, IL-8 appears to be a significant participant of neutrophil chemotaxis in AIDS-associated PCP, and may participate in the recruitment...

  15. Phenotypic changes in neutrophils related to anti-inflammatory therapy.

    Science.gov (United States)

    Barton, A E; Bayley, D L; Mikami, M; Llewellyn-Jones, C G; Stockley, R A

    2000-01-03

    Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.

  16. Evaluation of polymorphonuclear leukocyte chemotaxis of adult and neonatal rhesus monkeys using 51-chromium labeling method

    International Nuclear Information System (INIS)

    Kinoshita, Yo; Masuda, Kiyokazu; Kobayashi, Yohnosuke

    1987-01-01

    Chemotaxis of polymorphonuclear leukocytes (PMN) from heparinized venous blood of 8 adult rhesus monkeys (Macaca Mulatta) and 13 rhesus monkey neonates within 48 hours of birth were evaluated by using 51-chromium labeling method. PMNs were prepared by Ficoll-Hypaque gradient and dextran sedimentation procedures and the final 51-chromium uptake was 3.21 ± 1.27 % to original count. PMN chemotaxis was succeeded by using two different chemotaxis filters (Nuclepore filter on top of Millipore filter) with incubation at 37 deg C for 90 min. The mean value of target: non target ratio (CPM in lower filter with chemoattractant/CPM in lower filter without chemoattractant) of 3.56 ± 2.49 from neonates showed no significant difference from that of 4.44 ± 1.24 from adults. Only about 30 % of neonates showed an impaired chemotaxis, but others showed similar chemotactic activity as adults. The results show that the 51-chromium labeling method is useful to assess neutrophil functions in rhesus monkey species and suggest that host defense mechanism of the rhesus monkey may differ from that of human in neonatal period. (author)

  17. Human neutrophils in auto-immunity.

    Science.gov (United States)

    Thieblemont, Nathalie; Wright, Helen L; Edwards, Steven W; Witko-Sarsat, Véronique

    2016-04-01

    Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Furebring Christina

    2009-03-01

    Full Text Available Abstract Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS blocks the Complement fragment C5a receptor (C5aR and formylated peptide receptor (FPR and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies

  19. Cryptococcal capsular glucuronoxylomannan reduces ischaemia-related neutrophil influx

    NARCIS (Netherlands)

    Ellerbroek, PM; Schoemaker, RG; van Veghel, R; Hoepelman, AIM; Coenjaerts, FEJ

    Background The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans interferes with the chemotaxis and transendothelial migration of neutrophils. Intravenous administration of purified GXM has been shown to reduce the influx of inflammatory cells in an animal model of

  20. Cytoplasmic lipid bodies of human neutrophilic leukocytes

    International Nuclear Information System (INIS)

    Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

    1989-01-01

    The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

  1. Platelet modulation of human neutrophil functions

    Energy Technology Data Exchange (ETDEWEB)

    McGarrity, S.T.; Hyers, T.M.; Webster, R.O.

    1986-03-01

    The combined presence of platelets (PLTS) and neutrophils (PMN) at inflammatory sites has led to examination of the hypothesis that interaction of these cells modulates their responses to stimuli. Gel-filtered human PLTS (GFP) were found to inhibit N-formyl-met-leu-phe (FMLP) and phorbol myristate acetate (PMA) stimulated PMN O/sub 2//sup -/ generation in a concentration-dependent fashion. The heat-stable inhibitory activity was present in the supernatants of GFP after incubation with FMLP (10/sup -7/M), thrombin (0.5 U/ml) or ADP (20 ..mu..M), suggesting a role for PLT release products. PLT lysates added to PMN produced up to 80% inhibition of O/sub 2//sup -/ generation for PMA and 40% for FMLP. Like GFP, lysates failed to scavenge O/sub 2/..pi.. produced by xanthine oxidase-hypoxanthine. The inhibitory activity could not be ascribed to serotonin or adenosine. PLT lysates failed to compete with /sup 3/H-FMLP for binding to PMN. Sephadex G-200 fractionation of PLT lysates releaved two peaks of inhibitory activity with apparent Mr > 200,000 and < 14,000 Daltons. Pretreatment of PMN with PLT lysates also results in a concentration-dependent inhibition of degranulation provoked by FMLP (2 x 10/sup -7/M) or PMA (2 ng/ml) and PMN chemotaxis to FMLP (10/sup -8/M). These studies indicate that preformed PLT mediator(s) released in response to physiological stimuli may limit tissue damage by PMN at sites of inflammation.

  2. Interleukin-8: an expanding universe beyond neutrophil chemotaxis and activation.

    Science.gov (United States)

    Mukaida, N

    2000-12-01

    Since the discovery 13 years ago of interleukin (IL)-8 as a potent neutrophil chemotactic factor, accumulating evidence has established it as a crucial mediator in neutrophil-dependent acute inflammation. Numerous observations have demonstrated that various types of cells can produce a large amount of IL-8, either in response to various stimuli or constitutively, after malignant transformation. Recent studies of IL-8-mediated signaling have revealed that IL-8 activates a wide range of signaling molecules in a coordinate manner. IL-8 has been proven to have diverse actions on various types of leukocytic and nonleukocytic cells besides neutrophils. The author reviews recent progress in IL-8 signal transduction and biological actions on nonneutrophilic leukocytes, including T lymphocytes, monocytes, and hematopoietic progenitor cells. Potential involvement of IL-8 in viral infections and tumor progression is also discussed.

  3. The non-receptor tyrosine kinase Lyn controls neutrophil adhesion by recruiting the CrkL–C3G complex and activating Rap1 at the leading edge

    Science.gov (United States)

    He, Yuan; Kapoor, Ashish; Cook, Sara; Liu, Shubai; Xiang, Yang; Rao, Christopher V.; Kenis, Paul J. A.; Wang, Fei

    2011-01-01

    Establishing new adhesions at the extended leading edges of motile cells is essential for stable polarity and persistent motility. Despite recent identification of signaling pathways that mediate polarity and chemotaxis in neutrophils, little is known about molecular mechanisms governing cell–extracellular-matrix (ECM) adhesion in these highly polarized and rapidly migrating cells. Here, we describe a signaling pathway in neutrophils that is essential for localized integrin activation, leading edge attachment and persistent migration during chemotaxis. This pathway depends upon Gi-protein-mediated activation and leading edge recruitment of Lyn, a non-receptor tyrosine kinase belonging to the Src kinase family. We identified the small GTPase Rap1 as a major downstream effector of Lyn to regulate neutrophil adhesion during chemotaxis. Depletion of Lyn in neutrophil-like HL-60 cells prevented chemoattractant-induced Rap1 activation at the leading edge of the cell, whereas ectopic expression of Rap1 largely rescued the defects induced by Lyn depletion. Furthermore, Lyn controls spatial activation of Rap1 by recruiting the CrkL–C3G protein complex to the leading edge. Together, these results provide novel mechanistic insights into the poorly understood signaling network that controls leading edge adhesion during chemotaxis of neutrophils, and possibly other amoeboid cells. PMID:21628423

  4. RhoA determines disease progression by controlling neutrophil motility and restricting hyperresponsiveness

    DEFF Research Database (Denmark)

    Jennings, Richard T; Strengert, Monika; Hayes, Patti

    2014-01-01

    Neutrophil responses are central to host protection and inflammation. Neutrophil activation follows a two-step process where priming amplifies responses to activating stimuli. Priming is essential for life span extension, chemotaxis and respiratory burst activity. Here we show that the cytoskeletal...... organizer RhoA suppresses neutrophil priming via formins. Premature granule exocytosis in Rho-deficient neutrophils activated numerous signaling pathways and amplified superoxide generation. Deletion of Rho altered front-to-back coordination by simultaneously increasing uropod elongation, leading edge...... neutrophils exacerbated LPS-mediated lung injury, deleting Rho in innate immune cells was highly protective in Influenza A virus infection. Hence, Rho is a key regulator of disease progression by maintaining neutrophil quiescence and suppressing hyperresponsiveness....

  5. Effect of sevoflurane on human neutrophil apoptosis.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND AND OBJECTIVE: Both chronic occupational exposure to volatile anaesthetic agents and acute in vitro exposure of neutrophils to isoflurane have been shown to inhibit the rate of apoptosis of human neutrophils. It is possible that inhibition of neutrophil apoptosis arises through delaying mitochondrial membrane potential collapse. We assessed mitochondrial depolarization and apoptosis in unexposed neutrophils and neutrophils exposed to sevoflurane in vivo. METHODS: A total of 20 mL venous blood was withdrawn pre- and postinduction of anaesthesia, the neutrophils isolated and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. Mitochondrial depolarization was measured using the dual emission styryl dye JC-1. RESULTS: Apoptosis was significantly inhibited in neutrophils exposed to sevoflurane in vivo at 24 (exposed: 38 (12)% versus control: 28 (11)%, P = 0.001), but not at 1 or 12 h, in culture. Mitochondrial depolarization was not delayed in neutrophils exposed to sevoflurane. CONCLUSIONS: The most important findings are that sevoflurane inhibits neutrophil apoptosis in vivo and that inhibition is not mediated primarily by an effect on mitochondrial depolarization.

  6. Sphingosine 1-phosphate induces neutrophil chemoattractant IL-8: repression by steroids.

    Directory of Open Access Journals (Sweden)

    Md Mostafizur Rahman

    Full Text Available The bioactive sphingolipid sphingosine 1-phosphate (S1P is found in increased amounts in the airways of asthmatics. S1P can regulate airway smooth muscle functions associated with asthmatic inflammation and remodeling, including cytokine secretion. To date however, whether S1P induces secretion of an important chemokine responsible for neutrophilia in airway inflammation--IL-8--was unexplored. The aim of this study was to investigate whether S1P induces IL-8 gene expression and secretion to enhance neutrophil chemotaxis in vitro, as well as examine the molecular mechanisms responsible for repression by the corticosteroid dexamethasone. We show that S1P upregulates IL-8 secretion from ASM cells and enhance neutrophil chemotaxis in vitro. The corticosteroid dexamethasone significantly represses IL-8 mRNA expression and protein secretion in a concentration- and time-dependent manner. Additionally, we reveal that S1P-induced IL-8 secretion is p38 MAPK and ERK-dependent and that these key phosphoproteins act on the downstream effector mitogen- and stress-activated kinase 1 (MSK1 to control secretion of the neutrophil chemoattractant cytokine IL-8. The functional relevance of this in vitro data was demonstrated by neutrophil chemotaxis assays where S1P-induced effects can be significantly attenuated by pretreatment with dexamethasone, pharmacological inhibition of p38 MAPK- or ERK-mediated pathways, or by knocking down MSK-1 with siRNA. Taken together, our study reveals the molecular pathways responsible for IL-8 secretion from ASM cells in response to S1P and indicates ways in which the impact on IL-8-driven neutrophilia may be lessened.

  7. Biomaterial associated impairment of local neutrophil function.

    Science.gov (United States)

    Kaplan, S S; Basford, R E; Kormos, R L; Hardesty, R L; Simmons, R L; Mora, E M; Cardona, M; Griffith, B L

    1990-01-01

    The effect of biomaterials on neutrophil function was studied in vitro to determine if these materials activated neutrophils and to determine the subsequent response of these neutrophils to further stimulation. Two biomaterials--polyurethane, a commonly used substance, and Velcro pile (used in the Jarvik 7 heart)--were evaluated. Two control substances, polyethylene and serum-coated polystyrene, were used for comparison. Neutrophil superoxide release was measured following incubation with these materials for 10, 30, and 120 min in the absence of additional stimulation and after stimulation with formylmethionylleucylphenylalanine (fMLP) or phorbol myristate acetate (PMA). The authors observed that the incubation of neutrophils on both polyurethane and Velcro resulted in substantially increased superoxide release that was greater after the 10 min than after the 30 or 120 min association. These activated neutrophils exhibited a poor additional response to fMLP but responded well to PMA. The effect of implantation of the Novacor left ventricular assist device on peripheral blood neutrophil function was also evaluated. The peripheral blood neutrophils exhibited normal superoxide release and chemotaxis. These studies suggest that biomaterials may have a profound local effect on neutrophils, which may predispose the patient to periprosthetic infection, but that the reactivity of circulating neutrophils is unimpaired.

  8. Neutrophilic respiratory tract inflammation and peripheral blood neutrophilia after grain sorghum dust extract challenge.

    Science.gov (United States)

    Von Essen, S G; O'Neill, D P; McGranaghan, S; Olenchock, S A; Rennard, S I

    1995-11-01

    To determine if inhalation of grain sorghum dust in the laboratory would cause neutrophilic upper and lower respiratory tract inflammation in human volunteers, as well as systemic signs of illness. Prospective. University of Nebraska Medical Center. Thirty normal volunteers. Inhalation challenge with 20 mL of a nebulized solution of filter-sterilized grain sorghum dust extract (GSDE). One group received prednisone, 20 mg for 2 days, prior to the challenge. Bronchoscopy with bronchoalveolar lavage (BAL) was performed 24 h after challenge, with samples collected as bronchial and alveolar fractions. Findings included visible signs of airways inflammation, quantified as the bronchitis index. The percentage of bronchial neutrophils was significantly increased in those challenged with GSDE vs the control solution, Hanks' balanced salt solution (40.3 +/- 4.5% vs 14.3 +/- 5.1%, p grain dust extract. To explain the increase in peripheral blood neutrophil counts, the capacity of the peripheral blood neutrophils to migrate in chemotaxis experiments was examined. The results demonstrate an increase in peripheral blood neutrophils and an increase in chemotactic responsiveness. Inhalation challenge with a grain dust extract causes respiratory tract inflammation and a peripheral blood neutrophilia. One reason for this may be an increase in activated peripheral blood neutrophils.

  9. Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro

    Directory of Open Access Journals (Sweden)

    Sophia C. Dudte

    2017-08-01

    Full Text Available Yersinia pestis is a gram-negative, zoonotic, bacterial pathogen, and the causative agent of plague. The bubonic form of plague occurs subsequent to deposition of bacteria in the skin by the bite of an infected flea. Neutrophils are recruited to the site of infection within the first few hours and interactions between neutrophils and Y. pestis have been demonstrated in vivo. In contrast to macrophages, neutrophils have been considered non-permissive to Y. pestis intracellular survival. Several studies have shown killing of the vast majority of Y. pestis ingested by human neutrophils. However, survival of 10–15% of Y. pestis after phagocytosis by neutrophils is consistently observed. Furthermore, these surviving bacteria eventually replicate within and escape from the neutrophils. We set out to further characterize the interactions between Y. pestis and human neutrophils by (1 determining the effects of known Y. pestis virulence factors on bacterial survival after uptake by neutrophils, (2 examining the mechanisms employed by the neutrophil to kill the majority of intracellular Y. pestis, (3 determining the activation phenotype of Y. pestis-infected neutrophils, and (4 characterizing the Y. pestis-containing phagosome in neutrophils. We infected human neutrophils in vitro with Y. pestis and assayed bacterial survival and uptake. Deletion of the caf1 gene responsible for F1 capsule production resulted in significantly increased uptake of Y. pestis. Surprisingly, while the two-component regulator PhoPQ system is important for survival of Y. pestis within neutrophils, pre-induction of this system prior to infection did not increase bacterial survival. We used an IPTG-inducible mCherry construct to distinguish viable from non-viable intracellular bacteria and determined the association of the Y. pestis-containing phagosome with neutrophil NADPH-oxidase and markers of primary, secondary and tertiary granules. Additionally, we show that inhibition of

  10. Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

    Science.gov (United States)

    Lima, Tatiane S; Gov, Lanny; Lodoen, Melissa B

    2018-02-13

    Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii -infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii- infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T

  11. Induction of hyperresponsiveness in human airway tissue by neutrophils--mechanism of action.

    Science.gov (United States)

    Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

    1996-05-01

    The two main features of asthma are bronchial hyperresponsiveness and inflammation. The inflammatory response in asthma consists of infiltration and activation of a variety of inflammatory cells including neutrophils. Our previous studies have shown that stimulated neutrophil supernatants cause hyperresponsiveness of human bronchial tissue in vitro. To investigate the effect of the sensitization status of the tissue and the albumin concentration used to prepare supernatants on the response of human bronchial tissue to stimulated neutrophil supernatants. Neutrophil supernatants were prepared from human isolated blood in the presence of varying concentrations of albumin (0%, 0.1% and 4%). Neutrophil supernatants were added to sensitized and non-sensitized human isolated bronchial tissue which was stimulated with electrical field stimulation (EFS) (20 s every 4 min). Receptor antagonists specific for the prostaglandin and thromboxane (10(-7) M GR32191), platelet activating factor (10(-6) M WEB 2086), leukotriene D4 (10(-6) M MK-679) and neurokinin A (10(-7) M SR48968) receptors were used to identify neutrophil products responsible for the effects observed in the bronchial tissue. In non-sensitized human bronchial tissue, stimulated neutrophil supernatants induced a direct contraction in the presence of 0% and 0.1% but not 4% albumin. This contraction was due to leukotriene D4 as MK-679 completely inhibited the contraction. In contrast, stimulated neutrophil supernatants increased responsiveness of sensitized human bronchial tissue to EFS. The increased responsiveness was observed only in the presence of 0.1% albumin, with the site of modulation likely to be prejunctional on the parasympathetic nerve. The increased responsiveness was not inhibited by any of the antagonists tested. Sensitization status of the tissue and albumin concentration effect the responsiveness of human bronchial tissue to stimulated neutrophil supernatant. Our results suggest a possible role for

  12. Intracellular Secretory Leukoprotease Inhibitor Modulates Inositol 1,4,5-Triphosphate Generation and Exerts an Anti-Inflammatory Effect on Neutrophils of Individuals with Cystic Fibrosis and Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Emer P. Reeves

    2013-01-01

    Full Text Available Secretory leukoprotease inhibitor (SLPI is an anti-inflammatory protein present in respiratory secretions. Whilst epithelial cell SLPI is extensively studied, neutrophil associated SLPI is poorly characterised. Neutrophil function including chemotaxis and degranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca2+ levels which is mediated by production of inositol 1,4,5-triphosphate (IP3 in response to G-protein-coupled receptor (GPCR stimuli. The aim of this study was to investigate the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease. Neutrophils were isolated from healthy controls (n=10, individuals with cystic fibrosis (CF (n=5 or chronic obstructive pulmonary disease (COPD (n=5. Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP and interleukin(IL-8 induced neutrophil chemotaxis (P<0.05 and decreased degranulation of matrix metalloprotease-9 (MMP-9, hCAP-18, and myeloperoxidase (MPO (P<0.05. The mechanism of inhibition involved modulation of cytosolic IP3 production and downstream Ca2+ flux. The described attenuation of Ca2+ flux was overcome by inclusion of exogenous IP3 in electropermeabilized cells. Inhibition of IP3 generation and Ca2+ flux by SLPI may represent a novel anti-inflammatory mechanism, thus strengthening the attractiveness of SLPI as a potential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF, non-CF bronchiectasis, and COPD.

  13. Regulation of calcium homeostasis in activated human neutrophils ...

    African Journals Online (AJOL)

    Objectives. The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model ...

  14. Swell activated chloride channel function in human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Salmon, Michael D. [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom); Ahluwalia, Jatinder, E-mail: j.ahluwalia@uel.ac.uk [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  15. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

    Science.gov (United States)

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  16. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

    Directory of Open Access Journals (Sweden)

    Omar Rafael Alemán

    2016-01-01

    Full Text Available Neutrophils (PMN are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  17. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  18. The effect of interleukin-8 and granulocyte macrophage colony stimulating factor on the response of neutrophils to formyl methionyl leucyl phenylalanine.

    Science.gov (United States)

    Mikami, M; Llewellyn-Jones, C G; Stockley, R A

    1998-08-14

    Neutrophils isolated from patients with chronic bronchitis and emphysema have been shown to have enhanced responses to formyl peptides when assessed in vitro compared to age, sex matched controls. It is currently unclear whether the observed differences are due to a 'priming' effect by a second agent in vivo, or whether this is a primary difference in the neutrophils. We have studied the effects of interleukin-8, which is thought to be one of the major pro-inflammatory cytokines in chronic lung disease and granulocyte macrophage colony stimulating factor (GMCSF), in order to assess their effects on neutrophil chemotaxis and connective tissue degradation. In addition, we have assessed the effect of preincubation of these agents with neutrophils for 30 min followed by stimulation with F-Met-Leu-Phe (FMLP) to investigate any possible 'priming' effect that may be relevant to our clinical data. We report suppression of neutrophil chemotaxis to FMLP following incubation of the neutrophils with both IL-8 and GMCSF. However, we have observed an additive effect of IL-8 and FMLP for neutrophil degranulation leading to fibronectin degradation. The results suggest that IL-8 does not 'prime' neutrophils for subsequent FMLP stimulation as observed in vivo. Although the results for GMCSF were similar for the chemotactic response, the agent also had a synergistic effect on connective tissue degradation. However, it is concluded that neither agent could explain the enhanced neutrophil responses seen in our patients.

  19. Tissue-transglutaminase contributes to neutrophil granulocyte differentiation and functions.

    Science.gov (United States)

    Balajthy, Zoltán; Csomós, Krisztián; Vámosi, György; Szántó, Attila; Lanotte, Michel; Fésüs, László

    2006-09-15

    Promyelocytic NB4 leukemia cells undergo differentiation to granulocytes following retinoic acid treatment. Here we report that tissue transglutaminase (TG2), a protein cross-linking enzyme, was induced, then partially translocated into the nucleus, and became strongly associated with the chromatin during the differentiation process. The transglutaminase-catalyzed cross-link content of both the cytosolic and the nuclear protein fractions increased while NB4 cells underwent cellular maturation. Inhibition of cross-linking activity of TG2 by monodansylcadaverin in these cells led to diminished nitroblue tetrazolium (NBT) positivity, production of less superoxide anion, and decreased expression of GP91PHOX, the membrane-associated subunit of NADPH oxidase. Neutrophils isolated from TG2(-/-) mice showed diminished NBT reduction capacity, reduced superoxide anion formation, and down-regulation of the gp91phox subunit of NADPH oxidase, compared with wild-type cells. It was also observed that TG2(-/-) mice exhibited increased neutrophil phagocytic activity, but had attenuated neutrophil chemotaxis and impaired neutrophil extravasation with higher neutrophil counts in their circulation during yeast extract-induced peritonitis. These results clearly suggest that TG2 may modulate the expression of genes related to neutrophil functions and is involved in several intracellular and extracellular functions of extravasating neutrophil.

  20. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    Energy Technology Data Exchange (ETDEWEB)

    Williams, C. David; Bajt, Mary Lynn [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Sharpe, Matthew R. [Department of Internal Medicine, University of Kansas Hospital, Kansas City, KS (United States); McGill, Mitchell R. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

  1. Inhibitory Effects of Standardized Extracts of Phyllanthus amarus and Phyllanthus urinaria and Their Marker Compounds on Phagocytic Activity of Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Yuandani

    2013-01-01

    Full Text Available The standardized methanol extracts of Phyllanthus amarus and P. urinaria, collected from Malaysia and Indonesia, and their isolated chemical markers, phyllanthin and hypophyllanthin, were evaluated for their effects on the chemotaxis, phagocytosis and chemiluminescence of human phagocytes. All the plant extracts strongly inhibited the migration of polymorphonuclear leukocytes (PMNs with the Malaysian P. amarus showing the strongest inhibitory activity (IC50 value, 1.1 µg/mL. There was moderate inhibition by the extracts of the bacteria engulfment by the phagocytes with the Malaysian P. amarus exhibiting the highest inhibition (50.8% of phagocytizing cells. The Malaysian P. amarus and P. urinaria showed strong reactive oxygen species (ROS inhibitory activity, with both extracts exhibiting IC50 value of 0.7 µg/mL. Phyllanthin and hypophyllanthin exhibited relatively strong activity against PMNs chemotaxis, with IC50 values slightly lower than that of ibuprofen (1.4 µg/mL. Phyllanthin exhibited strong inhibitory activity on the oxidative burst with an IC50 value comparable to that of aspirin (1.9 µg/mL. Phyllanthin exhibited strong engulfment inhibitory activity with percentage of phagocytizing cells of 14.2 and 27.1% for neutrophils and monocytes, respectively. The strong inhibitory activity of the extracts was due to the presence of high amounts of phyllanthin and hypophyllanthin although other constituents may also contribute.

  2. Ascorbic acid transport and accumulation in human neutrophils

    International Nuclear Information System (INIS)

    Washko, P.; Rotrosen, D.; Levine, M.

    1989-01-01

    The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake

  3. Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils.

    Science.gov (United States)

    Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

    2016-01-01

    IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.

  4. Peptide secreted by human alveolar macrophages releases neutrophil granule contents

    International Nuclear Information System (INIS)

    MacArthur, C.K.; Miller, E.J.; Cohen, A.B.

    1987-01-01

    A monoclonal antibody was developed against an 8000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose, and by autoradiography. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-γ, tumor necrosis factor, or interleukin lα or 1β. The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes

  5. Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

    International Nuclear Information System (INIS)

    Grob, P.M.; David, E.; Warren, T.C.; DeLeon, R.P.; Farina, P.R.; Homon, C.A.

    1990-01-01

    Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons

  6. Neutrophil-induced human bronchial hyperresponsiveness in vitro--pharmacological modulation.

    Science.gov (United States)

    Hughes, J M; McKay, K O; Johnson, P R; Tragoulias, S; Black, J L; Armour, C L

    1993-04-01

    Although it has been postulated that inflammatory cells cause the bronchial hyperresponsiveness which is diagnostic of asthma, until recently there has been little direct evidence of such a link. We have recently shown that calcium ionophore-activated human neutrophils and eosinophils can induce a state of human airway hyperresponsiveness in vitro. In this study we have shown that the anti-inflammatory agent nedocromil sodium, 10(-7) M, inhibited the hyperresponsiveness induced by products released from ionophore activated neutrophils but did not inhibit the release of leukotriene B4 from the same cells. Neutrophil-induced bronchial hyperresponsiveness was also inhibited by pre-treatment of the bronchial tissues with a thromboxane A2 and prostaglandin receptor antagonist, GR32191, 10(-7) M. These findings indicate that cyclooxygenase products are involved in bronchial hyperresponsiveness induced by inflammatory cell products in vitro and that their release can be inhibited by nedocromil sodium.

  7. Gβ1 is required for neutrophil migration in zebrafish.

    Science.gov (United States)

    Ke, Wenfan; Ye, Ding; Mersch, Kacey; Xu, Hui; Chen, Songhai; Lin, Fang

    2017-08-01

    Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gβ1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gβγ signaling, neutrophils failed to migrate to wound sites. Although both the Gβ1 and Gβ4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gβ1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gβ1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gβ1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gβ1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gβ isoform in chemotaxis in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. A designated centre for people with disabilities operated by Kingsriver Community Holdings Ltd, Kilkenny

    LENUS (Irish Health Repository)

    Bergin, David A

    2010-12-01

    Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

  9. Connolly Hospital (Silver Birch & Woodland Units), Blanchardstown, Dublin 15.

    LENUS (Irish Health Repository)

    Bergin, David A

    2010-12-01

    Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

  10. An exploratory study of the effect of regular aquatic exercise on the function of neutrophils from women with fibromyalgia: role of IL-8 and noradrenaline.

    Science.gov (United States)

    Bote, M E; García, J J; Hinchado, M D; Ortega, E

    2014-07-01

    Fibromyalgia (FM) syndrome is associated with elevated systemic inflammatory and stress biomarkers, and an elevated innate cellular response mediated by monocytes and neutrophils. Exercise is accepted as a good non-pharmacological therapy for FM. We have previously found that regular aquatic exercise decreases the release of inflammatory cytokines by monocytes from FM patients. However, its effects on the functional capacity of neutrophils have not been studied. The aim of the present exploratory study was to evaluate, in 10 women diagnosed with FM, the effect of an aquatic exercise program (8months, 2sessions/week, 60min/session) on their neutrophils' function (phagocytic process), and on IL-8 and NA as potential inflammatory and stress mediators, respectively. A control group of 10 inactive FM patients was included in the study. After 4months of the exercise program, no significant changes were observed in neutrophil function (chemotaxis, phagocytosis, or fungicidal capacity) or in IL-8 and NA. However, at the end of the exercise program (8months), a neuro-immuno-endocrine adaptation was observed, manifested by a significant decrease to values below those in the basal state in neutrophil chemotaxis, IL-8, and NA. No significant seasonal changes in these parameters were observed during the same period in the group of non-exercised FM patients. After the 8months of the exercise program, the FM patients had lower concentrations of IL-8 and NA together with reduced chemotaxis of neutrophils compared with the values determined in the same month in the control group of non-exercised FM women. These results suggest that "anti-inflammatory" and "anti-stress" adaptations may be contributing to the symptomatic benefits that have been attributed to regular aquatic exercise in FM syndrome, as was corroborated in the present study by the scores on the Fibromyalgia Impact Questionnaire. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Depression of efficiency of neutrophils for Candida albicans phagocytosis in personnel working in radiation field

    International Nuclear Information System (INIS)

    Hassan, A.A.

    2000-01-01

    The neutrophil functions, chemotaxis (direct and random migration), phagocytosis using Candida albicans (percent, index), phagocytosis by NBT (percent, score) and adherence were studied on 55 persons working in radiation field (group I) and 40 persons as control (group II). The effect of radiation on blood picture of persons working in this field with special references to leucocytic counts and neutrophil functions was studied. White and red cells counts were 6.275 +- 1.723 and 5.475 +- 1.039 (group I) and 6.440 +- 1.556, 4.704 +- 0.734 for group II, respectively with no significant difference, while in neutrophil function there was a statistically significant difference in all functions between two groups (P < 0.01). This indicates the importance of neutrophil functions in following up persons working in radiation field

  12. Effect of fluticasone propionate on neutrophil chemotaxis, superoxide generation, and extracellular proteolytic activity in vitro.

    Science.gov (United States)

    Llewellyn-Jones, C G; Hill, S L; Stockley, R A

    1994-03-01

    Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue.

  13. The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes

    Science.gov (United States)

    Ferrante, Antonio

    2017-01-01

    The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies. PMID:29164154

  14. Effects of Acrolein on Leukotriene Biosynthesis in Human Neutrophils

    OpenAIRE

    Zemski Berry, Karin A.; Henson, Peter M.; Murphy, Robert C.

    2008-01-01

    Acrolein is a toxic, highly reactive α,β-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-LO products in addition to small amounts of COX produc...

  15. Measuring Borrelia burgdorferi Motility and Chemotaxis.

    Science.gov (United States)

    Zhang, Kai; Li, Chunhao

    2018-01-01

    Swimming plate, cell motion tracking, and capillary tube assays are very useful tools to quantitatively measure bacterial motility and chemotaxis. These methods were modified and applied to study Borrelia burgdorferi motility and chemotaxis. By using these methods, numerous motility and chemotaxis mutants have been characterized and several chemoattractants were identified. With the assistance of these tools, the role of motility and chemotaxis in the pathogenicity of B. burgdorferi has been established. In addition, these tools also facilitate the study of motility and chemotaxis in other spirochetes.

  16. Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

    DEFF Research Database (Denmark)

    Jensen, T; Aliouat, E M; Lundgren, B

    1998-01-01

    The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

  17. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis

    Science.gov (United States)

    Ong, Catherine W. M.; Elkington, Paul T.; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T.; Tezera, Liku B.; Pabisiak, Przemyslaw J.; Moores, Rachel C.; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H.; Porter, Joanna C.; Friedland, Jon S.

    2015-01-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease. PMID:25996154

  18. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

    Science.gov (United States)

    Ong, Catherine W M; Elkington, Paul T; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T; Tezera, Liku B; Pabisiak, Przemyslaw J; Moores, Rachel C; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H; Porter, Joanna C; Friedland, Jon S

    2015-05-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

  19. Enhancement by platelets of oxygen radical responses of human neutrophils

    International Nuclear Information System (INIS)

    McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

    1986-01-01

    When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O- 2 and H 2 O 2 . This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O- 2 generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O- 2 responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils

  20. Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters.

    Science.gov (United States)

    Taylor, R F; Price, T H; Schwartz, S M; Dale, D C

    1981-01-01

    We have developed a technique for growing endothelial monolayers on micropore filters. These monolayers demonstrate confluence by phase and electron microscopy and provide a functional barrier to passage of radiolabeled albumin. Neutrophils readily penetrate the monolayer in response to chemotaxin, whereas there is little movement in the absence of chemotaxin. This system offers unique advantages over available chemotaxis assays and may have wider applications in the study of endothelial function. Images PMID:7007441

  1. Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils.

    Directory of Open Access Journals (Sweden)

    Luísa M D Magalhães

    Full Text Available Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs associated with Chagas' disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively. Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host's immune response and favor parasite survival.

  2. A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

    Science.gov (United States)

    Kamoshida, Go; Tansho-Nagakawa, Shigeru; Kikuchi-Ueda, Takane; Nakano, Ryuichi; Hikosaka, Kenji; Nishida, Satoshi; Ubagai, Tsuneyuki; Higashi, Shouichi; Ono, Yasuo

    2016-12-01

    Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target. © Society for Leukocyte Biology.

  3. Enhancement by platelets of oxygen radical responses of human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

    1986-03-01

    When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O-/sub 2/ and H/sub 2/O/sub 2/. This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O-/sub 2/ generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O-/sub 2/ responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils.

  4. Solar ultraviolet irradiation induces decorin degradation in human skin likely via neutrophil elastase.

    Science.gov (United States)

    Li, Yong; Xia, Wei; Liu, Ying; Remmer, Henriette A; Voorhees, John; Fisher, Gary J

    2013-01-01

    Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.

  5. Tasting Pseudomonas aeruginosa biofilms.Human neutrophils express the bitter receptor T2R38 as sensor for the quorum sensing molecule N-(3-oxododecanoyl-L-homoserine lactone

    Directory of Open Access Journals (Sweden)

    Susanne eMaurer

    2015-07-01

    Full Text Available Bacteria communicate with each other via specialized signalling molecules, known as quorum sensing molecules or autoinducers. The Pseudomonas aeruginosa-derived quorum sensing molecule N-(3-oxododecanoyl-L-homoserine lactone (AHL-12, however, also activates mammalian cells. As shown previously, AHL-12 induced chemotaxis, up-regulated CD11b expression, and enhanced phagocytosis of polymorphonuclear neutrophils (PMN. Circumstantial evidence concurred with a receptor for AHL-12, which so far has been elusive. We investigated the bitter receptor T2R38 as a potential candidate. Although identified as a taste receptor, cells outside the gustatory system express T2R38, for example epithelial cells in the lung. We now detected T2R38 in peripheral blood neutrophils, monocytes and lymphocytes on the cell membrane, but also intracellular. In neutrophils, T2R38 was located in vesicles with characteristics of lipid droplets, and super-resolution microscopy showed a co-localisation with the lipid droplet membrane. Neutrophils take up AHL-12, and it co-localized with T2R38 as seen by laser scan microscopy. Binding of AHL-12 to T2R28 was confirmed by pull-down assays using biotin-coupled AHL-12 as bait. A commercially available antibody to T2R38 inhibited binding of AHL-12 to neutrophils, and this antibody by itself stimulated neutrophils, similarly to AHL-12. In conclusion, our data provide evidence for expression of functional T2R38 on neutrophils, and are compatible with the notion that T2R38 is the receptor for AHL-12 on neutrophils.

  6. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

    Science.gov (United States)

    Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

    2017-11-09

    CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

  7. Nucleobindin co-localizes and associates with cyclooxygenase (COX-2 in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Patrick Leclerc

    2008-05-01

    Full Text Available The inducible cyclooxygenase isoform (COX-2 is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc, a ubiquitous Ca(2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

  8. Indium-111 labeling of leukocytes: a detrimental effect on neutrophil and lymphocyte function and an improved method of cell labelling

    International Nuclear Information System (INIS)

    Segal, A.W.; Deteix, P.; Garcia, R.; Tooth, P.; Zanelli, G.D.; Allison, A.C.

    1978-01-01

    A technique for the labeling of cells with the gamma emitter indium-111 has recently been developed. In this study the effects of the labeling procedure on some in vitro functions of human neutrophils and lymphocytes were investigated. With the standard labeling procedure, neutrophil chemotaxis was reduced to approximately 50% of normal and lymphocytes lost surface receptors and failed to respond to stimulation with phytohemagglutinin. The 8-hydroxyquinoline that is used to chelate the indium is toxic to lymphocytes; accordingly the relationship between the quantity of oxine, the chelation of indium, and cell labeling were investigated. Optimal conditions for In-111 cell labeling were established: 100 million cells in 10 ml Hanks' balanced salt solution are mixed with 5 μg of oxine in a mixture of 50 μl of ethanol and 200 μl of saline; they are incubated at 37 0 C for 10 min and then washed. Initially, neutrophils and lymphocytes appear functionally normal, but after 24 to 48 hr lymphocyte function is impaired as a result of radiation damage. This toxicity may limit studies by external scanning on the distribution and kinetics of lymphocytes labeled with In-111

  9. Dictyostelium Chemotaxis studied with fluorescence fluctuation spectroscopy

    NARCIS (Netherlands)

    Ruchira, A.

    2005-01-01

    The movement of cells in the direction of a chemical gradient, also known as chemotaxis, is a vital biological process. During chemotaxis, minute extracellular signals are translated into complex cellular responses such as change in morphology and motility. To understand the chemotaxis mechanism at

  10. Effects of acrolein on leukotriene biosynthesis in human neutrophils.

    Science.gov (United States)

    Berry, Karin A Zemski; Henson, Peter M; Murphy, Robert C

    2008-12-01

    Acrolein is a toxic, highly reactive alpha,beta-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-lipoxygenase (5-LO) products in addition to small amounts of cyclooxygenase (COX) products were detected using LC/MS/MS. A dose-dependent decrease in the formation of 5-LO products was observed in GM-CSF/fMLP-stimulated neutrophils when acrolein (0-50 microM) was present with almost complete inhibition at > or = 25 microM acrolein. The production of COX products was not affected by acrolein in these cells. The effect of acrolein was examined on key parts of the eicosanoid pathway, such as arachidonic acid release, intracellular calcium ion concentration, and adenosine production. In addition, the direct effect of acrolein on 5-LO enzymatic activity was probed using a recombinant enzyme. Some of these factors were affected by acrolein but did not completely explain the almost complete inhibition of 5-LO product formation in GM-CSF/fMLP-treated cells with acrolein. In addition, the effect of acrolein on different stimuli that initiate the 5-LO pathway [platelet-activating factor (PAF)/fMLP, GM-CSF/PAF, opsonized zymosan, and A23187] was examined. Acrolein had no significant effect on the leukotriene production in neutrophils stimulated with PAF/fMLP, GM-CSF/ PAF, or OPZ. Additionally, 50% inhibition of the 5-LO pathway was observed in A23187-stimulated neutrophils. Our results suggest that acrolein has a profound effect on the 5-LO pathway in neutrophils, which may have implications in disease states, such as chronic obstructive pulmonary disease and other pulmonary disease, where both activated neutrophils and acrolein are

  11. Pharmacological intervention with oxidative burst in human neutrophils

    Czech Academy of Sciences Publication Activity Database

    Nosál, R.; Drábiková, K.; Jančinová, V.; Mačičková, T.; Pečivová, J.; Perečko, T.; Harmatha, Juraj

    2017-01-01

    Roč. 10, č. 2 (2017), s. 56-60 ISSN 1337-6853 Institutional support: RVO:61388963 Keywords : human neutrophils * oxidative burst * tharapeutical drugs * natural antioxidants Subject RIV: FR - Pharmacology ; Medidal Chemistry OBOR OECD: Pharmacology and pharmacy https://www.degruyter.com/downloadpdf/j/intox.2017.10.issue-2/intox-2017-0009/intox-2017-0009.pdf

  12. Products of neutrophils and eosinophils increase the responsiveness of human isolated bronchial tissue.

    Science.gov (United States)

    Hallahan, A R; Armour, C L; Black, J L

    1990-05-01

    This study examines the possibility that products of neutrophils and eosinophils could increase the responsiveness of human isolated bronchial tissue. Neutrophils and eosinophils were isolated from the peripheral blood of healthy volunteers. The cells were incubated with 1 microM calcium ionophore A23187 for 10-15 min then centrifuged, the supernatant collected and stored at -70 degrees C. Human bronchial rings (2-3 mm diameter, 3-4 mm long) were prepared from specimens resected at thoracotomy. The tissues were suspended in organ baths under a 1 g load and changes in tension measured isometrically. Stable contractions to bolus doses of histamine (0.1-10 microM) or to electrical field stimulation (40-100 V, 4-16 Hz, 1 ms for 20 s) were established. Supernatant from 106 neutrophils or 105 eosinophils was then added and tissue responsiveness reassessed. Neutrophil supernatant increased tissue responsiveness to histamine and electrical field stimulation by 54 +/- 17% (n = 5, p less than 0.05) and 18 +/- 7% (n = 6, p less than 0.05), respectively. Eosinophil supernatant increased the histamine response by 60 +/- 23% (n = 8, p less than 0.05) while tissue responsiveness to electrical field stimulation was unchanged (n = 3). Thus, as neutrophils and eosinophils can change the responsiveness of human bronchus in vitro it is possible that they do this in vivo and may not simply be temporally related to the development of bronchial hyperresponsiveness.

  13. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    International Nuclear Information System (INIS)

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-01-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51 Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51 Cr release from radiolabeled monolayers

  14. A novel antagonist of CRTH2 blocks eosinophil release from bone marrow, chemotaxis and respiratory burst

    DEFF Research Database (Denmark)

    Royer, J F; Schratl, P; Lorenz, S

    2007-01-01

    developed small molecule antagonist of CRTH2, Cay10471, on eosinophil function with respect to recruitment, respiratory burst and degranulation. METHODS: Chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils were determined using microBoyden chambers. Eosinophil release...... from bone marrow was investigated in the in situ perfused guinea pig hind limb preparation. Respiratory burst and degranulation were measured by flow cytometry. RESULTS: Cay10471 bound with high affinity to recombinant human and guinea pig CRTH2, but not DP, receptors. The antagonist prevented the PGD......(2)-induced release of eosinophils from guinea pig bone marrow, and inhibited the chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils. Pretreatment with PGD(2) primed eosinophils for chemotaxis towards eotaxin, and this effect was prevented by Cay10471. In contrast...

  15. A portable chemotaxis platform for short and long term analysis.

    Directory of Open Access Journals (Sweden)

    Chenjie Xu

    Full Text Available Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours and long-term (72 hours concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.

  16. A real time chemotaxis assay unveils unique migratory profiles amongst different primary murine macrophages.

    Directory of Open Access Journals (Sweden)

    Asif J Iqbal

    Full Text Available Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14(+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

  17. A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

    Science.gov (United States)

    Iqbal, Asif J.; Regan-Komito, Daniel; Christou, Ivy; White, Gemma E.; McNeill, Eileen; Kenyon, Amy; Taylor, Lewis; Kapellos, Theodore S.; Fisher, Edward A.; Channon, Keith M.; Greaves, David R.

    2013-01-01

    Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators. PMID:23516549

  18. tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori.

    Science.gov (United States)

    Cerda, Oscar A; Núñez-Villena, Felipe; Soto, Sarita E; Ugalde, José Manuel; López-Solís, Remigio; Toledo, Héctor

    2011-01-01

    About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.

  19. tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Oscar A Cerda

    2011-01-01

    Full Text Available About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.

  20. On the Pharmacology of Oxidative Burst of Human Neutrophils

    Czech Academy of Sciences Publication Activity Database

    Nosáľ, R.; Drábiková, K.; Jančinová, V.; Mačičková, T.; Pečivová, J.; Perečko, T.; Harmatha, Juraj; Šmidrkal, J.

    2015-01-01

    Roč. 64, Suppl 4 (2015), S445-S452 ISSN 0862-8408 Institutional support: RVO:61388963 Keywords : human neutrophils * oxidative burst * chemiluminescence * protein kinase C * apoptosis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.643, year: 2015 http://www.biomed.cas.cz/physiolres/pdf/64/64_S445.pdf

  1. Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis.

    Science.gov (United States)

    Sochalska, Maja; Potempa, Jan

    2017-01-01

    The pathogenesis of the chronic periodontal disease is associated with a skewed host inflammatory response to periodontal pathogens, such as Porphyromonas gingivalis , that accounts for the majority of periodontal tissue damage. Neutrophils are the most abundant leukocytes in periodontal pockets and depending on the stage of the disease, also plentiful PMNs are present in the inflamed gingival tissue and the gingival crevice. They are the most efficient phagocytes and eliminate pathogens by a variety of means, which are either oxygen-dependent or -independent. However, these secretory lethal weapons do not strictly discriminate between pathogens and host tissue. Current studies describe conflicting findings about neutrophil involvement in periodontal disease. On one hand literature indicate that hyper-reactive neutrophils are the main immune cell type responsible for this observed tissue damage and disease progression. Deregulation of neutrophil survival and functions, such as chemotaxis, migration, secretion of antimicrobial peptides or enzymes, and production of reactive oxygen species, contribute to observed tissue injury and the clinical signs of periodontal disease. On the other hand neutrophils deficiencies in patients and mice also result in periodontal phenotype. Therefore, P. gingivalis represents a periodontal pathogen that manipulates the immune responses of PMNs, employing several virulence factors, such as gingipains, serine proteases, lipid phosphatases, or fimbriae. This review will sum up studies devoted to understanding different strategies utilized by P. gingivalis to manipulate PMNs survival and functions in order to inhibit killing by a granular content, prolong inflammation, and gain access to nutrient resources.

  2. Attenuated, oncolytic, but not wild-type measles virus infection has pleiotropic effects on human neutrophil function.

    Science.gov (United States)

    Zhang, Yu; Patel, Bella; Dey, Aditi; Ghorani, Ehsan; Rai, Lena; Elham, Mohammed; Castleton, Anna Z; Fielding, Adele K

    2012-02-01

    We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

  3. Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

    Directory of Open Access Journals (Sweden)

    Matsunaga Kazuto

    2009-06-01

    Full Text Available Abstract Background Excessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro. Methods Human peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848 in the presence or absence of hydrogen peroxide (H2O2. Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed. Results Activation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H2O2 (p L-cysteine reversed this potentiation. The combination of H2O2 and R848 significantly potentiated NF-kB phosphorylation and IkBα degradation. The H2O2-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88, and tumor necrosis factor receptor-associated factor 6 (TRAF6 were not affected by H2O2. Conclusion TLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.

  4. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis.

    Science.gov (United States)

    Bonne-Année, Sandra; Kerepesi, Laura A; Hess, Jessica A; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B; Nolan, Thomas J; Abraham, David

    2014-06-01

    Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...... subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes....

  6. Staphylococcus epidermidis strategies to avoid killing by human neutrophils.

    Directory of Open Access Journals (Sweden)

    Gordon Y C Cheung

    2010-10-01

    Full Text Available Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs, including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS. These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.

  7. CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways.

    Science.gov (United States)

    Shahabuddin, Syed; Ji, Rong; Wang, Ping; Brailoiu, Eugene; Dun, Na; Yang, Yi; Aksoy, Mark O; Kelsen, Steven G

    2006-07-01

    Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 +/- 88% (mean +/- SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca(2+)] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5-10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

  8. Staphylococcus aureus panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils.

    Directory of Open Access Journals (Sweden)

    Bettina Löffler

    2010-01-01

    Full Text Available The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs, a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins, induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

  9. Deciphering chemotaxis pathways using cross species comparisons

    Directory of Open Access Journals (Sweden)

    Armitage Judith P

    2010-01-01

    Full Text Available Abstract Background Chemotaxis is the process by which motile bacteria sense their chemical environment and move towards more favourable conditions. Escherichia coli utilises a single sensory pathway, but little is known about signalling pathways in species with more complex systems. Results To investigate whether chemotaxis pathways in other bacteria follow the E. coli paradigm, we analysed 206 species encoding at least 1 homologue of each of the 5 core chemotaxis proteins (CheA, CheB, CheR, CheW and CheY. 61 species encode more than one of all of these 5 proteins, suggesting they have multiple chemotaxis pathways. Operon information is not available for most bacteria, so we developed a novel statistical approach to cluster che genes into putative operons. Using operon-based models, we reconstructed putative chemotaxis pathways for all 206 species. We show that cheA-cheW and cheR-cheB have strong preferences to occur in the same operon as two-gene blocks, which may reflect a functional requirement for co-transcription. However, other che genes, most notably cheY, are more dispersed on the genome. Comparison of our operons with shuffled equivalents demonstrates that specific patterns of genomic location may be a determining factor for the observed in vivo chemotaxis pathways. We then examined the chemotaxis pathways of Rhodobacter sphaeroides. Here, the PpfA protein is known to be critical for correct partitioning of proteins in the cytoplasmically-localised pathway. We found ppfA in che operons of many species, suggesting that partitioning of cytoplasmic Che protein clusters is common. We also examined the apparently non-typical chemotaxis components, CheA3, CheA4 and CheY6. We found that though variants of CheA proteins are rare, the CheY6 variant may be a common type of CheY, with a significantly disordered C-terminal region which may be functionally significant. Conclusions We find that many bacterial species potentially have multiple

  10. The Response of Macrophages and Neutrophils to Hypoxia in the Context of Cancer and Other Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Antje Egners

    2016-01-01

    Full Text Available Lack of oxygen (hypoxia is a hallmark of a multitude of acute and chronic diseases and can be either beneficial or detrimental for organ restitution and recovery. In the context of inflammation, hypoxia is particularly important and can significantly influence the course of inflammatory diseases. Macrophages and neutrophils, the chief cellular components of innate immunity, display distinct properties when exposed to hypoxic conditions. Virtually every aspect of macrophage and neutrophil function is affected by hypoxia, amongst others, morphology, migration, chemotaxis, adherence to endothelial cells, bacterial killing, differentiation/polarization, and protumorigenic activity. Prominent arenas of macrophage and neutrophil function, for example, acute/chronic inflammation and the microenvironment of solid tumors, are characterized by low oxygen levels, demonstrating the paramount importance of the hypoxic response for proper function of these cells. Members of the hypoxia-inducible transcription factor (HIF family emerged as pivotal molecular regulators of macrophages and neutrophils. In this review, we will summarize the molecular responses of macrophages and neutrophils to hypoxia in the context of cancer and other chronic inflammatory diseases and discuss the potential avenues for therapeutic intervention that arise from this knowledge.

  11. Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation

    NARCIS (Netherlands)

    Wanten, G. J.; Geijtenbeek, T. B.; Raymakers, R. A.; van Kooyk, Y.; Roos, D.; Jansen, J. B.; Naber, A. H.

    2000-01-01

    BACKGROUND: To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. METHODS: Neutrophils, isolated from the blood of 10 healthy volunteers, were

  12. Plant Natural Products Calycosin and Gallic Acid Synergistically Attenuate Neutrophil Infiltration and Subsequent Injury in Isoproterenol-Induced Myocardial Infarction: A Possible Role for Leukotriene B4 12-Hydroxydehydrogenase?

    Science.gov (United States)

    Cheng, Yuanyuan; Tse, Hung Fat; Le, X. Chris; Rong, Jianhui

    2015-01-01

    Leukotriene B4 12-hydroxydehydrogenase (LTB4DH) catalyzes the oxidation of proinflammatory LTB4 into less bioactive 12-oxo-LTB4. We recently discovered that LTB4DH was induced by two different natural products in combination. We previously isolated gallic acid from Radix Paeoniae through a bioactivity-guided fractionation procedure. The purpose of this study is to test the hypothesis that LTB4DH inducers may suppress neutrophil-mediated inflammation in myocardial infarction. We first isolated the active compound(s) from another plant, Radix Astragali, by the similar strategy. By evaluating LTB4DH induction, we identified calycosin and formononetin from Radix Astragali by HPLC-ESI-MS technique. We confirmed that gallic acid and commercial calycosin or formononetin could synergistically induce LTB4DH expression in HepG2 cells and human neutrophils. Moreover, calycosin and gallic acid attenuated the effects of LTB4 on the survival and chemotaxis of neutrophil cell culture. We further demonstrated that calycosin and gallic acid synergistically suppressed neutrophil infiltration and protected cardiac integrity in the isoproterenol-induced mice model of myocardial infarction. Calycosin and gallic acid dramatically suppressed isoproterenol-induced increase in myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Collectively, our results suggest that LTB4DH inducers (i.e., calycosin and gallic acid) may be a novel combined therapy for the treatment of neutrophil-mediated myocardial injury. PMID:26265982

  13. Strenuous physical exercise adversely affects monocyte chemotaxis

    DEFF Research Database (Denmark)

    Czepluch, Frauke S; Barres, Romain; Caidahl, Kenneth

    2011-01-01

    Physical exercise is important for proper cardiovascular function and disease prevention, but it may influence the immune system. We evaluated the effect of strenuous exercise on monocyte chemotaxis. Monocytes were isolated from blood of 13 young, healthy, sedentary individuals participating...... in a three-week training program which consisted of repeated exercise bouts. Monocyte chemotaxis and serological biomarkers were investigated at baseline, after three weeks training and after four weeks recovery. Chemotaxis towards vascular endothelial growth factor-A (VEGF-A) and transforming growth factor...

  14. Synchronisation of glycolytic oscillations in a suspension of human neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Poulsen, Allan K.; Olsen, Lars Folke

    Neutrophils are known to be able to synchronize their production of superoxide. We show that glycolysis is also synchronized in human neutrophils being in suspension and suggest that oscillations in glycolysis are driving the pulsatile production of superoxide. The synchronising agent remains so...... far unknown, however, much evident points to that it might be hydrogen peroxide or an intermediate in glycolysis....

  15. Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis

    Directory of Open Access Journals (Sweden)

    Maja Sochalska

    2017-05-01

    Full Text Available The pathogenesis of the chronic periodontal disease is associated with a skewed host inflammatory response to periodontal pathogens, such as Porphyromonas gingivalis, that accounts for the majority of periodontal tissue damage. Neutrophils are the most abundant leukocytes in periodontal pockets and depending on the stage of the disease, also plentiful PMNs are present in the inflamed gingival tissue and the gingival crevice. They are the most efficient phagocytes and eliminate pathogens by a variety of means, which are either oxygen-dependent or -independent. However, these secretory lethal weapons do not strictly discriminate between pathogens and host tissue. Current studies describe conflicting findings about neutrophil involvement in periodontal disease. On one hand literature indicate that hyper-reactive neutrophils are the main immune cell type responsible for this observed tissue damage and disease progression. Deregulation of neutrophil survival and functions, such as chemotaxis, migration, secretion of antimicrobial peptides or enzymes, and production of reactive oxygen species, contribute to observed tissue injury and the clinical signs of periodontal disease. On the other hand neutrophils deficiencies in patients and mice also result in periodontal phenotype. Therefore, P. gingivalis represents a periodontal pathogen that manipulates the immune responses of PMNs, employing several virulence factors, such as gingipains, serine proteases, lipid phosphatases, or fimbriae. This review will sum up studies devoted to understanding different strategies utilized by P. gingivalis to manipulate PMNs survival and functions in order to inhibit killing by a granular content, prolong inflammation, and gain access to nutrient resources.

  16. Radioassay of granulocyte chemotaxis. Studies of human granulocytes and chemotactic factors. [/sup 51/Cr tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Gallin, J I

    1974-01-01

    The above studies demonstrate that the /sup 51/Cr radiolabel chemotactic assay is a relatively simple and objective means for studying leukocyte chemotaxis in both normal and pathological conditions. Application of this method to studies of normal human chemotaxis revealed a relatively narrow range of normal and little day-to-day variability. Analysis of this variability revealed that there is more variability among the response of different granulocytes to a constant chemotactic stimulus than among the chemotactic activity of different sera to a single cell source. Utilizing the /sup 51/Cr radioassay, the abnormal granulocyte chemotactic behavior reported in Chediak-Higashi syndrome and a patient with recurrent pyogenic infections and mucocutaneous candidiasis has been confirmed. The /sup 51/Cr chemotactic assay has also been used to assess the generation of chemotactic activity from human serum and plasma. The in vitro generation of two distinct chemotactic factors were examined; the complement product (C5a) and kallikrein, an enzyme of the kinin-generating pathway. Kinetic analysis of complement-related chemotactic factor formation, utilizing immune complexes or endotoxin to activate normal sera in the presence or absence of EGTA as well as kinetic analysis of activation of C2-deficient human serum, provided an easy means of distinguishing the classical (antibody-mediated) complement pathway from the alternate pathway. Such kinetic analysis is necessary to detect clinically important abnormalities since, after 60 min of generation time, normal chemotactic activity may be present despite complete absence or inhibition of one complement pathway. The chemotactic factor generated by either pathway of complement activation appears to be predominately attributable to C5a.

  17. Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice

    Science.gov (United States)

    Suidan, Georgette L.; Demers, Melanie; Herr, Nadine; Carbo, Carla; Brill, Alexander; Cifuni, Stephen M.; Mauler, Maximilian; Cicko, Sanja; Bader, Michael; Idzko, Marco; Bode, Christoph

    2013-01-01

    The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1–deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1−/− mice. The velocity of rolling leukocytes was higher in Tph1−/− mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1−/− mice. Diminished rolling in Tph1−/− mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1−/− mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1−/− mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity. PMID:23243271

  18. In vivo study of indomethacin in bronchiectasis: effect on neutrophil function and lung secretion.

    Science.gov (United States)

    Llewellyn-Jones, C G; Johnson, M M; Mitchell, J L; Pye, A; Okafor, V C; Hill, S L; Stockley, R A

    1995-09-01

    Bronchiectasis is associated with sputum containing high levels of the proteolytic enzyme elastase, which is thought to be involved in the pathogenesis of the disease. Agents which inhibit neutrophil function and interfere with neutrophil elastase release may have a beneficial effect on the development and progression of such diseases. We have studied the effects of the nonsteroidal anti-inflammatory agent indomethacin on neutrophil function in nine patients with clinically stable bronchiectasis. All patients remained clinically stable during the study. We observed a significant reduction in peripheral neutrophil chemotaxis to 10 nmol.L-1 N-formyl-methionyl-leucyl-phenylalanine (FMLP) from a mean of 19.86 (SEM 1.35) to 8.46 (0.68) cells.field-1 after 4 weeks of therapy. There was also a significant reduction in fibronectin degradation both by resting and FMLP-stimulated neutrophils, from a mean of 1.90 (0.19) micrograms x 3 x 10(5) cells at the start of therapy to 0.87 (0.08) micrograms after 4 weeks, and from 3.17 (0.35) micrograms to 1.48 (0.05) micrograms, respectively. There was no effect on spontaneous or stimulated superoxide anion generation by neutrophils. Despite the marked changes in peripheral neutrophil function, no adverse effect was observed on viable bacterial load in the bronchial secretions. In addition, there was no difference in sputum albumin, elastase or myeloperoxidase levels, and only minor changes in the chemotactic activity of the sputum. These results suggest that nonsteroidal anti-inflammatory agents have a major effect on peripheral neutrophil function but do not appear to have an adverse effect on bacterial colonization of the airways.

  19. Sulfur mustard primes human neutrophils for increased degranulation and stimulates cytokine release via TRPM2/p38 MAPK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ham, Hwa-Yong [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Hong, Chang-Won, E-mail: chyj7983@hallym.ac.kr [Department of Chemical and Biological Warfare Research, The Armed Forces Medical Research Institute, Daejeon (Korea, Republic of); Lee, Si-Nae [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Kwon, Min-Soo [Department of Pharmacology, School of Medicine, CHA University, Seongnam (Korea, Republic of); Kim, Yeon-Ja [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Song, Dong-Keun, E-mail: dksong@hallym.ac.kr [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of)

    2012-01-01

    Sulfur mustard (2,2′-bis-chloroethyl-sulfide; SM) has been a military threat since the World War I. The emerging threat of bioterrorism makes SM a major threat not only to military but also to civilian world. SM injury elicits an inflammatory response characterized by infiltration of neutrophils. Although SM was reported to prime neutrophils, the mechanism has not been identified yet. In the present study, we investigated the mechanism of SM-induced priming in human neutrophils. SM increased [Ca{sup 2+}]{sub i} in human neutrophils in a concentration-dependent fashion. Transient receptor potential melastatin (TRPM) 2 inhibitors (clotrimazole, econazole and flufenamic acid) and silencing of TRPM2 by shRNA attenuated SM-induced [Ca{sup 2+}]{sub i} increase. SM primed degranulation of azurophil and specific granules in response to activation by fMLP as previously reported. SB203580, an inhibitor of p38 MAPK, inhibited SM-induced priming. Neither PD98057, an ERK inhibitor, nor SP600215, a JNK inhibitor, inhibited SM-induced priming. In addition, SM enhanced phosphorylation of NF-kB p65 and release of TNF-α, interleukin (IL)-6 and IL-8. SB203580 inhibited SM-induced NF-kB phosphorylation and cytokine release. These results suggest the involvement of TRPM2/p38 MAPK pathway in SM-induced priming and cytokines release in neutrophils. -- Highlights: ► SM increased [Ca{sup 2+}]{sub i} in human neutrophils through TPRM2-mediated calcium influx. ► SM primed degranulation of azurophil and specific granules. ► SM enhanced p38 MAPK and NF-κB p65 phosphorylation in human neutrophils. ► SM enhanced release of TNF-α, interleukin (IL)-6 and IL-8 from human neutrophils. ► SB203580 inhibited SM-induced priming, NF-κB p65 phosphorylation and cytokine release.

  20. A coupled chemotaxis-fluid model: Global existence

    KAUST Repository

    Liu, Jian-Guo; Lorz, Alexander

    2011-01-01

    We consider a model arising from biology, consisting of chemotaxis equations coupled to viscous incompressible fluid equations through transport and external forcing. Global existence of solutions to the Cauchy problem is investigated under certain conditions. Precisely, for the chemotaxis-Navier- Stokes system in two space dimensions, we obtain global existence for large data. In three space dimensions, we prove global existence of weak solutions for the chemotaxis-Stokes system with nonlinear diffusion for the cell density.© 2011 Elsevier Masson SAS. All rights reserved.

  1. A coupled chemotaxis-fluid model: Global existence

    KAUST Repository

    Liu, Jian-Guo

    2011-09-01

    We consider a model arising from biology, consisting of chemotaxis equations coupled to viscous incompressible fluid equations through transport and external forcing. Global existence of solutions to the Cauchy problem is investigated under certain conditions. Precisely, for the chemotaxis-Navier- Stokes system in two space dimensions, we obtain global existence for large data. In three space dimensions, we prove global existence of weak solutions for the chemotaxis-Stokes system with nonlinear diffusion for the cell density.© 2011 Elsevier Masson SAS. All rights reserved.

  2. Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

    Science.gov (United States)

    Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

    2005-01-01

    The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

  3. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Directory of Open Access Journals (Sweden)

    Zofia Sulowska

    2005-01-01

    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  4. Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Ma, Fang; Yi, Li; Yu, Ningwei; Wang, Guangyu; Ma, Zhe; Lin, Huixing; Fan, Hongjie

    2017-01-01

    Invasive infections caused by Streptococcus suis serotype 2 (SS2) has emerged as a clinical problem in recent years. Neutrophil extracellular traps (NETs) are an important mechanism for the trapping and killing of pathogens that are resistant to phagocytosis. Biofilm formation can protect bacteria from being killed by phagocytes. Until now, there have only been a few studies that focused on the interactions between bacterial biofilms and NETs. SS2 in both a biofilm state and a planktonic cell state were incubated with phagocytes and NETs, and bacterial survival was assessed. DNase I and cytochalasin B were used to degrade NET DNA or suppress phagocytosis, respectively. Extracellular DNA was stained with impermeable fluorescent dye to quantify NET formation. Biofilm formation increased up to 6-fold in the presence of neutrophils, and biofilms were identified in murine tissue. Both planktonic and biofilm cells induced neutrophils chemotaxis to the infection site, with neutrophils increasing by 85.1 and 73.8%, respectively. The bacteria in biofilms were not phagocytized. The bactericidal efficacy of NETs on the biofilms and planktonic cells were equal; however, the biofilm extracellular matrix can inhibit NET release. Although biofilms inhibit NETs release, NETs appear to be an important mechanism to eliminate SS2 biofilms. This knowledge advances the understanding of biofilms and may aid in the development of treatments for persistent infections with a biofilm component.

  5. Anti-inflammatory effect of Schinus terebinthifolius Raddi hydroalcoholic extract on neutrophil migration in zymosan-induced arthritis.

    Science.gov (United States)

    Rosas, Elaine Cruz; Correa, Luana Barbosa; Pádua, Tatiana de Almeida; Costa, Thadeu Estevam Moreira Maramaldo; Mazzei, José Luiz; Heringer, Alan Patrick; Bizarro, Carlos Alberto; Kaplan, Maria Auxiliadora Coelho; Figueiredo, Maria Raquel; Henriques, Maria G

    2015-12-04

    Schinus terebinthifolius is a species of plant from the Anacardiaceae family, which can be found in different regions of Brazil. Schinus is popularly known as aroeirinha, aroeira-vermelha, or Brazilian pepper. In folk medicine, S. terebinthifolius is used for several disorders, including inflammatory conditions, skin wounds, mucosal membrane ulcers, respiratory problems, gout, tumors, diarrhea and arthritis. According to chemical analyses, gallic acid, methyl gallate and pentagalloylglucose are the main components of hydroalcoholic extracts from S. terebinthifolius leaves. In the present study, we demonstrated the ability of a hydroalcoholic extract to inhibit cell migration in arthritis and investigated the mechanisms underlying this phenomenon. The anti-inflammatory effect of S. terebinthifolius hydroalcoholic leaf extract (ST-70) was investigated in a zymosan-induced experimental model of inflammation. Male Swiss and C57Bl/6 mice received zymosan (100 µg/cavity) via intra-thoracic (i.t.) or intra-articular (i.a.) injection after oral pre-treatment with ST-70. The direct action of ST-70 on neutrophils was evaluated via chemotaxis. ST-70 exhibited a dose-dependent effect in the pleurisy model. The median effective dose (ED50) was 100mg/kg, which inhibited 70% of neutrophil accumulation when compared with the control group. ST-70 reduced joint diameter and neutrophil influx for synovial tissues at 6h and 24h in zymosan-induced arthritis. Additionally, ST-70 inhibited synovial interleukin (IL)-6, IL-1β, keratinocyte-derived chemokine (CXCL1/KC) and Tumor Necrosis Factor (TNF)-α production at 6h and CXCL1/KC and IL-1β production at 24h. The direct activity of ST-70 on neutrophils was observed via the impairment of CXCL1/KC-induced chemotaxis in neutrophils. Oral administration of ST-70 did not induce gastric damage. Daily administration for twenty days did not kill any animals. In contrast, similar administrations of diclofenac induced gastric damage and killed

  6. Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion.

    Directory of Open Access Journals (Sweden)

    Zuopeng Wu

    2016-05-01

    Full Text Available Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1, which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.

  7. Aspiration of human neutrophils: effects of shear thinning and cortical dissipation.

    Science.gov (United States)

    Drury, J L; Dembo, M

    2001-12-01

    It is generally accepted that the human neutrophil can be mechanically represented as a droplet of polymeric fluid enclosed by some sort of thin slippery viscoelastic cortex. Many questions remain however about the detailed rheology and chemistry of the interior fluid and the cortex. To address these quantitative issues, we have used a finite element method to simulate the dynamics of neutrophils during micropipet aspiration using various plausible assumptions. The results were then systematically compared with aspiration experiments conducted at eight different combinations of pipet size and pressure. Models in which the cytoplasm was represented by a simple Newtonian fluid (i.e., models without shear thinning) were grossly incapable of accounting for the effects of pressure on the general time scale of neutrophil aspiration. Likewise, models in which the cortex was purely elastic (i.e., models without surface viscosity) were unable to explain the effects of pipet size on the general aspiration rate. Such models also failed to explain the rapid acceleration of the aspiration rate during the final phase of aspiration nor could they account for the geometry of the neutrophil during various phases of aspiration. Thus, our results indicate that a minimal mechanical model of the neutrophil needs to incorporate both shear thinning and surface viscosity to remain valid over a reasonable range of conditions. At low shear rates, the surface dilatation viscosity of the neutrophil was found to be on the order of 100 poise-cm, whereas the viscosity of the interior cytoplasm was on the order of 1000 poise. Both the surface viscosity and the interior viscosity seem to decrease in a similar fashion when the shear rate exceeds approximately 0.05 s(-1). Unfortunately, even models with both surface viscosity and shear thinning studied are still not sufficient to fully explain all the features of neutrophil aspiration. In particular, the very high rate of aspiration during the

  8. Coronin 1B regulates S1P-induced human lung endothelial cell chemotaxis: role of PLD2, protein kinase C and Rac1 signal transduction.

    Directory of Open Access Journals (Sweden)

    Peter V Usatyuk

    Full Text Available Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs with the bioactive lipid, sphingosine-1-phosphate (S1P rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA targeting coronin 1B (∼36%, PLD2 (∼45% or Rac1 (∼50% compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis.

  9. Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases - Anti-inflammatory Effects of Rutin on Neutrophils -

    Directory of Open Access Journals (Sweden)

    Bahareh Abd Nikfarjam

    2017-03-01

    Full Text Available Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO and myeloperoxidase (MPO. These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF-α productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI medium, pre-incubated with or without rutin (25 μM for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA. Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA, Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, and neutrophils were treated with various concentrations of rutin (1 - 100 μM, after which MTT was appended and incubated at 37ºC for 4 hour. Results: Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001. Also, MPO activity was significantly reduced by rutin (P < 0.001. Conclusion: In this in vitro study, rutin had an anti-inflammatory effect

  10. Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

    Science.gov (United States)

    Gazendam, Roel P; van Hamme, John L; Tool, Anton T J; Hoogenboezem, Mark; van den Berg, J Merlijn; Prins, Jan M; Vitkov, Ljubomir; van de Veerdonk, Frank L; van den Berg, Timo K; Roos, Dirk; Kuijpers, Taco W

    2016-02-01

    Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Chemotaxis by natural populations of coral reef bacteria.

    Science.gov (United States)

    Tout, Jessica; Jeffries, Thomas C; Petrou, Katherina; Tyson, Gene W; Webster, Nicole S; Garren, Melissa; Stocker, Roman; Ralph, Peter J; Seymour, Justin R

    2015-08-01

    Corals experience intimate associations with distinct populations of marine microorganisms, but the microbial behaviours underpinning these relationships are poorly understood. There is evidence that chemotaxis is pivotal to the infection process of corals by pathogenic bacteria, but this evidence is limited to experiments using cultured isolates under laboratory conditions. We measured the chemotactic capabilities of natural populations of coral-associated bacteria towards chemicals released by corals and their symbionts, including amino acids, carbohydrates, ammonium and dimethylsulfoniopropionate (DMSP). Laboratory experiments, using a modified capillary assay, and in situ measurements, using a novel microfabricated in situ chemotaxis assay, were employed to quantify the chemotactic responses of natural microbial assemblages on the Great Barrier Reef. Both approaches showed that bacteria associated with the surface of the coral species Pocillopora damicornis and Acropora aspera exhibited significant levels of chemotaxis, particularly towards DMSP and amino acids, and that these levels of chemotaxis were significantly higher than that of bacteria inhabiting nearby, non-coral-associated waters. This pattern was supported by a significantly higher abundance of chemotaxis and motility genes in metagenomes within coral-associated water types. The phylogenetic composition of the coral-associated chemotactic microorganisms, determined using 16S rRNA amplicon pyrosequencing, differed from the community in the seawater surrounding the coral and comprised known coral associates, including potentially pathogenic Vibrio species. These findings indicate that motility and chemotaxis are prevalent phenotypes among coral-associated bacteria, and we propose that chemotaxis has an important role in the establishment and maintenance of specific coral-microbe associations, which may ultimately influence the health and stability of the coral holobiont.

  12. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yang-Chang [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Lan, Yu-Hsuan [School of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Chang, Fang-Rong [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Wen [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

  13. Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Leonardo Iula

    2018-02-01

    AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

  14. Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device -- TAXIScan.

    Science.gov (United States)

    Nitta, Nao; Tsuchiya, Tomoko; Yamauchi, Akira; Tamatani, Takuya; Kanegasaki, Shiro

    2007-03-30

    We have reported previously the development of an optically accessible, horizontal chemotaxis apparatus, in which migration of cells in the channel from a start line can be traced with time-lapse intervals using a CCD camera (JIM 282, 1-11, 2003). To obtain statistical data of migrating cells, we have developed quantitative methods to calculate various parameters in the process of chemotaxis, employing human eosinophil and CXCL12 as a model cell and a model chemoattractant, respectively. Median values of velocity and directionality of each cell within an experimental period could be calculated from the migratory pathway data obtained from time-lapse images and the data were expressed as Velocity-Directionality (VD) plot. This plot is useful for quantitatively analyzing multiple migrating cells exposed to a certain chemoattractant, and can distinguish chemotaxis from random migration. Moreover precise observation of cell migration revealed that each cell had a different lag period before starting chemotaxis, indicating variation in cell sensitivity to the chemoattractant. Thus lag time of each cell before migration, and time course of increment of the migrating cell ratio at the early stages could be calculated. We also graphed decrement of still moving cell ratio at the later stages by calculating the duration time of cell migration of each cell. These graphs could distinguish different motion patterns of chemotaxis of eosinophils, in response to a range of chemoattractants; PGD(2), fMLP, CCL3, CCL5 and CXCL12. Finally, we compared parameters of eosinophils from normal volunteers, allergy patients and asthma patients and found significant difference in response to PGD(2). The quantitative methods described here could be applicable to image data obtained with any combination of cells and chemoattractants and useful not only for basic studies of chemotaxis but also for diagnosis and for drug screening.

  15. Travelling Waves in Hybrid Chemotaxis Models

    KAUST Repository

    Franz, Benjamin

    2013-12-18

    Hybrid models of chemotaxis combine agent-based models of cells with partial differential equation models of extracellular chemical signals. In this paper, travelling wave properties of hybrid models of bacterial chemotaxis are investigated. Bacteria are modelled using an agent-based (individual-based) approach with internal dynamics describing signal transduction. In addition to the chemotactic behaviour of the bacteria, the individual-based model also includes cell proliferation and death. Cells consume the extracellular nutrient field (chemoattractant), which is modelled using a partial differential equation. Mesoscopic and macroscopic equations representing the behaviour of the hybrid model are derived and the existence of travelling wave solutions for these models is established. It is shown that cell proliferation is necessary for the existence of non-transient (stationary) travelling waves in hybrid models. Additionally, a numerical comparison between the wave speeds of the continuum models and the hybrid models shows good agreement in the case of weak chemotaxis and qualitative agreement for the strong chemotaxis case. In the case of slow cell adaptation, we detect oscillating behaviour of the wave, which cannot be explained by mean-field approximations. © 2013 Society for Mathematical Biology.

  16. Candida albicans escapes from mouse neutrophils

    DEFF Research Database (Denmark)

    Ermert, David; Niemiec, Maria J; Röhm, Marc

    2013-01-01

    is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed...

  17. Grain dust induces IL-8 production from bronchial epithelial cells: effect on neutrophil recruitment.

    Science.gov (United States)

    Park, H S; Suh, J H; Kim, S S; Kwon, O J

    2000-06-01

    There have been several investigations suggesting an involvement of activated neutrophils in the development of grain dust (GD)-induced occupational asthma. Interleukin-8 in the sputa from GD-induced asthmatic patients increased significantly after the exposure to GD. To confirm IL-8 production from bronchial epithelial cells when exposed to GD, and to evaluate the role of IL-8 on neutrophil recruitment. We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four different concentrations ranging from 1 to 200 microg/mL of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production and neutrophil chemotaxis, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant derived from subjects with GD-induced asthma exposed to 10 microg/mL of GD, and then compared with those without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. Neutrophil chemotactic activity of the culture supernatant was determined by modified Boyden chamber method. Interleukin-8 production and neutrophil chemotactic activity from bronchial epithelial cells significantly increased with additions of GD in a dose-dependent manner (P < .05, respectively), and were significantly augmented with additions of PBMC supernatant (P < .05, respectively) at each concentration. Close correlation was noted between neutrophil chemotactic activity and IL-8 level (r = 0.87, P < .05). Compared with the untreated sample, pre-treatment of anti-IL-8 antibody induced a significant suppression (up to 67.2%) of neutrophil chemotactic activity in a dose-dependent manner. These results suggest that IL-8 produced from bronchial epithelial cells may be a major cytokine, which induces neutrophil migration into the airways when exposed to GD.

  18. Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

    International Nuclear Information System (INIS)

    Dowling, J.N.; Saha, A.K.; Glew, R.H.

    1987-01-01

    Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP 3 ) was explored. When neutrophil phosphoinositides were labeled with 32 P, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP 2 ) over 2 h. Treatment of [ 3 H]inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP 2 . Following fMLP stimulation, the fractional reduction in PIP 2 and the fractional increase in IP 3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP 3 was reduced by ACP pre-treatment. The reduction in IP 3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP 2 available for hydrolysis. However, some loss of IP 3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP 2 , the prognitor of IP 3 , and by hydrolyzing IP 3 itself

  19. Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils

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    Hytönen Jukka

    2006-10-01

    Full Text Available Abstract Background Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry. Results In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain. Conclusion These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The

  20. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang; Zhang, Huoming; Guo, Tiannan; Li, Wenying; Li, Huiyu; Zhu, Yi; Huang, Shiang

    2014-01-01

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang

    2014-06-11

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils.

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    Xiang-Qing Zhu

    Full Text Available Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170 expression levels were decreased, including chemokine ligand 23 (CCL23 and IFN-γ, while 11 cytokine (11/170 expression levels were increased, such as death receptor 6 (DR6. Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78 in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells.

  3. Visceral leishmaniasis patients display altered composition and maturity of neutrophils as well as impaired neutrophil effector functions

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    Endalew Yizengaw

    2016-11-01

    Full Text Available Immunologically, active visceral leishmaniasis (VL is characterised by profound immunosuppression, severe systemic inflammatory responses and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis, however, their role in human visceral leishmaniasis is poorly understood.In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase and elastase, all contained in neutrophils’ granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analysed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species and phagocytose bacterial particles, but not Leishmania parasites.Our results suggest that impaired effector functions, increased activation and immaturity of neutrophils play a key role in the pathogenesis of VL.

  4. Analysis of migration rate and chemotaxis of human adipose-derived mesenchymal stem cells in response to LPS and LTA in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Herzmann, Nicole; Salamon, Achim [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany); Fiedler, Tomas [Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany)

    2016-03-15

    Mesenchymal stem cells (MSC) are able to stimulate the regeneration of injured tissue. Since bacterial infections are common complications in wound healing, bacterial pathogens and their components come into direct contact with MSC. The interaction with bacterial structures influences the proliferation, differentiation and migratory activity of the MSC, which might be of relevance during regeneration. Studies on MSC migration in response to bacterial components have shown different results depending on the cell type. Here, we analyzed the migration rate and chemotaxis of human adipose-derived MSC (adMSC) in response to the basic cell-wall components lipopolysaccharide (LPS) of Gram-negative bacteria and lipoteichoic acid (LTA) of Gram-positive bacteria in vitro. To this end, we used transwell and scratch assays, as well as a specific chemotaxis assay combined with live-cell imaging. We found no significant influence of LPS or LTA on the migration rate of adMSC in transwell or scratch assays. Furthermore, in the µ-slide chemotaxis assay, the stimulation with LPS did not exert any chemotactic effect on adMSC. - Highlights: • LPS increased the release of IL-6 and IL-8 in adMSC significantly. • The migration rate of adMSC was not influenced by LPS or LTA. • LPS or LTA did not exert a chemotactic effect on adMSC.

  5. Analysis of migration rate and chemotaxis of human adipose-derived mesenchymal stem cells in response to LPS and LTA in vitro

    International Nuclear Information System (INIS)

    Herzmann, Nicole; Salamon, Achim; Fiedler, Tomas; Peters, Kirsten

    2016-01-01

    Mesenchymal stem cells (MSC) are able to stimulate the regeneration of injured tissue. Since bacterial infections are common complications in wound healing, bacterial pathogens and their components come into direct contact with MSC. The interaction with bacterial structures influences the proliferation, differentiation and migratory activity of the MSC, which might be of relevance during regeneration. Studies on MSC migration in response to bacterial components have shown different results depending on the cell type. Here, we analyzed the migration rate and chemotaxis of human adipose-derived MSC (adMSC) in response to the basic cell-wall components lipopolysaccharide (LPS) of Gram-negative bacteria and lipoteichoic acid (LTA) of Gram-positive bacteria in vitro. To this end, we used transwell and scratch assays, as well as a specific chemotaxis assay combined with live-cell imaging. We found no significant influence of LPS or LTA on the migration rate of adMSC in transwell or scratch assays. Furthermore, in the µ-slide chemotaxis assay, the stimulation with LPS did not exert any chemotactic effect on adMSC. - Highlights: • LPS increased the release of IL-6 and IL-8 in adMSC significantly. • The migration rate of adMSC was not influenced by LPS or LTA. • LPS or LTA did not exert a chemotactic effect on adMSC.

  6. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Jacobsen, Lars C; Rørvig, Sara

    2011-01-01

    Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associate...... significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema....

  7. Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation.

    Science.gov (United States)

    Rangé, Hélène; Labreuche, Julien; Louedec, Liliane; Rondeau, Philippe; Planesse, Cynthia; Sebbag, Uriel; Bourdon, Emmanuel; Michel, Jean-Baptiste; Bouchard, Philippe; Meilhac, Olivier

    2014-10-01

    Epidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens. Carotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium. Intraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers. This study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Campylobacter jejuni transducer like proteins: Chemotaxis and beyond.

    Science.gov (United States)

    Chandrashekhar, Kshipra; Kassem, Issmat I; Rajashekara, Gireesh

    2017-07-04

    Chemotaxis, a process that mediates directional motility toward or away from chemical stimuli (chemoeffectors/ligands that can be attractants or repellents) in the environment, plays an important role in the adaptation of Campylobacter jejuni to disparate niches. The chemotaxis system consists of core signal transduction proteins and methyl-accepting-domain-containing Transducer like proteins (Tlps). Ligands binding to Tlps relay a signal to chemotaxis proteins in the cytoplasm which initiate a signal transduction cascade, culminating into a directional flagellar movement. Tlps facilitate substrate-specific chemotaxis in C. jejuni, which plays an important role in the pathogen's adaptation, pathobiology and colonization of the chicken gastrointestinal tract. However, the role of Tlps in C. jejuni's host tissue specific colonization, physiology and virulence remains not completely understood. Based on recent studies, it can be predicted that Tlps might be important targets for developing strategies to control C. jejuni via vaccines and antimicrobials.

  9. The Pseudomonas aeruginosa chemotaxis methyltransferase CheR1 impacts on bacterial surface sampling.

    Directory of Open Access Journals (Sweden)

    Juliane Schmidt

    Full Text Available The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.

  10. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

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    N.E. Gomes

    2010-09-01

    Full Text Available Lipopolysaccharide (LPS activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R and smooth (S forms signal through Toll-like receptor 4 (TLR4, but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS and nitric oxide (NO generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

  11. Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes

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    Brzezinska Agnieszka A

    2006-12-01

    Full Text Available Abstract Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

  12. Localization and Functionality of the Inflammasome in Neutrophils

    DEFF Research Database (Denmark)

    Bakele, Martina; Joos, Melanie; Burdi, Sofia

    2014-01-01

    Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality...... of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein...... and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases...

  13. The effect of N-nitrosodimethylamine (NDMA) on Bax and Mcl-1 expression in human neutrophils.

    Science.gov (United States)

    Jablonski, Jakub; Jablonska, Ewa; Leonik, Agnieszka

    2011-12-01

    In the present study we examined a role of pro-apoptotic Bax and anti-apoptotic Mcl-1 proteins, participating in the regulation of intrinsic apoptosis pathway in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA), the environmental xenobiotic. For the purpose comparison, the same studies were conducted in autologous peripheral blood mononuclear cells (PBMCs). The production of cytochrome c by PMNs was also determined. A deficit of anti-apoptotic Mcl-1 and overexpression of the pro-apoptotic protein Bax suggest that the apoptosis process in human neutrophils exposed to NDMA is dependent on changes in the expression of these proteins. PMNs were more sensitive to NDMA than PBMCs.

  14. Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

    DEFF Research Database (Denmark)

    Christensen, Hanna B; Gloriam, David E; Pedersen, Daniel Sejer

    2017-01-01

    INTRODUCTION: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors...... (FPRs) in human neutrophils. METHODS: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist...... potencies. RESULTS: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H...

  15. Regulation of neutrophil senescence by microRNAs.

    Directory of Open Access Journals (Sweden)

    Jon R Ward

    2011-01-01

    Full Text Available Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.

  16. Severe exercise and exercise training exert opposite effects on human neutrophil apoptosis via altering the redox status.

    Directory of Open Access Journals (Sweden)

    Guan-Da Syu

    Full Text Available Neutrophil spontaneous apoptosis, a process crucial for immune regulation, is mainly controlled by alterations in reactive oxygen species (ROS and mitochondria integrity. Exercise has been proposed to be a physiological way to modulate immunity; while acute severe exercise (ASE usually impedes immunity, chronic moderate exercise (CME improves it. This study aimed to investigate whether and how ASE and CME oppositely regulate human neutrophil apoptosis. Thirteen sedentary young males underwent an initial ASE and were subsequently divided into exercise and control groups. The exercise group (n = 8 underwent 2 months of CME followed by 2 months of detraining. Additional ASE paradigms were performed at the end of each month. Neutrophils were isolated from blood specimens drawn at rest and immediately after each ASE for assaying neutrophil spontaneous apoptosis (annexin-V binding on the outer surface along with redox-related parameters and mitochondria-related parameters. Our results showed that i the initial ASE immediately increased the oxidative stress (cytosolic ROS and glutathione oxidation, and sequentially accelerated the reduction of mitochondrial membrane potential, the surface binding of annexin-V, and the generation of mitochondrial ROS; ii CME upregulated glutathione level, retarded spontaneous apoptosis and delayed mitochondria deterioration; iii most effects of CME were unchanged after detraining; and iv CME blocked ASE effects and this capability remained intact even after detraining. Furthermore, the ASE effects on neutrophil spontaneous apoptosis were mimicked by adding exogenous H(2O(2, but not by suppressing mitochondrial membrane potential. In conclusion, while ASE induced an oxidative state and resulted in acceleration of human neutrophil apoptosis, CME delayed neutrophil apoptosis by maintaining a reduced state for long periods of time even after detraining.

  17. Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; White, Mitchell R.; Tecle, Tesfaldet

    2006-01-01

    Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study...

  18. Specific receptor for inositol-1,4,5-trisphosphate in permeabilized rabbit neutrophils

    International Nuclear Information System (INIS)

    Bradford, P.G.; Spat, A.; Rubin, R.P.

    1986-01-01

    Neutrophil chemotaxis and degranulation are resultant, in part, from the mobilization of intracellular calcium by inositol-1,4,5-trisphosphate [(1,4,5)IP 3 ], one of the products of chemoattractant-stimulated phospholipase C activity. High specific activity (ca. 40 Ci/mmol) [ 32 P](1,4,5)IP 3 was prepared from [γ- 32 P]ATP-labeled human erythrocyte ghosts and was used in binding assays with saponin-permeabilized rabbit peritoneal neutrophils. At 4 0 C and in the presence of inhibitors of the IP 3 5-phosphomonoesterase, [ 32 P](1,4,5)IP 3 rapidly associated with a specific binding component which saturated within 60s. Nonspecific binding, taken as the residual binding in the presence of 10 μM (1,4,5)IP 3 , was 15% of the total. No specific binding was detected using intact cells. The specific binding to permeable cells was reversible (t/sup 1/2/ ∼ 60s) and could be inhibited in a dose-dependent manner by (1,4,5)IP 3 (EC 50 = 30 nM) and by other calcium mobilizing inositol phosphates [(2,4,5)IP 3 ] but not by inactive analogs [(1,4)IP 2 , (4,5)IP 2 , (1)IP]. The dose-responses of (1,4,5)IP 3 and (2,4,5)IP 3 in inhibiting [ 32 P](1,4,5)IP 3 specific binding correlated well with their abilities to release Ca 2+ from nonmitochondrial vesicular stores in the same preparation of cells, suggesting that the authors have identified the physiological receptor for (1,4,5)IP 3

  19. Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from X-irradiated human peripheral blood hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

    2009-01-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34 + hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34 + cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34 + cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34 + cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34 + cells. (author)

  20. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

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    Chen Zhang

    2013-01-01

    Full Text Available Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis. In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

  1. Transducer Like Proteins of Campylobacter jejuni 81-176: Role in chemotaxis and colonization of the chicken gastrointestinal tract

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    Gireesh eRajashekara

    2015-05-01

    Full Text Available Transducer Like Proteins (Tlps, also known as Methyl accepting chemotaxis proteins (MCP, enable enteric pathogens to respond to changing nutrient levels in the environment by mediating taxis towards or away from specific chemoeffector molecules such as nutrients. Despite recent advances in the characterization of chemotaxis responses in Campylobacter jejuni, the impact of Tlps on the adaptation of this pathogen to disparate niches and hosts is not fully characterized. The latter is particularly evident in the case of C. jejuni 81-176, a strain that is known to be highly invasive. Furthermore, the cytoplasmic group C Tlps (Tlp5, 6, and 8 was not extensively evaluated. Here, we investigated the role of C. jejuni 81-176 Tlps in chemotaxis towards various substrates, biofilm formation, in vitro interaction with human intestinal cells, and chicken colonization. We found that the ∆tlp6 and ∆tlp10 mutants exhibited decreased chemotaxis towards aspartate whereas the ∆tlp6 mutant displayed a decreased chemotaxis towards Tri-Carboxylic Acid (TCA cycle intermediates such as pyruvate, isocitrate, and succinate. Our findings also corroborated that more than one Tlp is involved in mediating chemotaxis towards the same nutrient. The deletion of tlps affected important phenotypes such as motility, biofilm formation, and invasion of human intestinal epithelial cells (INT-407. The ∆tlp8 mutant displayed increased motility in soft agar and showed decreased biofilm formation. The ∆tlp8 and ∆tlp9 mutants were significantly defective in invasion in INT-407 cells. The ∆tlp10 mutant was defective in colonization of the chicken proximal and distal gastrointestinal tract, while the ∆tlp6 and ∆tlp8 mutants showed reduced colonization of the duodenum and jejunum. Our results highlight the importance of Tlps in C. jejuni’s adaptation and pathobiology.

  2. Effects of exogenous IL-37 on the biological characteristics of human lung adenocarcinoma A549 cells and the chemotaxis of regulatory T cells.

    Science.gov (United States)

    Chen, Yu-Hua; Zhou, Bi-Yun; Wu, Guo-Cai; Liao, De-Quan; Li, Jing; Liang, Si-Si; Wu, Xian-Jin; Xu, Jun-Fa; Chen, Yong-Hua; Di, Xiao-Qing; Lin, Qiong-Yan

    2018-02-14

    This study aims to investigate the effects of exogenous interleukin (IL)-37 on the biological characteristics of human lung adenocarcinoma A549 cells and the chemotaxis of regulatory T (Treg) cells. After isolating the CD4+ CD25+ Treg cells from the peripheral blood, flow cytometry was used to detect the purity of the Treg cells. A549 cells were divided into blank (no transfection), empty plasmid (transfection with pIRES2-EGFP empty plasmid) or IL-37 group (transfection with pIRES2-EGFP-IL-37 plasmid). RT-PCR was used to detect mRNA expression of IL-37 and ELISA to determine IL-37 and MMP-9 expressions. Western blotting was applied to detect the protein expressions of PCNA, Ki-67, Cyclin D1, CDK4, cleaved caspase-3 and cleaved caspase-9. MTT assay, flow cytometry, scratch test and transwell assay were performed to detect cell proliferation, cycle, apoptosis, migration and invasion. Effect of exogenous IL-37 on the chemotaxis of Treg cells was measured through transwell assay. Xenograft models in nude mice were eastablished to detect the impact of IL-37 on A549 cells. The IL-37 group had a higher IL-37 expression, cell apoptosis in the early stage and percentage of cells in the G0/G1 phase than the blank and empty plasmid groups. The IL-37 group had a lower MMP-9 expression, optical density (OD), percentage of cells in the S and G2/M phases, migration, invasion and chemotaxis of CD4+CD25+ Foxp3+ Treg cells. The xenograft volume and weight of nude mice in the IL-37 group were lower than those in the blank and empty plasmid groups. Compared with the blank and empty plasmid groups, the IL-37 group had significantly reduced expression of PCNA, Ki-67, Cyclin D1 and CDK4 but elevated expression of cleaved caspase-3 and cleaved caspase-9. Therefore, exogenous IL-37 inhibits the proliferation, migration and invasion of human lung adenocarcinoma A549 cells as well as the chemotaxis of Treg cells while promoting the apoptosis of A549 cells.

  3. Azospirillum brasilense Chemotaxis Depends on Two Signaling Pathways Regulating Distinct Motility Parameters.

    Science.gov (United States)

    Mukherjee, Tanmoy; Kumar, Dhivya; Burriss, Nathan; Xie, Zhihong; Alexandre, Gladys

    2016-06-15

    The genomes of most motile bacteria encode two or more chemotaxis (Che) systems, but their functions have been characterized in only a few model systems. Azospirillum brasilense is a motile soil alphaproteobacterium able to colonize the rhizosphere of cereals. In response to an attractant, motile A. brasilense cells transiently increase swimming speed and suppress reversals. The Che1 chemotaxis pathway was previously shown to regulate changes in the swimming speed, but it has a minor role in chemotaxis and root surface colonization. Here, we show that a second chemotaxis system, named Che4, regulates the probability of swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization. Experimental evidence indicates that Che1 and Che4 are functionally linked to coordinate changes in the swimming motility pattern in response to attractants. The effect of Che1 on swimming speed is shown to enhance the aerotactic response of A. brasilense in gradients, likely providing the cells with a competitive advantage in the rhizosphere. Together, the results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis pathways regulating distinct motility parameters to alter movement in gradients and enhance the chemotactic advantage. Chemotaxis provides motile bacteria with a competitive advantage in the colonization of diverse niches and is a function enriched in rhizosphere bacterial communities, with most species possessing at least two chemotaxis systems. Here, we identify the mechanism by which cells may derive a significant chemotactic advantage using two chemotaxis pathways that ultimately regulate distinct motility parameters. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Protectin DX, a double lipoxygenase product of DHA, inhibits both ROS production in human neutrophils and cyclooxygenase activities

    Science.gov (United States)

    Liu, Miao; Boussetta, Tarek; Makni-Maalej, Karama; Fay, Michèle; Driss, Fathi; El-Benna, Jamel; Lagarde, Michel; Guichardant, Michel

    2014-01-01

    Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits with an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX) - a docosahexaenoic acid (DHA) di-hydroxylated product which inhibits blood platelet aggregation - on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides (LPS)-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B4 (LTB4). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases. PMID:24254970

  5. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  6. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Japanese encephalitis virus (JEV) induces human peripheral blood monocytes to secrete a chemotactic cytokine [human macrophage-derived factor (hMDF)] which causes chemotaxis of neutrophils. The only known assay for hMDF cannot quantify its level in samples, so an enzyme immunoassay has been standardized for ...

  7. Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura.

    Science.gov (United States)

    Cao, Wenjing; Pham, Huy P; Williams, Lance A; McDaniel, Jenny; Siniard, Rance C; Lorenz, Robin G; Marques, Marisa B; Zheng, X Long

    2016-11-01

    Acquired thrombotic thrombocytopenic purpura is primarily caused by the deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13. However, ADAMTS13 deficiency alone is often not sufficient to cause acute thrombotic thrombocytopenic purpura. Infections or systemic inflammation may precede acute bursts of the disease, but the underlying mechanisms are not fully understood. Herein, 52 patients with acquired autoimmune thrombotic thrombocytopenic purpura and 30 blood donor controls were recruited for the study. The plasma levels of human neutrophil peptides 1-3 and complement activation fragments (i.e. Bb, iC3b, C4d, and sC5b-9) were determined by enzyme-linked immunosorbent assays. Univariate analyses were performed to determine the correlation between each biomarker and clinical outcomes. We found that the plasma levels of human neutrophil peptides 1-3 and Bb in patients with acute thrombotic thrombocytopenic purpura were significantly higher than those in the control (Ppurpura patients and the control. We conclude that innate immunity, i.e. neutrophil and complement activation via the alternative pathway, may play a role in the pathogenesis of acute autoimmune thrombotic thrombocytopenic purpura, and a therapy targeted at these pathways may be considered in a subset of these patients. Copyright© Ferrata Storti Foundation.

  8. High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma

    DEFF Research Database (Denmark)

    Arshid, Samina; Tahir, Muhammad; Fontes, Belchor

    2017-01-01

    and surgical trauma rats in this study. Extracted proteins were analyzed using nano liquid chromatography coupled to tandem mass spectrometry. A total of 2924 rat neutrophil proteins were identified in our analysis, of which 393 were found differentially regulated between control and trauma groups. By using...... functional pathways analysis of the 190 proteins up-regulated in surgical trauma we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome...... degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations were then confirmed by in silico protein-protein interaction analysis. Collectively, our results reveal...

  9. Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils.

    Science.gov (United States)

    Docherty, J C; Wilson, T W

    1987-10-29

    Arachidonic acid metabolism in human neutrophils stimulated in vitro with the calcium ionophore A23187 was studied using combined HPLC and radioimmunoassays. Indomethacin (0.1 and 1.0 microM) caused a 300% increase in LTB4 formation in neutrophils stimulated with A23187. 5-, 12- and 15-HETE levels were also increased. In the presence of exogenous arachidonic acid 1.0 microM Indomethacin caused a 37% increase in LTB4 formation. Acetyl Salicylic Acid and Ibuprofen had no effect on the formation of lipoxygenase metabolites. The effect of indomethacin on LTB4 formation does not appear to be due to a simple redirection of substrate arachidonic acid from the cyclooxygenase to the lipoxygenase pathways.

  10. Effects of ghrelin on the apoptosis of human neutrophils in vitro

    Science.gov (United States)

    Li, Bin; Zeng, Mian; Zheng, Haichong; Huang, Chunrong; He, Wanmei; Lu, Guifang; Li, Xia; Chen, Yanzhu; Xie, Ruijie

    2016-01-01

    Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation and the diffuse infiltration of neutrophils into the alveolar space. Neutrophils are abundant, short-lived leukocytes that play a key role in immune defense against microbial infections. These cells die via apoptosis following the activation and uptake of microbes, and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter pathogens. Apoptosis is essential for the removal of neutrophils from inflamed tissues and for the timely resolution of neutrophilic inflammation. Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue receptor, produced and secreted mainly from the stomach. Previous studies have reported that ghrelin exerts anti-inflammatory effects in lung injury through the regulation of the apoptosis of different cell types; however, the ability of ghrelin to regulate alveolar neutrophil apoptosis remains largely undefined. We hypothesized that ghrelin may have the ability to modulate neutrophil apoptosis. In this study, to examine this hypothesis, we investigated the effects of ghrelin on freshly isolated neutrophils in vitro. Our findings demonstrated a decrease in the apoptotic ratio (as shown by flow cytometry), as well as in the percentage of cells with decreased mitochondrial membrane potential (ΔΨm) and in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling-positive rate, accompanied by an increased B-cell lymphoma 2/Bax ratio and the downregulation of cleaved caspase-3 in neutrophils following exposure to lipopolysaccharide (100 ng/ml). However, pre-treatment with ghrelin at a physiological level (100 nM) did not have a notable influence on the neutrophils in all the aforementioned tests. Our findings suggest that ghrelin may not possess the ability to modulate the neutrophil lifespan in vitro. PMID:27431014

  11. Effects of gadolinium oxide nanoparticles on the oxidative burst from human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Abrikossova, Natalia; Skoglund, Caroline; Ahrén, Maria; Uvdal, Kajsa; Bengtsson, Torbjörn

    2012-01-01

    We have previously shown that gadolinium oxide (Gd 2 O 3 ) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd 2 O 3 nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd 2 O 3 nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd 2 O 3 nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd 2 O 3 nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes. (paper)

  12. Role of ERK1/2 kinase in the expression of iNOS by NDMA in human neutrophils.

    Science.gov (United States)

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr

    2013-01-01

    Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.

  13. Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; White, Mitchell R.; Tecle, Tesfaldet

    2006-01-01

    Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characte......Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study...... was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered...... with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some...

  14. Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

    International Nuclear Information System (INIS)

    Hiraiwa, Tetsuya; Nishikawa, Masatoshi; Shibata, Tatsuo; Nagamatsu, Akihiro; Akuzawa, Naohiro

    2014-01-01

    Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold. (paper)

  15. Doxycycline induced photodamage to human neutrophils and tryptophan

    International Nuclear Information System (INIS)

    Sandberg, S.; Glette, J.; Hopen, G.; Solberg, C.O.

    1984-01-01

    Neutrophil function were studied following irradiation (340-380 nm) of the cells in the presence of 22 μM doxycycline. At increasing light fluence the locomotion, chemiluminescence and glucose oxidation (by the hexose monophosphate shunt) of the neutrophils steadily decreased. The photodamage increased with increasing preincubation temperature and time and was enhanced in D 2 O, reduced in azide and abolished in anaerobiosis. Superoxide dismutase, catalase or mannitol did not influence the photodamage. Photooxidation of tryptophan in the presence of doxycycline was increased 9-10-fold in D 2 O and nearly abolished in the presence of 0.25 mM NaN 3 , indicating that singlet oxygen is the most important reactive oxygen species in the doxycycline-induced photodamage. The results may explain some of the features of tetracycline-induced photosensitivity and why other authors have obtained diverging results when studying the influence of tetracyclines on neutrophil functions. (author)

  16. Chemotaxis on the Move – Active Learning Teaching Tool

    Directory of Open Access Journals (Sweden)

    Ann H. Williams

    2010-11-01

    Full Text Available In Microbiology courses, concepts such as chemotaxis can be difficult to visualize for students. Described here is a short visual playacting activity where students simulate E.coli moving towards an attractant source using a biased random walk. This short interactive activity is performed in the lecture course of General Microbiology that contains mostly Biology major juniors or seniors prior to the lecture on the subject of chemotaxis and flagellar movements. It is utilized to help students (class of 30–40 understand and visualize the process of chemotaxis and the concepts of random walk, biased random walk, runs, tumbles and directed movement of flagella in response to attractants and repellents.

  17. Feedback control architecture and the bacterial chemotaxis network.

    Directory of Open Access Journals (Sweden)

    Abdullah Hamadeh

    2011-05-01

    Full Text Available Bacteria move towards favourable and away from toxic environments by changing their swimming pattern. This response is regulated by the chemotaxis signalling pathway, which has an important feature: it uses feedback to 'reset' (adapt the bacterial sensing ability, which allows the bacteria to sense a range of background environmental changes. The role of this feedback has been studied extensively in the simple chemotaxis pathway of Escherichia coli. However it has been recently found that the majority of bacteria have multiple chemotaxis homologues of the E. coli proteins, resulting in more complex pathways. In this paper we investigate the configuration and role of feedback in Rhodobacter sphaeroides, a bacterium containing multiple homologues of the chemotaxis proteins found in E. coli. Multiple proteins could produce different possible feedback configurations, each having different chemotactic performance qualities and levels of robustness to variations and uncertainties in biological parameters and to intracellular noise. We develop four models corresponding to different feedback configurations. Using a series of carefully designed experiments we discriminate between these models and invalidate three of them. When these models are examined in terms of robustness to noise and parametric uncertainties, we find that the non-invalidated model is superior to the others. Moreover, it has a 'cascade control' feedback architecture which is used extensively in engineering to improve system performance, including robustness. Given that the majority of bacteria are known to have multiple chemotaxis pathways, in this paper we show that some feedback architectures allow them to have better performance than others. In particular, cascade control may be an important feature in achieving robust functionality in more complex signalling pathways and in improving their performance.

  18. Highlighting the role of Ras and Rap during Dictyostelium chemotaxis

    NARCIS (Netherlands)

    Kortholt, Arjan; van Haastert, Peter J. M.

    Chemotaxis, the directional movement towards a chemical compound, is an essential property of many cells and has been linked to the development and progression of many diseases. Eukaryotic chemotaxis is a complex process involving gradient sensing, cell polarity, remodelling of the cytoskeleton and

  19. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  20. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  1. Ir-LBP, an ixodes ricinus tick salivary LTB4-binding lipocalin, interferes with host neutrophil function.

    Directory of Open Access Journals (Sweden)

    Jérôme Beaufays

    Full Text Available BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: We previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: +/-1 nM, similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the tick's ability to interfere with proper neutrophil function in inflammation. CONCLUSIONS/SIGNIFICANCE: These elements suggest that Ir-LBP is a "scavenger" of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.

  2. Influence of exogenous leptin on redox homeostasis in neutrophils and lymphocytes cultured in synovial fluid isolated from patients with rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Michał Gajewski

    2016-07-01

    Full Text Available Objectives : Leptin is an adipose cells derived hormone that regulates energy homeostasis within the body. Energy metabolism of immune cells influences their activity within numerous pathological states, but the effect of leptin on these cells in unclear. On the one hand, it was observed that leptin induces neutrophils chemotaxis and modulates phagocytosis. On the other hand, neutrophils exposed to leptin did not display detectable Ca 2+ ions mobilization or β 2 -integrin upregulation. In this study, we investigated the effect of leptin on the redox homeostasis in lymphocytes and neutrophils. Material and methods : Neutrophils and lymphocytes were isolated by density-gradient centrifugation of blood from healthy volunteers. Cells were cultured with or without leptin (100 ng/ml for lymphocytes and 500 ng/ml for neutrophils or with or without synovial fluid (85% for 0–72 h. Culture media were not changed during incubation. Cells were homogenized and homogenate was frozen until laboratory measurements. Redox homeostasis was assessed by the reduced glutathione (GSH vs. oxidized glutathione (GSSG ratio and membrane lipid peroxidation evaluation. Results : Lymphocytes cultured with leptin and synovial fluid showed a significant increase of the GSSG level. The GSSG/GSH ratio increased by 184 ±37%. In neutrophils incubated in a similar environment, the GSSG/GSH ratio increased by just 21 ±7%, and the effect was observed irrespectively of whether they were exposed to leptin or synovial fluid or both together. Neither leptin nor synovial fluid influenced lipid peroxidation in neutrophils, but in lymphocytes leptin intensified lipid peroxidation. Conclusions : Leptin altered the lymphocytes, but not neutrophils redox state. Because firstly neutrophils are anaerobic cells and have just a few mitochondria and secondly lymphocytes have typical aerobic metabolism, the divergence of our data supports the hypothesis that leptin induces oxidative stress by

  3. DMPD: Cellular signaling in macrophage migration and chemotaxis. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11073096 Cellular signaling in macrophage migration and chemotaxis. Jones GE. J Leu...koc Biol. 2000 Nov;68(5):593-602. (.png) (.svg) (.html) (.csml) Show Cellular signaling in macrophage migration... and chemotaxis. PubmedID 11073096 Title Cellular signaling in macrophage migration and chemotaxis. Autho

  4. Local anesthetic-induced inhibition of human neutrophil priming: the influence of structure, lipophilicity, and charge

    NARCIS (Netherlands)

    Picardi, Susanne; Cartellieri, Sibylle; Groves, Danja; Hahnenekamp, Klaus; Gerner, Peter; Durieux, Marcel E.; Stevens, Markus F.; Lirk, Philipp; Hollmann, Markus W.

    2013-01-01

    Local anesthetics (LAs) are widely known for inhibition of voltage-gated sodium channels underlying their antiarrhythmic and antinociceptive effects. However, LAs have significant immunomodulatory properties and were shown to affect human neutrophil functions independent of sodium-channel blockade.

  5. Sepsis Induces a Dysregulated Neutrophil Phenotype That Is Associated with Increased Mortality

    Directory of Open Access Journals (Sweden)

    Jaimin M. Patel

    2018-01-01

    Full Text Available Background. Neutrophil dysfunction in sepsis has been implicated in the pathogenesis of multiorgan failure; however, the role of neutrophil extracellular traps (NETs remains uncertain. We aimed to determine the sequential changes in ex vivo NETosis and its relationship with mortality in patients with sepsis and severe sepsis. Methods. This was a prospective observational cohort study enrolling 21 healthy age-matched controls and 39 sepsis and 60 severe sepsis patients from acute admissions to two UK hospitals. Patients had sequential bloods for the ex vivo assessment of NETosis in response to phorbol-myristate acetate (PMA using a fluorometric technique and chemotaxis using time-lapse video microscopy. Continuous data was tested for normality, with appropriate parametric and nonparametric tests, whilst categorical data was analysed using a chi-squared test. Correlations were performed using Spearman’s rho. Results. Ex vivo NETosis was reduced in patients with severe sepsis, compared to patients with sepsis and controls (p=0.002. PMA NETosis from patients with septic shock was reduced further (p<0.001 compared to controls. The degree of metabolic acidosis correlated with reduced NETosis (p<0.001, and this was replicated when neutrophils from healthy donors were incubated in acidotic media. Reduced NETosis at baseline was associated with an increased 30-day (p=0.002 and 90-day mortality (p=0.014 in sepsis patients. These findings were accompanied by defects in neutrophil migration and delayed apoptosis. Resolution of sepsis was not associated with the return to baseline levels of NETosis or migration. Conclusions. Sepsis induces significant changes in neutrophil function with the degree of dysfunction corresponding to the severity of the septic insult which persists beyond physiological recovery from sepsis. The changes induced lead to the failure to effectively contain and eliminate the invading pathogens and contribute to sepsis

  6. Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

    Science.gov (United States)

    Kamoshida, Go; Kikuchi-Ueda, Takane; Nishida, Satoshi; Tansho-Nagakawa, Shigeru; Ubagai, Tsuneyuki; Ono, Yasuo

    2018-01-01

    Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections. PMID:29467765

  7. Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

    Directory of Open Access Journals (Sweden)

    Go Kamoshida

    2018-02-01

    Full Text Available Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs, has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA, and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

  8. Drosophila chemotaxis: a first look with neurogenetics.

    Science.gov (United States)

    Gao, Xiaojing J

    2014-01-01

    Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. In our original article, we combined a quantitative behavioral assay with genetic manipulations to dissect the neural substrate for chemotaxis. In this Extra View article, we offer a more chronological narration of the findings leading to our key conclusion that aversion engages specific motor-related circuits and kinematics. We speculate on the separation and crosstalk between aversion and attraction circuits in the brain and the ventral nerve cord, and the implication for valence encoding in the olfactory system.

  9. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

    National Research Council Canada - National Science Library

    Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fullter, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

    2006-01-01

    .... Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory...

  10. Differential effector responses by circulating/blood and tissue/peritoneal neutrophils following burn combined with Enterococcus faecalis infection.

    Science.gov (United States)

    Fazal, Nadeem; Shelip, Alla; Siddiqui, Erum; Ali, Ashraf; Azim, Anser C; Al-Ghoul, Walid M

    2012-03-01

    Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  11. Tamoxifen induces apoptotic neutrophil efferocytosis in horses.

    Science.gov (United States)

    Olave, C; Morales, N; Uberti, B; Henriquez, C; Sarmiento, J; Ortloff, A; Folch, H; Moran, G

    2018-03-01

    Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.

  12. Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

    Science.gov (United States)

    Robertson, James D; Ward, Jon R; Avila-Olias, Milagros; Battaglia, Giuseppe; Renshaw, Stephen A

    2017-05-01

    Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In this study, we deliver therapeutic compounds to neutrophils using biocompatible, nanometer-sized synthetic vesicles, or polymersomes, which are internalized by binding to scavenger receptors and subsequently escape the early endosome through a pH-triggered disassembly mechanism. This allows polymersomes to deliver molecules into the cell cytosol of neutrophils without causing cellular activation. After optimizing polymersome size, we show that polymersomes can deliver the cyclin-dependent kinase inhibitor (R)-roscovitine into human neutrophils to promote apoptosis in vitro. Finally, using a transgenic zebrafish model, we show that encapsulated (R)-roscovitine can speed up inflammation resolution in vivo more efficiently than the free drug. These results show that polymersomes are effective intracellular carriers for drug delivery into neutrophils. This has important consequences for the study of neutrophil biology and the development of neutrophil-targeted therapeutics. Copyright © 2017 The Authors.

  13. Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis.

    Directory of Open Access Journals (Sweden)

    Rie Ibusuki

    Full Text Available Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1.Transgenic (TG mice expressing HNP-1 under the control of a β-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes in vitro.After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner in vitro.HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.

  14. Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

    Science.gov (United States)

    Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

    2017-09-01

    Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

  15. Dynamic interactions of neutrophils and biofilms

    Directory of Open Access Journals (Sweden)

    Josefine Hirschfeld

    2014-12-01

    Full Text Available Background: The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design: In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion: Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown.

  16. Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

    Science.gov (United States)

    Dalli, Jesmond; Montero-Melendez, Trinidad; Norling, Lucy V; Yin, Xiaoke; Hinds, Charles; Haskard, Dorian; Mayr, Manuel; Perretti, Mauro

    2013-01-01

    Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. PMID:23660474

  17. Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.

    Science.gov (United States)

    Jančinová, Viera; Pažoureková, Silvia; Lucová, Marianna; Perečko, Tomáš; Mihalová, Danica; Bauerová, Katarína; Nosáľ, Radomír; Drábiková, Katarína

    2015-09-01

    Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCβII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  19. Damage to Aspergillus fumigatus and Rhizopus oryzae Hyphae by Oxidative and Nonoxidative Microbicidal Products of Human Neutrophils In Vitro

    OpenAIRE

    Diamond, Richard D.; Clark, Robert A.

    1982-01-01

    Our previous studies established that human neutrophils could damage and probably kill hyphae of Aspergillus fumigatus and Rhizopus oryzae in vitro, primarily by oxygen-dependent mechanisms active at the cell surface. These studies were extended, again quantitating hyphal damage by reduction in uptake of 14C-labeled uracil or glutamine. Neither A. fumigatus nor R. oryzae hyphae were damaged by neutrophils from patients with chronic granulomatous disease, confirming the importance of oxidative...

  20. Differentiation-inducing factor-1 and -2 function also as modulators for Dictyostelium chemotaxis.

    Directory of Open Access Journals (Sweden)

    Hidekazu Kuwayama

    Full Text Available BACKGROUND: In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2 were originally identified as the factors (chlorinated alkylphenones that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase and a decrease in the intracellular cGMP concentration ([cGMP](i. DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase and an increase in [cGMP](i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules for chemotaxis having differentiation-inducing activity.

  1. Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide.

    Science.gov (United States)

    Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Li, Yixuan; Wada, Satoshi; Yamaguchi, Masaya; Sumitomo, Tomoko; Hayashi, Mikako; Kawabata, Shigetada

    2017-01-01

    Streptococcus is the dominant bacterial genus in the human oral cavity and a leading cause of infective endocarditis. Streptococcus sanguinis belongs to the mitis group of streptococci and produces hydrogen peroxide (H2O2) by the action of SpxB, a pyruvate oxidase. In this study, we investigated the involvement of SpxB in survival of S. sanguinis in human blood and whether bacterial H2O2 exhibits cytotoxicity against human neutrophils. Results of a bactericidal test with human whole blood revealed that the spxB mutation in S. sanguinis is detrimental to its survival in blood. When S. sanguinis strains were exposed to isolated neutrophils, the bacterial survival rate was significantly decreased by spxB deletion. Furthermore, human neutrophils exposed to the S. sanguinis wild-type strain, in contrast to those exposed to an spxB mutant strain, underwent cell death with chromatin de-condensation and release of web-like extracellular DNA, reflecting induction of neutrophil extracellular traps (NETs). Since reactive oxygen species-mediated NET induction requires citrullination of arginine residues in histone proteins and subsequent chromatin de-condensation, we examined citrullination levels of histone in infected neutrophils. It is important to note that the citrullinated histone H3 was readily detected in neutrophils infected with the wild-type strain, as compared to infection with the spxB mutant strain. Moreover, decomposition of streptococcal H2O2 with catalase reduced NET induction. These results suggest that H2O2 produced by S. sanguinis provokes cell death of neutrophils and NET formation, thus potentially affecting bacterial survival in the bloodstream.

  2. Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide.

    Directory of Open Access Journals (Sweden)

    Ryuichi Sumioka

    Full Text Available Streptococcus is the dominant bacterial genus in the human oral cavity and a leading cause of infective endocarditis. Streptococcus sanguinis belongs to the mitis group of streptococci and produces hydrogen peroxide (H2O2 by the action of SpxB, a pyruvate oxidase. In this study, we investigated the involvement of SpxB in survival of S. sanguinis in human blood and whether bacterial H2O2 exhibits cytotoxicity against human neutrophils. Results of a bactericidal test with human whole blood revealed that the spxB mutation in S. sanguinis is detrimental to its survival in blood. When S. sanguinis strains were exposed to isolated neutrophils, the bacterial survival rate was significantly decreased by spxB deletion. Furthermore, human neutrophils exposed to the S. sanguinis wild-type strain, in contrast to those exposed to an spxB mutant strain, underwent cell death with chromatin de-condensation and release of web-like extracellular DNA, reflecting induction of neutrophil extracellular traps (NETs. Since reactive oxygen species-mediated NET induction requires citrullination of arginine residues in histone proteins and subsequent chromatin de-condensation, we examined citrullination levels of histone in infected neutrophils. It is important to note that the citrullinated histone H3 was readily detected in neutrophils infected with the wild-type strain, as compared to infection with the spxB mutant strain. Moreover, decomposition of streptococcal H2O2 with catalase reduced NET induction. These results suggest that H2O2 produced by S. sanguinis provokes cell death of neutrophils and NET formation, thus potentially affecting bacterial survival in the bloodstream.

  3. GROUP B STREPTOCOCCUS CIRCUMVENTS NEUTROPHILS AND NEUTROPHIL EXTRACELLULAR TRAPS DURING AMNIOTIC CAVITY INVASION AND PRETERM LABOR

    Science.gov (United States)

    Boldenow, Erica; Gendrin, Claire; Ngo, Lisa; Bierle, Craig; Vornhagen, Jay; Coleman, Michelle; Merillat, Sean; Armistead, Blair; Whidbey, Christopher; Alishetti, Varchita; Santana-Ufret, Veronica; Ogle, Jason; Gough, Michael; Srinouanprachanh, Sengkeo; MacDonald, James W; Bammler, Theo K; Bansal, Aasthaa; Liggitt, H. Denny; Rajagopal, Lakshmi; Waldorf, Kristina M Adams

    2016-01-01

    Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion of the amniotic cavity (MIAC) is associated with the majority of early preterm births, the temporal events that occur during MIAC and preterm labor are not known. Group B Streptococci (GBS) are β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur during MIAC, we utilized a unique chronically catheterized nonhuman primate model that closely emulates human pregnancy. This model allows monitoring of uterine contractions, timing of MIAC and immune responses during pregnancy-associated infections. Here, we show that adverse outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during infection with hemolytic GBS when compared to nonhemolytic GBS. Although MIAC was associated with systematic progression in chorioamnionitis beginning with chorionic vasculitis and progressing to neutrophilic infiltration, the ability of the GBS hemolytic pigment toxin to induce neutrophil cell death and subvert killing by neutrophil extracellular traps (NETs) in placental membranes in vivo facilitated MIAC and fetal injury. Furthermore, compared to maternal neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired phagocytic functions to GBS. Collectively, our studies demonstrate how a unique bacterial hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to induce fetal injury and preterm labor. PMID:27819066

  4. Noradrenaline increases the expression and release of Hsp72 by human neutrophils.

    Science.gov (United States)

    Giraldo, E; Multhoff, G; Ortega, E

    2010-05-01

    The blood concentration of extracellular 72kDa heat shock protein (eHsp72) increases under conditions of stress, including intense exercise. However, the signal(s), source(s), and secretory pathways in its release into the bloodstream have yet to be clarified. The aim of the present study was to evaluate the role of noradrenaline (NA) as a stress signal on the expression and release of Hsp72 by circulating neutrophils (as a source), all within a context of the immunophysiological regulation during exercise-induced stress in sedentary and healthy young (21-26years) women. The expression of Hsp72 on the surface of isolated neutrophils was determined by flow cytometry, and its release by cultured isolated neutrophils was determined by ELISA. Incubation with cmHsp70-FITC showed that neutrophils express Hsp72 on their surface under basal conditions. In addition, cultured isolated neutrophils (37 degrees C and 5% CO(2)) also released Hsp72 under basal conditions, with this release increasing from 10min to 24h in the absence of cell damage. NA at 10(-9)-10(-5)M doubled the percentage of neutrophils expressing Hsp72 after 60min and 24h incubation. NA also stimulated (by about 20%) the release of Hsp72 after 10min of incubation. (1) Hsp72 is expressed on the surface of isolated neutrophils under basal conditions, and this expression is augmented by NA. (2) Isolated neutrophils can also release Hsp72 under cultured basal conditions in the absence of cell death, and NA can increase this release. These results may contribute to confirming the hypothesis that NA can act as a "stress signal" for the increased eHsp72 in the context of exercise stress, with a role for neutrophils as a source for the expression and, to a lesser degree, the release of Hsp72 after activation by NA. Copyright 2010 Elsevier Inc. All rights reserved.

  5. Effect of the dimetilsulfoxido in the response chemiluminescent and the consumption of oxygen of neutrophils activated human

    International Nuclear Information System (INIS)

    Garcia, J.

    2001-01-01

    Dimethylsulfoxide (DMSO), a hydroxyl radical scavenger, exerted a dose dependent inhibition on the luminol and lucigenin-enhanced chemiluminescent responses of human neutrophils activated with soluble and particulate stimulants. DMSO inhibition of the luminol chemiluminescense induced by calcium ionophore A23187 was probably due to OH scavenging, whereas inhibition of the lucigenin chemiluminescence suggested DMSO negatively affects the NADPH-dependent membrane oxidase of neutrophils. In agreement with this, DMSO moderately inhibited O2 consumption in PMN suspensions stimulated with chemotactic peptide and opsonized zymosan-induced luminol chemiluminescense was observed only when added before or in conjunction with stimulants, whereas A23187-induced chemiluminescense was inhibited by DMSO regardless of time of addition. Washing of DMSO-treated PMN resulted in increased luminol enhanced chemiluminescense in response to chemotactic peptide and opsonized zymosan. This is consistent with the idea that DMSO may be interfering with activation of the membrane subunits of the oxidase by translocation and docking of the cytoplasmic, regulatory subunits. These data imply that DMSO inhibits neutrophil chemiluminescense both by OH scavenging and interfering with oxidase activation. Key words:Dimethylsulfoxide, chemiluminescent, luminol, lucigenin,neutrophils [es

  6. Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function

    Directory of Open Access Journals (Sweden)

    Antommattei Frances M

    2008-10-01

    Full Text Available Abstract Background Geobacter species are δ-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production. Recent studies in other bacteria have demonstrated that signaling pathways homologous to the paradigm established for Escherichia coli chemotaxis can regulate type IV pili-dependent motility, the synthesis of flagella and type IV pili, the production of extracellular matrix material, and biofilm formation. The classification of these pathways by comparative genomics improves the ability to understand how Geobacter thrives in natural environments and better their use in microbial fuel cells. Results The genomes of G. sulfurreducens, G. metallireducens, and G. uraniireducens contain multiple (~70 homologs of chemotaxis genes arranged in several major clusters (six, seven, and seven, respectively. Unlike the single gene cluster of E. coli, the Geobacter clusters are not all located near the flagellar genes. The probable functions of some Geobacter clusters are assignable by homology to known pathways; others appear to be unique to the Geobacter sp. and contain genes of unknown function. We identified large numbers of methyl-accepting chemotaxis protein (MCP homologs that have diverse sensing domain architectures and generate a potential for sensing a great variety of environmental signals. We discuss mechanisms for class-specific segregation of the MCPs in the cell membrane, which serve to maintain pathway specificity and diminish crosstalk. Finally, the regulation of gene expression in Geobacter differs from E. coli. The sequences of predicted promoter elements suggest that the alternative sigma factors

  7. Mycobacterium tuberculosis Cell Wall Fragments Released upon Bacterial Contact with the Human Lung Mucosa Alter the Neutrophil Response to Infection.

    Science.gov (United States)

    Scordo, Julia M; Arcos, Jesús; Kelley, Holden V; Diangelo, Lauren; Sasindran, Smitha J; Youngmin, Ellie; Wewers, Mark D; Wang, Shu-Hua; Balada-Llasat, Joan-Miquel; Torrelles, Jordi B

    2017-01-01

    In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis ( M.tb ) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site.

  8. Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

    Directory of Open Access Journals (Sweden)

    Massimo Conese

    Full Text Available In seeking more specific biomarkers of the cystic fibrosis (CF lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, hydrogen voltage-gated channel 1 (HVCN1, and β-arrestin 1 (ARRB1. The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils

  9. Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

    Science.gov (United States)

    Conese, Massimo; Castellani, Stefano; Lepore, Silvia; Palumbo, Orazio; Manca, Antonio; Santostasi, Teresa; Polizzi, Angela Maria; Copetti, Massimiliano; Di Gioia, Sante; Casavola, Valeria; Guerra, Lorenzo; Diana, Anna; Montemurro, Pasqualina; Mariggiò, Maria Addolorata; Gallo, Crescenzio; Maffione, Angela Bruna; Carella, Massimo

    2014-01-01

    In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and

  10. Activation of bovine neutrophils by Brucella spp.

    Science.gov (United States)

    Keleher, Lauren L; Skyberg, Jerod A

    2016-09-01

    Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. The Impact of Odor--Reward Memory on Chemotaxis in Larval "Drosophila"

    Science.gov (United States)

    Schleyer, Michael; Reid, Samuel F.; Pamir, Evren; Saumweber, Timo; Paisios, Emmanouil; Davies, Alexander; Gerber, Bertram; Louis, Matthieu

    2015-01-01

    How do animals adaptively integrate innate with learned behavioral tendencies? We tackle this question using chemotaxis as a paradigm. Chemotaxis in the "Drosophila" larva largely results from a sequence of runs and oriented turns. Thus, the larvae minimally need to determine (i) how fast to run, (ii) when to initiate a turn, and (iii)…

  12. The effect of cigarette smoking on neutrophil kinetics in human lungs [see comments

    International Nuclear Information System (INIS)

    MacNee, W.; Wiggs, B.; Belzberg, A.S.; Hogg, J.C.

    1989-01-01

    Neutrophils may play a part in the pathogenesis of the centrilobular emphysema associated with cigarette smoking. The capillary bed of the lungs concentrates neutrophils approximately 100-fold with respect to erythrocytes, producing a large pool of marginated cells. We examined the effect of cigarette smoking on the kinetics of this pool of cells, using 99mTc-labeled erythrocytes to measure regional blood velocity and 111In-labeled neutrophils to measure the removal of neutrophils during the first passage through the pulmonary circulation, their subsequent washout from the lungs, and the effect of local blood velocity on the number of neutrophils retained in each lung region. We observed no difference in these measurements between subjects who had never smoked (n = 6) and smokers who did not smoke during the study (n = 12). However, subjects who did smoke during the study (n = 12) had a significantly slower rate of washout of radiolabeled neutrophils from the lung (0.08 +/- 0.04 of the total per minute, as compared with 0.13 +/- 0.06 in smokers who did not smoke during the experiment and 0.14 +/- 0.08 in non-smokers) (P = 0.02). We also observed an increase in the regional retention of labeled neutrophils with respect to blood velocity in 5 of the 12 subjects who smoked during the study, but in none of the other subjects. We conclude that the presence of cigarette smoke in the lungs of some subjects increases the local concentration of neutrophils, and suggest that the lesions that characterize emphysema may be a result of the destruction of lung tissue by neutrophils that remain within pulmonary microvessels

  13. Protein phosphorylation and bacterial chemotaxis

    International Nuclear Information System (INIS)

    Hess, J.F.; Bourret, R.B.; Oosawa, K.; Simon, M.I.; Matsumura, P.

    1988-01-01

    Bacteria are able to respond to changes in concentration of a large variety of chemicals and to changes in physical parameters, including viscosity, osmolarity, and temperature, by swimming toward a more favorable location (for review, see Stewart and Dahlquist 1987). Most chemotactic responses are mediated by a series of transmembrane receptor proteins that interact with or bind specific chemicals and thus monitor environmental conditions. Over the past 10 years, work in a number of laboratories has resulted in the identification and characterization of many of the genes and proteins required for the signal transduction process. The authors postulated that rapid and transient covalent modification of the chemotaxis gene products could function to transmit information from the receptor by regulating protein-protein interaction between the chemotaxis gene products. To test this idea, the authors purified the proteins corresponding to the cheA, cheY, cheZ, cheW, and cheB genes and tested the purified polypeptides to determine whether they could be covalently modified and whether they would interact with each other in vitro

  14. Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential

    NARCIS (Netherlands)

    Duijn, Bert van; Vogelzang, Sake A.; Ypey, Dirk L.; Molen, Loek G. van der; Haastert, Peter J.M. van

    1990-01-01

    We examined a possible role for the plasma membrane potential in signal transduction during cyclic AMP-induced chemotaxis in the cellular slime mold Dictyostelium discoideum. Chemotaxis, cyclic GMP and cyclic AMP responses in cells with a depolarized membrane potential were measured. Cells can be

  15. Ectoenzymes and innate immunity: the role of human CD157 in leukocyte trafficking.

    Science.gov (United States)

    Funaro, Ada; Ortolan, Erika; Bovino, Paola; Lo Buono, Nicola; Nacci, Giulia; Parrotta, Rossella; Ferrero, Enza; Malavasi, Fabio

    2009-01-01

    CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.

  16. Pneumovirus-Induced Lung Disease in Mice Is Independent of Neutrophil-Driven Inflammation

    NARCIS (Netherlands)

    Cortjens, Bart; Lutter, René; Boon, Louis; Bem, Reinout A.; van Woensel, Job B. M.

    2016-01-01

    The human pneumovirus respiratory syncytial virus (RSV) is the most common pathogen causing lower respiratory tract disease in young children worldwide. A hallmark of severe human RSV infection is the strong neutrophil recruitment to the airways and lungs. Massive neutrophil activation has been

  17. High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions.

    Science.gov (United States)

    Rice, W G; Kinkade, J M; Parmley, R T

    1986-08-01

    Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA

  18. Neutrophils are not less sensitive than other blood leukocytes to the genomic effects of glucocorticoids.

    Directory of Open Access Journals (Sweden)

    Gaelle Hirsch

    Full Text Available Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out.We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes.Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL alone or combined with hydrocortisone, prednisolone or dexamethasone (10(-8 M and 10(-6 M. IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations.We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils.Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some

  19. Neutrophils Are Not Less Sensitive Than Other Blood Leukocytes to the Genomic Effects of Glucocorticoids

    Science.gov (United States)

    Hirsch, Gaelle; Lavoie-Lamoureux, Anouk; Beauchamp, Guy; Lavoie, Jean-Pierre

    2012-01-01

    Background Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out. Objective We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes. Methods Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10−8 M and 10−6 M). IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations. Results We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Conclusions Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to

  20. In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumour necrosis factor

    Directory of Open Access Journals (Sweden)

    Anne Silvestre

    2015-07-01

    Full Text Available Background: Entamoeba histolytica cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction. The tissue inflammation associated with tumour necrosis factor (TNF secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. Methods: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. Results: an antibody against human TNF receptor 1 (TNFR1 stained the E. histolytica trophozoite surface and (on immunoblots binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as "cell surface protein", CSP, in view of its cellular location. Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. Conclusions: there is a clear link between TNF chemotaxis, CSP and pathogenesis.

  1. The selective estrogen receptor modulator raloxifene inhibits neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Roxana Flores

    2016-12-01

    Full Text Available Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effect on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA, a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs. Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Like raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation but not reactive oxygen species (ROS production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production.

  2. Relation between chemotaxis and consumption of amino acids in bacteria

    Science.gov (United States)

    Yang, Yiling; M. Pollard, Abiola; Höfler, Carolin; Poschet, Gernot; Wirtz, Markus; Hell, Rüdiger

    2015-01-01

    Summary Chemotaxis enables bacteria to navigate chemical gradients in their environment, accumulating toward high concentrations of attractants and avoiding high concentrations of repellents. Although finding nutrients is likely to be an important function of bacterial chemotaxis, not all characterized attractants are nutrients. Moreover, even for potential nutrients, the exact relation between the metabolic value of chemicals and their efficiency as chemoattractants has not been systematically explored. Here we compare the chemotactic response of amino acids with their use by bacteria for two well‐established models of chemotactic behavior, E scherichia coli and B acillus subtilis. We demonstrate that in E . coli chemotaxis toward amino acids indeed strongly correlates with their utilization. However, no such correlation is observed for B . subtilis, suggesting that in this case, the amino acids are not followed because of their nutritional value but rather as environmental cues. PMID:25807888

  3. Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors

    Directory of Open Access Journals (Sweden)

    Karin eChristenson

    2013-04-01

    Full Text Available Most notable among the acute phase proteins is serum amyloid A (SAA, levels of which can increase 1000-fold during infections, aseptic inflammation, and/or trauma. Chronically elevated SAA levels are associated with a wide variety of pathological conditions, including obesity and rheumatic diseases. Using a recombinant hybrid of the two human SAA isoforms (SAA1 and 2 that does not exist in vivo, numerous in vitro studies have given rise to the notion that acute phase SAA is a pro-inflammatory molecule with cytokine-like properties. It is however unclear whether endogenous acute phase SAA per se mediates pro-inflammatory effects. We tested this in samples from patients with inflammatory arthritis and in a transgenic mouse model that expresses human SAA1. Endogenous human SAA did not drive production of pro-inflammatory IL-8/KC in either of these settings. Human neutrophils derived from arthritis patients displayed no signs of activation, despite being exposed to severely elevated SAA levels in circulation, and SAA-rich sera also failed to activate cells in vitro. In contrast, two recombinant SAA variants (the hybrid SAA and SAA1 both activated human neutrophils, inducing L-selectin shedding, production of reactive oxygen species, and production of IL-8. The hybrid SAA was approximately 100-fold more potent than recombinant SAA1. Recombinant hybrid SAA and SAA1 activated neutrophils through different receptors, with recombinant SAA1 being a ligand for formyl peptide receptor 2 (FPR2. We conclude that even though recombinant SAAs can be valuable tools for studying neutrophil activation, they do not reflect the nature of the endogenous protein.

  4. Microbiological, immunological and genetic factors in family members with periodontitis as a manifestation of systemic disease, associated with hematological disorders.

    Science.gov (United States)

    Okada, Mitsugi; Awane, Saori; Suzuki, Junji; Hino, Takamune; Takemoto, Toshinobu; Kurihara, Hidemi; Miura, Kazuo

    2002-08-01

    The microflora, immunological profiles of host defence functions, and human leukocyte antigen (HLA) findings are reported for a mother, son and daughter who were diagnosed as having 'periodontitis as a manifestation of systemic diseases, associated with hematological disorders'. Examinations were made of the bacterial flora from the periodontal pocket, neutrophil chemotaxis, neutrophil phagocytosis, and the genotypes (DQB1) and serotypes (DR locus) of HLA class II antigens. Phenotypic analyses of the peripheral lymphocytes were also conducted. The subgingival microflora from the mother was dominated by Gram-negative rods, especially Porphyromonas endodontalis, Prevotella intermedia/Prevotella nigrescens and Fusobacterium nucleatum. Subgingival microflora samples from the son and daughter were dominated by Gram-positive cocci and Gram-positive rods. Through the use of polymerase chain reaction, Campylobacter rectus and Capnocytophaga gingivalis were detected in all subjects, whereas Porphyromonas gingivalis, P. intermedia, and Treponema denticola were not detected in any subjects. All three subjects showed a remarkable level of depressed neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine, although their phagocyte function levels were normal, in comparison to healthy control subjects. Each subject had the same genotype, HLA-DQB1*0601, while the mother had HLA-DR2 and HLA-DR8, and the son and daughter had HLA-DR2 only. In summary, the members of this family showed a similar predisposition to periodontitis with regard to certain host defence functions. It is suggested that the depressed neutrophil chemotaxis that was identified here could be a significant risk factor for periodontitis in this family.

  5. Different migration patterns of sea urchin and mouse sperm revealed by a microfluidic chemotaxis device.

    Directory of Open Access Journals (Sweden)

    Haixin Chang

    Full Text Available Chemotaxis refers to a process whereby cells move up or down a chemical gradient. Sperm chemotaxis is known to be a strategy exploited by marine invertebrates such as sea urchins to reach eggs efficiently in moving water. Less is understood about how or whether chemotaxis is used by mammalian sperm to reach eggs, where fertilization takes place within the confinement of a reproductive tract. In this report, we quantitatively assessed sea urchin and mouse sperm chemotaxis using a recently developed microfluidic model and high-speed imaging. Results demonstrated that sea urchin Arbacia punctulata sperm were chemotactic toward the peptide resact with high chemotactic sensitivity, with an average velocity Vx up the chemical gradient as high as 20% of its average speed (238 μm/s, while mouse sperm displayed no statistically significant chemotactic behavior in progesterone gradients, which had been proposed to guide mammalian sperm toward eggs. This work demonstrates the validity of a microfluidic model for quantitative sperm chemotaxis studies, and reveals a biological insight that chemotaxis up a progesterone gradient may not be a universal strategy for mammalian sperm to reach eggs.

  6. Chemotaxis-growth under the influence of lateral inhibition in a three-component reaction–diffusion system

    International Nuclear Information System (INIS)

    Kawaguchi, Satoshi

    2011-01-01

    In this study, we consider the effects of chemotaxis and lateral inhibition on an activator in a three-component reaction–diffusion system. Simulation results show that spot, planar and travelling front solutions in two dimensions are destabilized to form multibranch patterns. In order to analyse the stability of stationary solutions, a singular perturbation method is employed. The bifurcation diagrams suggest that chemotaxis and lateral inhibition cooperatively result in the destabilization of the stationary solutions. Our three-component model is compared with the two-component chemotaxis-growth model. Furthermore, the conditions for observing the cooperative effects of chemotaxis and lateral inhibition on an activator in experiments are inferred from the model

  7. Postprandial triglyceride-rich lipoproteins promote lipid accumulation and apolipoprotein B-48 receptor transcriptional activity in human circulating and murine bone marrow neutrophils in a fatty acid-dependent manner.

    Science.gov (United States)

    Ortega-Gómez, Almudena; Varela, Lourdes M; López, Sergio; Montserrat de la Paz, Sergio; Sánchez, Rosario; Muriana, Francisco J G; Bermúdez, Beatriz; Abia, Rocío

    2017-09-01

    Postprandial triglyceride-rich lipoproteins (TRLs) promote atherosclerosis. Recent research points the bone marrow (BM) as a primary site in atherosclerosis. We elucidated how the acute administration of monounsaturated fatty acids (MUFAs) MUFAs, omega-3 polyunsaturated fatty acids (PUFAs) PUFAs and saturated fatty acids (SFAs) affects human circulating and murine BM neutrophil lipid accumulation and functionality. Postprandial hypertriglyceridemia was induced in healthy subjects and Apoe -/- mice by the acute administration of dietary fats enriched in MUFAs, PUFAs, or SFAs. Postprandial hypertriglyceridemia increased apolipoprotein-B48 receptor (ApoB48R) transcriptional activity that was linearly correlated with intracellular triglycerides (TGs) TGs accumulation in human circulating and murine BM neutrophils. MUFA and omega-3 PUFAs attenuated ApoB48R gene expression and intracellular TG accumulation compared to SFAs. TRLs induced apoB48R-dependent TG accumulation in human neutrophils ex vivo. Murine BM neutrophils showed a decrease in surface L-selectin and an increase in TNF-α and IL-1β mRNA expressions only after SFAs administration. TRLs enriched in SFAs induced BM neutrophil degranulation ex vivo suggesting cell priming/activation. Postprandial TRLs disrupts the normal biology and function of circulating and BM neutrophils. MUFA- and omega-3 PUFA-rich dietary fats such as virgin olive oil or fish oil has the potential to prevent excessive neutrophil lipid accumulation and activation by targeting the fatty acid composition of TRLs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime

    Directory of Open Access Journals (Sweden)

    M. Mathy-Hartert

    1995-01-01

    Full Text Available We investigated the effects of the antibiotic ceftazidime (CAZ on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−. In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC were capable of taking up active MPO. In presence of H2O2 (10−4 M, this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC. Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2 was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon. So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

  9. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, Kirley M.; Silveira, Edilberto R., E-mail: edil@ufc.br [Universidade Federal do Ceara (UFCE), Fortaleza, CE (Brazil). Departamento de Quimica Organica e Inorganica; Leal, Luzia K.A.M.; Lopes, Amanda A. [Universidade Federal do Ceara (CEFAC/UFCE), Fortaleza, CE (Brazil). Centro de Estudos Farmaceuticos e Cosmeticos. Departamento de Farmacia; Coleman, Christina M.; Ferreira, Daneel [Department of Pharmacognosy and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS (United States)

    2014-04-15

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL{sup -1}). At concentration higher than 25 μg mL{sup -1}, both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  10. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    International Nuclear Information System (INIS)

    Canuto, Kirley M.; Silveira, Edilberto R.

    2014-01-01

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL -1 ). At concentration higher than 25 μg mL -1 , both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  11. Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

    LENUS (Irish Health Repository)

    Hong, Ying

    2012-01-01

    The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

  12. Chronic neutrophilic leukemia.

    Science.gov (United States)

    Bredeweg, Arthur; Burch, Micah; Krause, John R

    2018-01-01

    Chronic neutrophilic leukemia is a rare myeloproliferative disorder characterized by a sustained peripheral blood neutrophilia, absence of the BCR/ABL oncoprotein, bone marrow hypercellularity with less than 5% myeloblasts and normal neutrophil maturation, and no dysplasia. This leukemia has been associated with mutations in the colony-stimulating factor 3 receptor (CSF3R) that may activate this receptor, leading to the proliferation of neutrophils that are the hallmark of chronic neutrophilic leukemia. We present a case of chronic neutrophilic leukemia and discuss the criteria for diagnosis and the significance of mutations found in this leukemia.

  13. Biomaterial-induced alterations of neutrophil superoxide production.

    Science.gov (United States)

    Kaplan, S S; Basford, R E; Mora, E; Jeong, M H; Simmons, R L

    1992-08-01

    Because periprosthetic infection remains a vexing problem for patients receiving implanted devices, we evaluated the effect of several materials on neutrophil free radical production. Human peripheral blood neutrophils were incubated with several sterile, lipopolysaccharide (LPS)-free biomaterials used in surgically implantable prosthetic devices: polyurethane, woven dacron, and velcro. Free radical formation as the superoxide (O2-) anion was evaluated by cytochrome c reduction in neutrophils that were exposed to the materials and then removed and in neutrophils allowed to remain in association with the materials. Neutrophils exposed to polyurethane or woven dacron for 30 or 60 min and then removed consistently exhibited an enhanced release of O2- after simulation via receptor engagement with formyl methionyl-leucyl-phenylalanine. Enhanced reactivity to stimulation via protein kinase C with phorbol myristate acetate, however, was not consistently observed. The cells evaluated for O2- release during continuous association with the biomaterials showed enhanced metabolic activity during short periods of association (especially with polyurethane and woven dacron). Although O2- release by neutrophils in association with these materials decreased with longer periods of incubation, it was not obliterated. These studies, therefore, show that several commonly used biomaterials activate neutrophils soon after exposure and that this activated state diminishes with prolonged exposure but nevertheless remains measurable. The diminishing level of activity with prolonged exposure, however, suggests that ultimately a depletion of reactivity may occur and may result in increased susceptibility to periprosthetic infection.

  14. Neutrophilic dermatosis resembling pyoderma gangrenosum in a dog with polyarthritis.

    Science.gov (United States)

    Bardagí, M; Lloret, A; Fondati, A; Ferrer, L

    2007-04-01

    This report describes a case of neutrophilic dermatosis in a dog, with a number of clinical and pathological similarities to human pyoderma gangrenosum. A seven-year-old, female German shepherd dog with a history of non-erosive idiopathic polyarthritis was presented with severe facial swelling, bilateral erosivoulcerative lesions on the muzzle and multiple, eroded, dermal-subcutaneous nodules on the cranial trunk. Histopathological examination of skin biopsies revealed a necrotising neutrophilic dermatitis. No infectious agents could be detected using specific stains, immunohistochemistry, serology and bacterial aerobic, anaerobic or fungal cultures. A sterile neutrophilic dermatosis resembling human pyoderma gangrenosum was presumptively diagnosed, and the patient showed an excellent response to treatment with prednisone and ciclosporin.

  15. Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

    NARCIS (Netherlands)

    Maianski, N. A.; Geissler, J.; Srinivasula, S. M.; Alnemri, E. S.; Roos, D.; Kuijpers, T. W.

    2004-01-01

    Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly

  16. TLR9 and NF-κB are partially involved in activation of human neutrophils by Helicobacter pylori and its purified DNA.

    Directory of Open Access Journals (Sweden)

    Lourdes Alvarez-Arellano

    Full Text Available Helicobacter pylori infection represents one of the most common bacterial infections worldwide. The inflammatory response to this bacterium involves a large influx of neutrophils to the lamina propria of the gastric mucosa. However, little is known about the receptors and molecular mechanisms involved in activation of these neutrophils. In this study, we aimed to determine the role of toll-like receptor 9 (TLR9 in the response of human neutrophils to H. pylori and purified H. pylori DNA (Hp-DNA. Neutrophils were isolated from the blood of adult volunteers and challenged with either H. pylori or Hp-DNA. We found that both, H. pylori and Hp-DNA induced increased expression and release of IL-8. Furthermore, we showed that TLR9 is involved in the induction of IL-8 production by H. pylori and Hp-DNA. IL-8 production induced by H. pylori but not by Hp-DNA was partially mediated by NF-κB. In conclusion, this study showed for first time that both, H. pylori and Hp-DNA activate TLR9 and induce a different inflammatory response that leads to activation of neutrophils.

  17. Global Solutions to the Coupled Chemotaxis-Fluid Equations

    KAUST Repository

    Duan, Renjun

    2010-08-10

    In this paper, we are concerned with a model arising from biology, which is a coupled system of the chemotaxis equations and the viscous incompressible fluid equations through transport and external forcing. The global existence of solutions to the Cauchy problem is investigated under certain conditions. Precisely, for the Chemotaxis-Navier-Stokes system over three space dimensions, we obtain global existence and rates of convergence on classical solutions near constant states. When the fluid motion is described by the simpler Stokes equations, we prove global existence of weak solutions in two space dimensions for cell density with finite mass, first-order spatial moment and entropy provided that the external forcing is weak or the substrate concentration is small. © Taylor & Francis Group, LLC.

  18. Divergent effects of tumor necrosis factor alpha on apoptosis of human neutrophils

    NARCIS (Netherlands)

    van den Berg, J. M.; Weyer, S.; Weening, J. J.; Roos, D.; Kuijpers, T. W.

    2001-01-01

    Apoptosis of neutrophils is a key mechanism to control the intensity of the acute inflammatory response. Previously, the cytokine tumor necrosis factor alpha (TNF-alpha) was reported by some to have pro-apoptotic and by others to have antiapoptotic effects on neutrophils. The aim of this study was

  19. Reduction of Monocyte Chemoattractant Protein-1 and Interleukin-8 Levels by Ticlopidine in TNF-α Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Chaur-Jong Hu

    2009-01-01

    Full Text Available Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1 is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8, a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-α-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1 in human umbilical vein endothelial cells (HUVECs. Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-α stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-α induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

  20. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, Kirley M.; Silveira, Edilberto R., E-mail: edil@ufc.br [Universidade Federal do Ceara (UFCE), Fortaleza, CE (Brazil). Departamento de Quimica Organica e Inorganica; Leal, Luzia K.A.M.; Lopes, Amanda A. [Universidade Federal do Ceara (CEFAC/UFCE), Fortaleza, CE (Brazil). Centro de Estudos Farmaceuticos e Cosmeticos. Departamento de Farmacia; Coleman, Christina M.; Ferreira, Daneel [Department of Pharmacognosy and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS (United States)

    2014-04-15

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL{sup -1}). At concentration higher than 25 μg mL{sup -1}, both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  1. Immune modulation by neutrophil subsets

    NARCIS (Netherlands)

    Kamp, V.M.

    2013-01-01

    We show that human neutrophils can suppress T-cell proliferation in acute systemic inflammation and thus have anti-inflammatory functions, next to their well-known pro-inflammatory functions. The suppression is mediated by ROS production and integrin MAC-1, which are also important for the

  2. Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection.

    Directory of Open Access Journals (Sweden)

    Martin S Davey

    2011-05-01

    Full Text Available Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8. In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN-γ and tumor necrosis factor (TNF-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1, and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in

  3. Human Neutrophil Clearance of Bacterial Pathogens Triggers Anti-Microbial γδ T Cell Responses in Early Infection

    Science.gov (United States)

    Roberts, Gareth W.; Heuston, Sinéad; Brown, Amanda C.; Chess, James A.; Toleman, Mark A.; Gahan, Cormac G. M.; Hill, Colin; Parish, Tanya; Williams, John D.; Davies, Simon J.; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Eberl, Matthias

    2011-01-01

    Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early

  4. N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils.

    Science.gov (United States)

    Mora-Cartín, Ricardo; Chacón-Díaz, Carlos; Gutiérrez-Jiménez, Cristina; Gurdián-Murillo, Stephany; Lomonte, Bruno; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; Moreno, Edgardo

    2016-06-01

    Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Neutrophils at work

    DEFF Research Database (Denmark)

    Nauseef, William M; Borregaard, Niels

    2014-01-01

    In this Review we discuss data demonstrating recently recognized aspects of neutrophil homeostasis in the steady state, granulopoiesis in 'emergency' conditions and interactions of neutrophils with the adaptive immune system. We explore in vivo observations of the recruitment of neutrophils from ...

  6. Neutrophils in critical illness.

    Science.gov (United States)

    McDonald, Braedon

    2018-03-01

    During critical illness, dramatic alterations in neutrophil biology are observed including abnormalities of granulopoeisis and lifespan, cell trafficking and antimicrobial effector functions. As a result, neutrophils transition from powerful antimicrobial protectors into dangerous mediators of tissue injury and organ dysfunction. In this article, the role of neutrophils in the pathogenesis of critical illness (sepsis, trauma, burns and others) will be explored, including pathological changes to neutrophil function during critical illness and the utility of monitoring aspects of the neutrophil phenotype as biomarkers for diagnosis and prognostication. Lastly, we review findings from clinical trials of therapies that target the harmful effects of neutrophils, providing a bench-to-bedside perspective on neutrophils in critical illness.

  7. MicroRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment.

    Science.gov (United States)

    Dorhoi, Anca; Iannaccone, Marco; Farinacci, Maura; Faé, Kellen C; Schreiber, Jörg; Moura-Alves, Pedro; Nouailles, Geraldine; Mollenkopf, Hans-Joachim; Oberbeck-Müller, Dagmar; Jörg, Sabine; Heinemann, Ellen; Hahnke, Karin; Löwe, Delia; Del Nonno, Franca; Goletti, Delia; Capparelli, Rosanna; Kaufmann, Stefan H E

    2013-11-01

    The molecular mechanisms that control innate immune cell trafficking during chronic infection and inflammation, such as in tuberculosis (TB), are incompletely understood. During active TB, myeloid cells infiltrate the lung and sustain local inflammation. While the chemoattractants that orchestrate these processes are increasingly recognized, the posttranscriptional events that dictate their availability are unclear. We identified microRNA-223 (miR-223) as an upregulated small noncoding RNA in blood and lung parenchyma of TB patients and during murine TB. Deletion of miR-223 rendered TB-resistant mice highly susceptible to acute lung infection. The lethality of miR-223(–/–) mice was apparently not due to defects in antimycobacterial T cell responses. Exacerbated TB in miR-223(–/–) animals could be partially reversed by neutralization of CXCL2, CCL3, and IL-6, by mAb depletion of neutrophils, and by genetic deletion of Cxcr2. We found that miR-223 controlled lung recruitment of myeloid cells, and consequently, neutrophil-driven lethal inflammation. We conclude that miR-223 directly targets the chemoattractants CXCL2, CCL3, and IL-6 in myeloid cells. Our study not only reveals an essential role for a single miRNA in TB, it also identifies new targets for, and assigns biological functions to, miR-223. By regulating leukocyte chemotaxis via chemoattractants, miR-223 is critical for the control of TB and potentially other chronic inflammatory diseases.

  8. Disentangling the effects of tocilizumab on neutrophil survival and function.

    Science.gov (United States)

    Gaber, Timo; Hahne, Martin; Strehl, Cindy; Hoff, Paula; Dörffel, Yvonne; Feist, Eugen; Burmester, Gerd-Rüdiger; Buttgereit, Frank

    2016-06-01

    The synovial tissue in rheumatoid arthritis (RA) represents a hypoxic environment with up-regulated pro-inflammatory cytokines and cellular infiltrates including neutrophils. Although inhibition of the interleukin (IL)6 receptor pathway by tocilizumab is a potent treatment option for RA, it may also cause adverse effects such as an occasionally high-grade neutropenia. We analysed the impact of tocilizumab on survival, mediator secretion, oxidative burst, phagocytosis and energy availability of high-dose toll-like receptor (TLR)2/4-stimulated neutrophils (to mimic an arthritis flare) under normoxic versus hypoxic conditions. Human neutrophils were purified, pre-treated with varying doses of tocilizumab, dexamethasone or human IgG1 and high-dose-stimulated with lipopolysaccharide (LPS) alone-triggering TLR2/4-, LPS plus IL6, or left unstimulated. Cells were then incubated under normoxic (18 % O2) or hypoxic (1 % O2) conditions and subsequently analysed. Neutrophil survival and energy availability were significantly decreased by tocilizumab in a dose-dependent manner in high-dose TLR2/4-stimulated cells, but to a greater extent under normoxia as compared to hypoxia. We also found high-dose LPS-stimulated oxidative burst and phagocytosis of neutrophils to be higher under hypoxic versus normoxic conditions, but this difference was reduced by tocilizumab. Finally, we observed that tocilizumab affected neutrophil mediator secretion as a function of oxygen availability. Tocilizumab is known for both beneficial effects and a higher incidence of neutropenia when treating RA patients. Our results suggest that both effects can at least in part be explained by a reduction in neutrophil survival, a dose-dependent inhibition of hypoxia-induced NADPH oxidase-mediated oxidative burst and phagocytosis of infiltrating hypoxic neutrophils and an alteration of mediator secretion.

  9. Chemotaxis of nurse shark leukocytes.

    Science.gov (United States)

    Obenauf, S D; Smith, S H

    1985-01-01

    Studies were conducted to determine the ability of leukocytes from the nurse shark to migrate in an in vitro micropore filter chemotaxis assay and to determine optimal assay conditions and suitable attractants for such an assay. A migratory response was seen with several attractants: activated rat serum, activated shark plasma, and a pool of shark complement components. Only the response to activated rat serum was chemotactic, as determined by the checkerboard assay.

  10. Dispatch. Dictyostelium chemotaxis: fascism through the back door?

    Science.gov (United States)

    Insall, Robert

    2003-04-29

    Aggregating Dictyostelium cells secrete cyclic AMP to attract their neighbours by chemotaxis. It has now been shown that adenylyl cyclase is enriched in the rear of cells, and this localisation is required for normal aggregation.

  11. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    Science.gov (United States)

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Oxidative burst of circulating neutrophils following traumatic brain injury in human.

    Directory of Open Access Journals (Sweden)

    Yiliu Liao

    Full Text Available Besides secondary injury at the lesional site, Traumatic brain injury (TBI can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91(phox in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected

  13. Neutrophil extracellular traps - the dark side of neutrophils

    DEFF Research Database (Denmark)

    Sørensen, Ole E.; Borregaard, Niels

    2016-01-01

    Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those ori...

  14. Tumor Associated Neutrophils in Human Lung Cancer

    Science.gov (United States)

    2016-10-01

    tumor innate immune response. anti-tumor adaptive immune response, neutrophil and T cell interaction. ACCOMPLISHMENTS There were no significant...and by producing factors to recruit and acti- vate cells of the innate and adaptive immune system (Mantovani et al., 2011). Given these varying effects...vivo effects on neutro- phil activation (Figure 2, A and B) and cleavage of myeloid and lymphoid cell markers (Supplemental Figure 1, C–G). Once opti

  15. Proinflammatory mediators stimulate neutrophil-directed angiogenesis.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Vascular endothelial growth factor (VEGF; vascular permeability factor) is one of the most potent proangiogenic cytokines, and it plays a central role in mediating the process of angiogenesis or new blood vessel formation. Neutrophils (PMNs) recently have been shown to produce VEGF. HYPOTHESIS: The acute inflammatory response is a potent stimulus for PMN-directed angiogenesis. METHODS: Neutrophils were isolated from healthy volunteers and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and anti-human Fas monoclonal antibody. Culture supernatants were assayed for VEGF using enzyme-linked immunosorbent assays. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs were then added to human umbilical vein endothelial cells and human microvessel endothelial cells and assessed for endothelial cell proliferation using 5-bromodeoxyuridine labeling. Tubule formation was also assessed on MATRIGEL basement membrane matrix. Neutrophils were lysed to measure total VEGF release, and VEGF expression was detected using Western blot analysis. RESULTS: Lipopolysaccharide and TNF-alpha stimulation resulted in significantly increased release of PMN VEGF (532+\\/-49 and 484+\\/-80 pg\\/mL, respectively; for all, presented as mean +\\/- SEM) compared with control experiments (32+\\/-4 pg\\/mL). Interleukin 6 and Fas had no effect. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs also resulted in significant increases (P<.005) in macrovascular and microvascular endothelial cell proliferation and tubule formation. Adding anti-human VEGF-neutralizing polyclonal antibody to stimulated PMN supernatant inhibited these effects. Total VEGF release following cell lysis and Western blot analysis suggests that the VEGF is released from an intracellular store. CONCLUSION: Activated human PMNs are directly angiogenic by releasing VEGF, and this has important implications for inflammation, capillary leak syndrome

  16. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    International Nuclear Information System (INIS)

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H.

    1991-01-01

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC

  17. Antimicrobial Peptide Human Neutrophil Peptide 1 as a Potential Link Between Chronic Inflammation and Ductal Adenocarcinoma of the Pancreas.

    Science.gov (United States)

    Pausch, Thomas; Adolph, Sarah; Felix, Klaus; Bauer, Andrea S; Bergmann, Frank; Werner, Jens; Hartwig, Werner

    Defensins are antimicrobial peptides playing a role in innate immunity, in epithelial cell regeneration, and in carcinogenesis of inflammation-triggered malignancies. We analyzed this role in pancreatic ductal adenocarcinoma (PDAC) in the context of its association with chronic pancreatitis (CP). Human tissue of healthy pancreas, CP, and PDAC was screened for defensins by immunohistochemistry. Defensin α 1 (human neutrophil peptide 1 [HNP-1]) expression was validated using mass spectrometry and microarray analysis. Human neutrophil peptide 1 expression and influences of proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, and interferon γ) were studied in human pancreatic cancer cells (Colo 357, T3M4, PANC-1) and normal human pancreatic duct epithelial cells (HPDE). Accumulation of HNP-1 in malignant pancreatic ductal epithelia was seen. Spectrometry showed increased expression of HNP-1 in CP and even more in PDAC. At RNA level, no significant regulation was found. In cancer cells, HNP-1 expression was significantly higher than in HPDE. Proinflammatory cytokines significantly led to increased HNP-1 levels in culture supernatants and decreased levels in lysates of cancer cells. In HPDE cytokines significantly decreased HNP-1 levels. Inflammatory regulation of HNP-1 in PDAC tissue and cells indicates that HNP-1 may be a link between chronic inflammation and malignant transformation in the pancreas.

  18. Aspirin-triggered lipoxin A4 and lipoxin A4 up-regulate transcriptional corepressor NAB1 in human neutrophils.

    Science.gov (United States)

    Qiu, F H; Devchand, P R; Wada, K; Serhan, C N

    2001-12-01

    Aspirin-triggered 15-epi-lipoxin A4 (ATL) is an endogenous lipid mediator that mimics the actions of native lipoxin A4, a putative "stop signal" involved in regulating resolution of inflammation. A metabolically more stable analog of ATL, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 analog (ATLa), inhibits neutrophil recruitment in vitro and in vivo and displays potent anti-inflammatory actions. ATLa binds with high affinity to the lipoxin A4 receptor, a G protein-coupled receptor on the surface of leukocytes. In this study, we used freshly isolated human neutrophils to examine ATLa's potential for initiating rapid nuclear responses. Using differential display reverse transcription polymerase chain reaction, we identified a subset of genes that was selectively up-regulated upon short exposure of polymorphonuclear leukocytes to ATLa but not to the chemoattractant leukotriene B4 or vehicle alone. We further investigated ATLa regulation of one of the genes, NAB1, a transcriptional corepressor identified previously as a glucocorticoid-responsive gene in hamster smooth muscle cells. Treatment of human neutrophils with pertussis toxin blocked ATLa up-regulation of NAB1. In addition, ATLa stimulated NAB1 gene expression in murine lung vascular smooth muscle in vivo. These findings provide evidence for rapid transcriptional induction of a cassette of genes via an ATLa-stimulated G protein-coupled receptor pathway that is potentially protective and overlaps with the anti-inflammatory glucocorticoid regulatory circuit.

  19. Chemotaxis toward carbohydrates and peptides by mixed ruminal protozoa when fed, fasted, or incubated with polyunsaturated fatty acids.

    Science.gov (United States)

    Diaz, H L; Karnati, S K R; Lyons, M A; Dehority, B A; Firkins, J L

    2014-01-01

    In contrast to the well-characterized chemotaxis and migratory behavior between the dorsal and ventral locations of the rumen by isotrichids, we hypothesized that chemotaxis toward soluble nutrients maintains entodiniomorphid protozoa in the particulate fraction. The objectives of these experiments were to compare the dose-responsive chemotaxis (1) toward different glucose concentrations when ruminal samples were harvested from fed versus fasted cows; (2) toward increasing concentrations of glucose compared with xylose when protozoa were harvested from a fed cow; (3) toward peptides of bacterial, protozoal, and soy origin; and (4) toward glucose when mixed ruminal protozoa were previously incubated for 0, 3, or 6h in the presence of emulsified polyunsaturated fatty acids (PUFA; Liposyn II, Hospira, Lake Forest, IL). In experiment 1, isotrichid protozoa decreased chemotaxis toward increasing glucose concentration when cows were fasted. Entodiniomorphids exhibited chemotaxis to similar concentrations of glucose as did isotrichids, but to a lesser magnitude of response. In experiment 2, xylose was chemotactic to both groups. Xylose might draw fibrolytic entodiniomorphid protozoa toward newly ingested feed. In contrast, even though isotrichids should not use xylose as an energy source, they were highly chemoattracted to xylose. In experiment 3, entodiniomorphids were not selectively chemoattracted toward bacterial or protozoal peptides compared with soy peptides. In experiment 4, despite isotrichid populations decreasing in abundance with increasing time of incubation in PUFA, chemotaxis to glucose remained unchanged. In contrast, entodiniomorphids recovered chemotaxis to glucose with increased time of PUFA incubation. Current results support isotrichid chemotaxis to sugars but also our hypothesis that a more moderate chemotaxis toward glucose and peptides explains how they swim in the fluid but pass from the rumen with the potentially digestible fraction of

  20. Targeting neutrophilic inflammation in severe neutrophilic asthma : can we target the disease-relevant neutrophil phenotype?

    NARCIS (Netherlands)

    Bruijnzeel, Piet L B; Uddin, Mohib; Koenderman, Leo

    2015-01-01

    In severe, neutrophilic asthma, neutrophils are thought to have an important role in both the maintenance of the disease and during exacerbations. These patients often display excessive, mucosal airway inflammation with unresolving neutrophilia. Because this variant of asthma is poorly controlled by

  1. Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients

    DEFF Research Database (Denmark)

    Hjortø, Gertrud Malene; Olsen, Mark Holm; Svane, Inge Marie

    2015-01-01

    Dendritic cell chemotaxis is known to follow chemoattractant concentration gradients through tissue of heterogeneous pore sizes, but the dependence of migration velocity on pore size and gradient steepness is not fully understood. We enabled chemotaxis studies for at least 42 hours at confinement...

  2. Exact solutions of certain nonlinear chemotaxis diffusion reaction ...

    Indian Academy of Sciences (India)

    constructed coupled differential equations. The results obtained ... Nonlinear diffusion reaction equation; chemotaxis; auxiliary equation method; solitary wave solutions. ..... fact limits the scope of applications of the derived results. ... Research Fellowship and AP acknowledges DU and DST for PURSE grant for financial.

  3. Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate

    Directory of Open Access Journals (Sweden)

    Tsang Jennifer LY

    2010-07-01

    Full Text Available Abstract Background Diisopropyl fluorophosphate (DFP is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst. Methods We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM, and evaluated neutrophil elastase (NE activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS, processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release. Results Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner. Conclusion We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

  4. Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Madison Floyd

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also

  5. Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils

    International Nuclear Information System (INIS)

    Hanazono, Y.; Hosoi, T.; Kuwaki, T.; Matsuki, S.; Miyazono, K.; Miyagawa, K.; Takaku, F.

    1990-01-01

    We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism

  6. The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

    Full Text Available Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

  7. The effects of beta 2-agonists and methylxanthines on neutrophil function in vitro.

    Science.gov (United States)

    Llewellyn-Jones, C G; Stockley, R A

    1994-08-01

    Therapeutic agents which affect polymorphonuclear neutrophil (PMN) functions have the potential to reduce or increase PMN activation and, hence, influence the progression of lung inflammation. We have assessed the effects of the beta 2-agonist, terbutaline, and the methylxanthine, aminophylline, on PMN functions in vitro at both therapeutic and higher concentrations. At therapeutic levels, both agents increased PMN chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP) in a dose-dependent manner from a control value of 22.5 +/- 3.58 cells.field-1 to 26.1 +/- 4.73 cells.field-1 with 4 mg.l-1 terbutaline, and to 26.3 +/- 4.49 cells.field-1 with 20 mg.l-1 aminophylline. When the cells were preincubated with higher doses of the agents in separate experiments there was inhibition of chemotaxis from a control value of 31.1 +/- 2.06 cells.field-1 to 18.3 +/- 0.82 cells.field-1 at 160 mg.l-1 terbutaline, and to 16.1 +/- 0.77 cells.field-1 at 400 mg.l-1 aminophylline. A similar effect was seen when the PMNs were preincubated with terbutaline and aminophylline prior to assessment of superoxide anion generation, with stimulation of superoxide release at therapeutic levels of the drugs and inhibition at higher doses (19% increase from resting control cells at terbutaline 4 mg.l-1 and 53% reduction at 160 mg.l-1; 28% increase with aminophylline 20 mg.l-1 and 22% reduction at 400 mg.l-1). Both terbutaline and aminophylline had no effect on PMN degranulation, as assessed by the degradation of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Activated Protein C Attenuates Severe Inflammation by Targeting VLA-3high Neutrophil Subpopulation in Mice.

    Science.gov (United States)

    Sarangi, Pranita P; Lee, Hyun-Wook; Lerman, Yelena V; Trzeciak, Alissa; Harrower, Eric J; Rezaie, Alireza R; Kim, Minsoo

    2017-10-15

    The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3 high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1 high CD11b high VLA-3 high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities. Copyright © 2017 by The American Association of Immunologists, Inc.

  9. The Beta-2-Adrenoreceptor Agonists, Formoterol and Indacaterol, but Not Salbutamol, Effectively Suppress the Reactivity of Human Neutrophils In Vitro

    Directory of Open Access Journals (Sweden)

    Ronald Anderson

    2014-01-01

    Full Text Available The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious possibly due to differences in their molecular structures and agonist activities. The current study has compared the effects of 3 different categories of β2-agonists, namely, salbutamol (short-acting, formoterol (long-acting and indacaterol (ultra-long-acting, at concentrations of 1–1000 nM, with human blood neutrophils in vitro. Neutrophils were activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 µM or platelet-activating factor (PAF, 200 nM in the absence and presence of the β2-agonists followed by measurement of the generation of reactive oxygen species and leukotriene B4, release of elastase, and expression of the β2-integrin, CR3, using a combination of chemiluminescence, ELISA, colorimetric, and flow cytometric procedures respectively. These were correlated with alterations in the concentrations of intracellular cyclic-AMP and cytosolic Ca2+. At the concentrations tested, formoterol and indacaterol caused equivalent, significant (P<0.05 at 1–10 nM dose-related inhibition of all of the pro-inflammatory activities tested, while salbutamol was much less effective (P<0.05 at 100 nM and higher. Suppression of neutrophil reactivity was accompanied by elevations in intracellular cAMP and accelerated clearance of Ca2+ from the cytosol of activated neutrophils. These findings demonstrate that β2-agonists vary with respect to their suppressive effects on activated neutrophils.

  10. Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

    Energy Technology Data Exchange (ETDEWEB)

    Sugino, Noriko [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Yao, Hisayuki [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Iwasa, Masaki; Fujishiro, Aya [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Division of Gastroenterology and Hematology, Shiga University of Medical Science, Shiga 520-2192 (Japan); Fujii, Sumie [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Hirai, Hideyo [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Takaori-Kondo, Akifumi [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Ichinohe, Tatsuo [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Maekawa, Taira [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan)

    2016-01-22

    Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment

  11. Neutrophil evasion strategies by Streptococcus pneumoniae and Staphylococcus aureus.

    Science.gov (United States)

    Lewis, Megan L; Surewaard, Bas G J

    2018-03-01

    Humans are well equipped to defend themselves against bacteria. The innate immune system employs diverse mechanisms to recognize, control and initiate a response that can destroy millions of different microbes. Microbes that evade the sophisticated innate immune system are able to escape detection and could become pathogens. The pathogens Streptococcus pneumoniae and Staphylococcus aureus are particularly successful due to the development of a wide variety of virulence strategies for bacterial pathogenesis and they invest significant efforts towards mechanisms that allow for neutrophil evasion. Neutrophils are a primary cellular defense and can rapidly kill invading microbes, which is an indispensable function for maintaining host health. This review compares the key features of Streptococcus pneumoniae and Staphylococcus aureus in epidemiology, with a specific focus on virulence mechanisms utilized to evade neutrophils in bacterial pathogenesis. It is important to understand the complex interactions between pathogenic bacteria and neutrophils so that we can disrupt the ability of pathogens to cause disease.

  12. The emerging role of neutrophils in thrombosis – The journey of TF through NETs

    Directory of Open Access Journals (Sweden)

    Konstantinos eKambas

    2012-12-01

    Full Text Available The production of TF by neutrophils and their contribution in thrombosis was until recently a matter of scientific debate. Experimental data suggested the de novo TF production by neutrophils under inflammatory stimuli, while others proposed that these cells acquired microparticle-derived TF. Recent experimental evidence revealed the critical role of neutrophils in thrombotic events. Neutrophil derived TF has been implicated in this process in several human and animal models. Additionally, neutrophil extracellular trap (NET release has emerged as a major contributor in neutrophil-driven thrombogenicity in disease models including sepsis, deep venous thrombosis and malignancy. It is suggested that NETs provide the scaffold for fibrin deposition and platelet entrapment and subsequent activation. The recently reported autophagy-dependent extracellular delivery of TF in NETs further supports the involvement of neutrophils in thrombosis. Herein, we seek to review novel data regarding the role of neutrophils in thrombosis, emphasizing the implication of TF and NETs.

  13. Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

    Directory of Open Access Journals (Sweden)

    Nauder Faraday

    Full Text Available Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

  14. Bothrops asper snake venom and its metalloproteinase BaP–1 activate the complement system. Role in leucocyte recruitment

    Directory of Open Access Journals (Sweden)

    Sandra H. P. Farsky

    2000-01-01

    Full Text Available The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins – phospholipases and metalloproteinase – activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP–1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1 did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s which can cause direct activation

  15. Oral neutrophil responses to acute prolonged exercise may not be representative of blood neutrophil responses.

    Science.gov (United States)

    Davison, Glen; Jones, Arwel Wyn

    2015-03-01

    Neutrophil numbers and function (oxidative burst) were assessed in peripheral blood and oral samples before and after prolonged exercise. Blood neutrophil count increased (∼3.5-fold, P < 0.001) and function decreased (30% ± 19% decrease, P = 0.005) postexercise. Oral neutrophil count (P = 0.392) and function (P = 0.334) were unchanged. Agreement between oral and blood neutrophil function responses to exercise was poor. These findings highlight the importance of studying neutrophils within various compartments/sample types.

  16. Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

    Science.gov (United States)

    Wanten, G J; Geijtenbeek, T B; Raymakers, R A; van Kooyk, Y; Roos, D; Jansen, J B; Naber, A H

    2000-01-01

    To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

  17. Spontaneous neutrophil activation in HTLV-1 infected patients

    Directory of Open Access Journals (Sweden)

    Jaqueline B. Guerreiro

    Full Text Available Human T cell lymphotropic Virus type-1 (HTLV-1 induces lymphocyte activation and proliferation, but little is known about the innate immune response due to HTLV-1 infection. We evaluated the percentage of neutrophils that metabolize Nitroblue tetrazolium (NBT to formazan in HTLV-1 infected subjects and the association between neutrophil activation and IFN-gamma and TNF-alpha levels. Blood was collected from 35 HTLV-1 carriers, from 8 patients with HAM/TSP (HTLV-1- associated myelopathy; 22 healthy individuals were evaluated for spontaneous and lipopolysaccharide (LPS-stimulated neutrophil activity (reduction of NBT to formazan. The production of IFN-gamma and TNF-alpha by unstimulated mononuclear cells was determined by ELISA. Spontaneous NBT levels, as well as spontaneous IFN-gamma and TNF-alpha production, were significantly higher (p<0.001 in HTLV-1 infected subjects than in healthy individuals. A trend towards a positive correlation was noted, with increasing percentage of NBT positive neutrophils and levels of IFN-gamma. The high IFN-gamma producing HTLV-1 patient group had significantly greater NBT than healthy controls, 43±24% and 17±4.8% respectively (p< 0.001, while no significant difference was observed between healthy controls and the low IFN-gamma-producing HTLV-1 patient group (30±20%. Spontaneous neutrophil activation is another marker of immune perturbation resulting from HTLV-1 infection. In vivo activation of neutrophils observed in HTLV-1 infected subjects is likely to be the same process that causes spontaneous IFN-gamma production, or it may partially result from direct IFN-gamma stimulation.

  18. Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Gullett, Jessica M; Bible, Amber; Alexandre, Gladys

    2017-07-01

    Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense , Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms. IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular

  19. The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants

    Directory of Open Access Journals (Sweden)

    Mário F. Neto

    2016-08-01

    Full Text Available A simple, affordable diagnostic test for pulmonary tuberculosis (TB is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while trained honeybees respond to three of the volatile organic compounds (VOCs detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, which are outside the scope of most disease testing facilities. Here, we report that the innate olfactory behavioral response of the roundworm nematode Caenorhabditis elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. Dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. We propose that exposing naive dauer larvae to TB-related VOCs and recording their response in this behavioral assay could lead to the development of a new method for TB diagnostics using breath as the sample type. Keywords: Tuberculosis, Caenorhabditis elegans, Chemotaxis, Volatile organic compounds, Diagnostics, Odorants

  20. COUPLED CHEMOTAXIS FLUID MODEL

    KAUST Repository

    LORZ, ALEXANDER

    2010-06-01

    We consider a model system for the collective behavior of oxygen-driven swimming bacteria in an aquatic fluid. In certain parameter regimes, such suspensions of bacteria feature large-scale convection patterns as a result of the hydrodynamic interaction between bacteria. The presented model consist of a parabolicparabolic chemotaxis system for the oxygen concentration and the bacteria density coupled to an incompressible Stokes equation for the fluid driven by a gravitational force of the heavier bacteria. We show local existence of weak solutions in a bounded domain in d, d = 2, 3 with no-flux boundary condition and in 2 in the case of inhomogeneous Dirichlet conditions for the oxygen. © 2010 World Scientific Publishing Company.

  1. Heterogeneity of neutrophil antibodies in patients with primary Sjögren's syndrome.

    Science.gov (United States)

    Lamour, A; Le Corre, R; Pennec, Y L; Cartron, J; Youinou, P

    1995-11-01

    Our aims were to determine the prevalence of neutrophil antibodies in patients with primary Sjögren's syndrome (pSS), identify their target antigen(s), and evaluate their functional significance. Neutrophil antibodies were detected using an indirect immunofluorescence (IIIF) test and an enzyme-linked immunosorbent assay (ELISA), using recombinant human Fc-gamma receptor (Fc gamma RIIIb) as a capture agent. Luminol-dependent chemiluminescence was then measured by an established technique. Antibodies to neutrophils were detected in 30 of 66 patients (45%) and categorized on the basis of positivity for the two assays: IIF+/ELISA+ (group A: five patients), IIF+/ELISA- (group B: five patients), and IFF-/ELISA+ (group C: 20 patients). All positive sera contained antibodies directed to the neutrophil specific Fc gamma RIIIb, and none of them bound to NAnull neutrophils. The titer of neutrophil-reactive antibodies (groups A and B) showed no correlation with the neutrophil count, but these autoantibodies did reduce the cell ability to generate a respiratory burst. Thus, neutrophil antibodies are common in patients with pSS. Their main target appears to be Fc gamma RIII, and this may partly account for the dysfunction in Fc gamma R-mediated clearance by the reticuloendothelial system reported in these patients.

  2. Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils--a promising candidate formulation for amnioinfusion fluid.

    Science.gov (United States)

    Uchida, Toshiyuki; Itoh, Hiroaki; Nakamura, Yuki; Kobayashi, Yukiko; Hirai, Kyuya; Suzuki, Kazunao; Sugihara, Kazuhiro; Kanayama, Naohiro; Hiramatsu, Mitsuo

    2010-06-01

    Oligohydramnios is often caused by the premature rupturing of membranes and subsequent intrauterine infections, such as chorioamnionitis, in which event oxidative stress is hypothesized to be closely associated with the damage to the fetal organs. The clinical efficiency of amnioinfusion using warmed saline in cases of premature rupture of membranes is still controversial, especially concerning the prognosis for the fetus. In the present study, we found that human amniotic fluid per se suppresses the release of superoxide from cultured human neutrophils, suggesting an acute or chronic shortage of amniotic fluid in cases of premature rupture of membranes can affect the shielding of intrauterine organs from oxidative stress. The aim of this study was to propose a formula of zinc and magnesium ions in saline for amnioinfusion, by assessing antioxidative activities. A combination of 5 microM zinc and 5mM magnesium in saline synergistically inhibited superoxide production by cultured human neutrophils, equivalent to human amniotic fluid. The intraperitoneal administration of this formula significantly improved the survival rate in a rat model of peritonitis compared to the saline control (46.7% vs. 10%). The combination of these metals with saline may thus be a promising formula for an amnioinfusion fluid with the capacity to protect fetal organs from oxidative stress. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  3. Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5

    NARCIS (Netherlands)

    Alard, Jean-Eric; Ortega-Gomez, Almudena; Wichapong, Kanin; Bongiovanni, Dario; Horckmans, Michael; Megens, Remco T. A.; Leoni, Giovanna; Ferraro, Bartolo; Rossaint, Jan; Paulin, Nicole; Ng, Judy; Ippel, Hans; Suylen, Dennis; Hinkel, Rabea; Blanchet, Xavier; Gaillard, Fanny; D'Amico, Michele; von Hundelshausen, Phillipp; Zarbock, Alexander; Scheiermann, Christoph; Hackeng, Tilman M.; Steffens, Sabine; Kupatt, Christian; Nicolaes, Gerry A. F.; Weber, Christian; Soehnlein, Oliver

    2015-01-01

    In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human

  4. The lipidated peptidomimetic Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 is a novel formyl peptide receptor 2 agonist that activates both human and mouse neutrophil NADPH-oxidase

    DEFF Research Database (Denmark)

    Holdfeldt, Andre; Skovbakke, Sarah Line; Winther, Malene

    2016-01-01

    Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists......2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated......2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases....

  5. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  6. Insulin regulates multiple signaling pathways leading to monocyte/macrophage chemotaxis into the wound tissue

    Directory of Open Access Journals (Sweden)

    Yan Liu

    2018-01-01

    Full Text Available Wound healing is a complex process that involves sequential phases that overlap in time and space and affect each other dynamically at the gene and protein levels. We previously showed that insulin accelerates wound healing by stimulating faster and regenerative healing. One of the processes that insulin stimulates is an increase in monocyte/macrophage chemotaxis. In this study, we performed experiments in vivo and in vitro to elucidate the signaling transduction pathways that are involved in insulin-induced monocyte/macrophage chemotaxis. We found that insulin stimulates THP-1 cell chemotaxis in a dose-dependent and insulin receptor-dependent manner. We also show that the kinases PI3K-Akt, SPAK/JNK, and p38 MAPK are key molecules in the insulin-induced signaling pathways that lead to chemoattraction of the THP-1 cell. Furthermore, both PI3K-Akt and SPAK/JNK signaling involve Rac1 activation, an important molecule in regulating cell motility. Indeed, topical application of Rac1 inhibitor at an early stage during the healing process caused delayed and impaired healing even in the presence of insulin. These results delineate cell and molecular mechanisms involved in insulin-induced chemotaxis of monocyte/macrophage, cells that are critical for proper healing.

  7. Oxidative burst of human neutrophils is suppressed by N-feruloylserotonin isolated from seeds of Leuzea carthamoides (Wild) DC

    Czech Academy of Sciences Publication Activity Database

    Nosáľ, R.; Perečko, T.; Jančinová, V.; Drábiková, K.; Harmatha, Juraj; Sviteková, K.

    2010-01-01

    Roč. 3, č. 3 (2010), A70-A71 ISSN 1337-6853. [Toxcon 2010, Borderless Toxicology. 15th Interdisciplinary Toxicological Conference & Advanced Toxicological Course. 06.09.-10.09.2010, Stará Lesná - Hotel Academia] R&D Projects: GA ČR(CZ) GA203/07/1227 Institutional research plan: CEZ:AV0Z40550506 Keywords : N-feruloylserotonin * human neutrophils * Leuzea carthamoides Subject RIV: CC - Organic Chemistry

  8. Localized Subcutaneous Acute Febrile Neutrophilic Dermatosis in a Dog

    Directory of Open Access Journals (Sweden)

    Karolin Schoellhorn

    2012-01-01

    Full Text Available A two-year-old spayed female mixed-breed dog was presented with a five-day history of hemorrhagic gastroenteritis and fever. On physical examination, the dog was lethargic and clinically dehydrated. The skin of the entire ventral abdomen extending to both flanks was erythematous, swollen and painful on palpation. Histopathological examination of skin biopsies revealed a severe diffuse neutrophilic dermatitis and panniculitis, resembling the subcutaneous form of Sweet’s syndrome in humans. A large part of the skin lesion developed full-thickness necrosis. After intensive care, three surgical wound debridements and wound adaptations, the wound healed by secondary intention within ten weeks. In the absence of infection of the skin or neoplasia, a diagnosis of neutrophilic dermatosis and panniculitis, resembling the subcutaneous form of acute febrile neutrophilic dermatosis, was made.

  9. Effect of progesterone receptor status on maspin synthesis via nitric oxide production in neutrophils in human breast cancer.

    Science.gov (United States)

    Ganguly Bhattacharjee, Karabi; Bhattacharyya, Mau; Halder, Umesh Chandra; Jana, Pradipta; Sinha, Asru K

    2014-09-01

    Although progesterone receptor (PR) status, similarly to estrogen receptor status, is of prognostic importance in breast cancer, the involvement of the PR in breast cancer remains obscure. Studies were conducted to determine the function of the PR in neutrophils in the nitric oxide-induced synthesis of maspin, an anti-breast-cancer protein produced in nonmalignant mammary cells and in neutrophils in the circulation. PR status was determined by immunohistochemistry. Maspin synthesis was determined by in-vitro translation of messenger RNA and quantified by enzyme-linked immunosorbent assay. Nitric oxide was determined by the methemoglobin method. It was found that PR status in neutrophils was identical with that in malignant breast tissues. A Scatchard plot for progesterone binding to normal and PR-positive (PR+) neutrophils revealed that whereas normal neutrophils had 11.5 × 10(10) PR sites/cell with K d = 47.619 nM, PR+ neutrophils had 6.6 × 10(10) PR sites/cell with K d = 47.619 nM. The progesterone negative (PR-) neutrophils failed to bind to progesterone. Incubation of normal and PR+ neutrophils with 25 nM progesterone produced 1.317 μM NO and 2.329 nM maspin; the PR+ neutrophils produced 0.72 μM NO and 1.138 nM maspin. The PR- neutrophils failed to produce any NO or maspin in the presence of progesterone. Inhibition of progesterone-induced NO synthesis led to complete inhibition of maspin synthesis in all neutrophils. These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.

  10. Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Magdalena Riedl

    2017-01-01

    Discussion: Therefore, our findings of (i neutrophils adhering to complement-activated endothelial cells, (ii the formation of neutrophil-platelet aggregates on endothelial cells, and (iii the ability of aHUS serum to induce similar effects identify a possible role for neutrophils in aHUS manifestation.

  11. Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

    Directory of Open Access Journals (Sweden)

    Elodie Parzy

    Full Text Available Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4 cells per milliliter, well under the physiological neutrophils concentrations.These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

  12. Simulation of Paramecium Chemotaxis Exposed to Calcium Gradients.

    Science.gov (United States)

    Sarvestani, Ali N; Shamloo, Amir; Ahmadian, Mohammad Taghi

    2016-06-01

    Paramecium or other ciliates have the potential to be utilized for minimally invasive surgery systems, making internal body organs accessible. Paramecium shows interesting responses to changes in the concentration of specific ions such as K(+), Mg(2+), and Ca(2+) in the ambient fluid. Some specific responses are observed as, changes in beat pattern of cilia and swimming toward or apart from the ion source. Therefore developing a model for chemotactic motility of small organisms is necessary in order to control the directional movements of these microorganisms before testing them. In this article, we have developed a numerical model, investigating the effects of Ca(2+) on swimming trajectory of Paramecium. Results for Ca(2+)-dependent chemotactic motility show that calcium gradients are efficient actuators for controlling the Paramecium swimming trajectory. After applying a very low Ca(2+) gradient, a directional chemotaxis of swimming Paramecium is observable in this model. As a result, chemotaxis is shown to be an efficient method for controlling the propulsion of these small organisms.

  13. Feeding ducks, bacterial chemotaxis, and the Gini index

    Science.gov (United States)

    Peaudecerf, François J.; Goldstein, Raymond E.

    2015-08-01

    Classic experiments on the distribution of ducks around separated food sources found consistency with the "ideal free" distribution in which the local population is proportional to the local supply rate. Motivated by this experiment and others, we examine the analogous problem in the microbial world: the distribution of chemotactic bacteria around multiple nearby food sources. In contrast to the optimization of uptake rate that may hold at the level of a single cell in a spatially varying nutrient field, nutrient consumption by a population of chemotactic cells will modify the nutrient field, and the uptake rate will generally vary throughout the population. Through a simple model we study the distribution of resource uptake in the presence of chemotaxis, consumption, and diffusion of both bacteria and nutrients. Borrowing from the field of theoretical economics, we explore how the Gini index can be used as a means to quantify the inequalities of uptake. The redistributive effect of chemotaxis can lead to a phenomenon we term "chemotactic levelling," and the influence of these results on population fitness are briefly considered.

  14. Obesity is associated with more activated neutrophils in African American male youth.

    Science.gov (United States)

    Xu, X; Su, S; Wang, X; Barnes, V; De Miguel, C; Ownby, D; Pollock, J; Snieder, H; Chen, W; Wang, X

    2015-01-01

    There is emerging evidence suggesting the role of peripheral blood leukocytes in the pathogenesis of obesity and related diseases. However, few studies have taken a genome-wide approach to investigating gene expression profiles in peripheral leukocytes between obese and lean individuals with the consideration of obesity-related shifts in leukocyte types. We conducted this study in 95 African Americans (AAs) of both genders (age 14-20 years, 46 lean and 49 obese). Complete blood count with differential test (CBC) was performed in whole blood. Genome-wide gene expression analysis was obtained using the Illumina HumanHT-12 V4 Beadchip with RNA extracted from peripheral leukocytes. Out of the 95 participants, 64 had neutrophils stored. The validation study was based on real-time PCR with RNA extracted from purified neutrophils. CBC test suggested that, in males, obesity was associated with increased neutrophil percentage (P=0.03). Genome-wide gene expression analysis showed that, in males, the majority of the most differentially expressed genes were related to neutrophil activation. Validation of the gene expression levels of ELANE (neutrophil elastase) and MPO (myeloperoxidase) in purified neutrophils demonstrated that the expression of these two genes--important biomarkers of neutrophils activation--were significantly elevated in obese males (P=0.01 and P=0.02, respectively). The identification of increased neutrophil percentage and activation in obese AA males suggests that neutrophils have an essential role in the pathogenesis of obesity-related disease. Further functional and mechanistic studies on neutrophils may contribute to the development of novel intervention strategies reducing the burden associated with obesity-related health problems.

  15. Interleukin-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

    Science.gov (United States)

    Liu, Rebecca; Lauridsen, Holly M.; Amezquita, Robert A.; Pierce, Richard W.; Jane-wit, Dan; Fang, Caodi; Pellowe, Amanda S.; Kirkiles-Smith, Nancy C.; Gonzalez, Anjelica L.; Pober, Jordan S.

    2016-01-01

    A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multi-step process that involves sequential cell-cell interactions of circulating leukocytes with interleukin (IL)-1- or tumor necrosis factor-α (TNF)-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a pro-inflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, while neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA-seq analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media (CM) from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and also stimulate neutrophil production of pro-inflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17-activated PCs but not ECs can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondria outer membrane permeabilization and caspase 9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by CM from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space. PMID:27534549

  16. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  17. Nimesulide inhibits platelet-activating factor synthesis in activated human neutrophils

    NARCIS (Netherlands)

    Verhoeven, A. J.; Tool, A. T.; Kuijpers, T. W.; Roos, D.

    1993-01-01

    In an inflammatory locus, products of activated neutrophils may be toxic both to the micro-organisms to be eliminated and to the surrounding tissue. In several models of inflammation, nimesulide possesses marked anti-inflammatory properties. The present study was undertaken to further investigate

  18. Epithelial Cell-Neutrophil Interactions in the Alimentary Tract: A Complex Dialog in Mucosal Surveillance and Inflammation

    Directory of Open Access Journals (Sweden)

    Sean P. Colgan

    2002-01-01

    Full Text Available Inflammatory diseases of mucosal organs as diverse as the lung, kidney, and intestine, inevitably require the intimate interactions of neutrophils with columnar epithelia. The physiologic consequences of such interactions often determine endpoint organ function, and for this reason, much recent interest has developed in identifying mechanisms and novel targets for the treatment of mucosal inflammation. Elegant in vitro model systems incorporating purified human neutrophils and human epithelial cells grown in physiologic orientations have aided in discovery of new and insightful pathways to define basic inflammatory pathways. Here, we will review the recent literature regarding the interactions between columnar epithelial cells and neutrophils, with an emphasis on intestinal epithelial cells, structural aspects of neutrophil transepithelial migration, molecular determinants of neutrophil-epithelial cell interactions, as well as modulation of these pathways. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

  19. Signal transduction and chemotaxis in mast cells

    Czech Academy of Sciences Publication Activity Database

    Dráber, Petr; Hálová, Ivana; Polakovičová, Iva; Kawakami, T.

    2016-01-01

    Roč. 778, jaro (2016), s. 11-23 ISSN 0014-2999 R&D Projects: GA ČR(CZ) GA14-09807S; GA ČR(CZ) GBP302/12/G101; GA ČR(CZ) GA14-00703S Institutional support: RVO:68378050 Keywords : Mast cell * IgE receptor * KIT receptor * Signal transduction * Chemotaxis * Plasma membrane Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.896, year: 2016

  20. CXCL1 is a negative regulator of mast cell chemotaxis to airway smooth muscle cell products in vitro.

    Science.gov (United States)

    Alkhouri, H; Moir, L M; Armour, C L; Hughes, J M

    2014-03-01

    Activated mast cells (MC) numbers on airway smooth muscle (ASM) are increased in eosinophilic asthma. In vitro, asthmatic cytokine-stimulated ASM cell-conditioned medium (CM) induces more MC chemotaxis than CM from nonasthmatic ASM cells. Intriguingly the nonasthmatic ASM CM inhibits MC chemotaxis to the asthmatic ASM CM. However, the inhibitory factor(s) in the nonasthmatic ASM CM is still to be identified. To identify the factor(s) released by nonasthmatic ASM cells that inhibits MC chemotaxis. Confluent, serum-starved ASM cells from donors with and without asthma were stimulated with IL-1β and T-helper (Th)1 (TNFα and IFNγ) or Th2 (IL-4, IL-13) cytokines, or left unstimulated. CM samples were collected after 24 h, and a potential inhibitory factor identified using cytokine protein arrays. Its production was assessed using ELISA and RT-PCR and inhibitory role investigated in MC chemotaxis and Ca(2+) mobilization assays. Only CXCL1 was produced in greater amounts by nonasthmatic than asthmatic ASM cells following Th1 and Th2 cytokine stimulation. CXCL1 mRNA expression was also increased. Exogenous rh-CXCL1 significantly inhibited MC intracellular Ca(2+) mobilization and chemotaxis to either CXCL10, CXCL8 or CM collected from asthmatic ASM cells following Th1 or Th2 cytokine stimulation. Neutralizing CXCL1 in nonasthmatic ASM CM or blocking its receptor significantly promoted MC chemotaxis. CXCL1 was a major factor regulating MC chemotaxis in vitro. Its differential release by ASM cells may explain the differences observed in MC localization to the ASM of people with and without asthma. CXCL1 inhibition of MC recruitment to the ASM may lead to new targets to limit asthma pathophysiology. © 2013 John Wiley & Sons Ltd.

  1. A tick mannose-binding lectin inhibits the vertebrate complement cascade to enhance transmission of the Lyme disease agent

    OpenAIRE

    Schuijt, Tim J.; Coumou, Jeroen; Narasimhan, Sukanya; Dai, Jianfeng; DePonte, Kathleen; Wouters, Diana; Brouwer, Mieke; Oei, Anneke; Roelofs, Joris J.T.H.; van Dam, Alje P.; van der Poll, Tom; van ’t Veer, Cornelis; Hovius, Joppe W.; Fikrig, Erol

    2011-01-01

    The Lyme disease agent, Borrelia burgdorferi, is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8 that reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade resulting in impaired neutrophil phagocytosis and chemotaxis, and diminished Borrelia lysi...

  2. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    Science.gov (United States)

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras

  3. Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa

    Science.gov (United States)

    Loo, Shining; Kam, Antony; Xiao, Tianshu; Nguyen, Giang K. T.; Liu, Chuan Fa; Tam, James P.

    2016-01-01

    Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases. PMID:27991569

  4. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  5. The role of cGMP and the rear of the cell in Dictyostelium chemotaxis and cell streaming

    NARCIS (Netherlands)

    Veltman, Douwe M.; van Haastert, Peter J. M.

    2008-01-01

    During chemotaxis, pseudopod extensions lead the cell towards the source of attractant. The role of actin-filled pseudopodia at the front of the cell is well recognized, whereas the function of the rear of the cell in chemotaxis and cell-cell interactions is less well known. Dictyostelium cell

  6. (±)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Aam, B.B.; Fonnum, F.

    2006-01-01

    (±)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic

  7. Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

    DEFF Research Database (Denmark)

    Guarino, Carla; Hamon, Yveline; Croix, Cécile

    2017-01-01

    cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites....... These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases...

  8. Neutrophil extracellular traps in patients with pulmonary tuberculosis

    NARCIS (Netherlands)

    van der Meer, Anne Jan; Zeerleder, Sacha; Blok, Dana C.; Kager, Liesbeth M.; Lede, Ivar O.; Rahman, Wahid; Afroz, Rumana; Ghose, Aniruddha; Visser, Caroline E.; Zahed, Abu Shahed Md; Husain, Md Anwar; Alam, Khan Mashrequl; Barua, Pravat Chandra; Hassan, Mahtabuddin; Tayab, Md Abu; Dondorp, Arjen M.; van der Poll, Tom

    2017-01-01

    Tuberculosis is a devastating infectious disease causing many deaths worldwide. Recent investigations have implicated neutrophil extracellular traps (NETs) in the host response to tuberculosis. The aim of the current study was to obtain evidence for NETs release in the circulation during human

  9. Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-16-1-0615 TITLE: Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy PRINCIPAL...29 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy...infection or cigarette smoke enhanced pulmonary metastasis from breast cancer in humans and mice. Similarly, autoimmune arthritis, characterized by

  10. Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis

    Science.gov (United States)

    Mercer, Frances; Ng, Shek Hang; Brown, Taylor M.; Boatman, Grace; Johnson, Patricia J.

    2018-01-01

    T. vaginalis, a human-infective parasite, causes the most common nonviral sexually transmitted infection (STI) worldwide and contributes to adverse inflammatory disorders. The immune response to T. vaginalis is poorly understood. Neutrophils (polymorphonuclear cells [PMNs]) are the major immune cell present at the T. vaginalis–host interface and are thought to clear T. vaginalis. However, the mechanism of PMN clearance of T. vaginalis has not been characterized. We demonstrate that human PMNs rapidly kill T. vaginalis in a dose-dependent, contact-dependent, and neutrophil extracellular trap (NET)-independent manner. In contrast to phagocytosis, we observed that PMN killing of T. vaginalis involves taking “bites” of T. vaginalis prior to parasite death, using trogocytosis to achieve pathogen killing. Both trogocytosis and parasite killing are dependent on the presence of PMN serine proteases and human serum factors. Our analyses provide the first demonstration, to our knowledge, of a mammalian phagocyte using trogocytosis for pathogen clearance and reveal a novel mechanism used by PMNs to kill a large, highly motile target. PMID:29408891

  11. Impairment of Several Immune Functions and Redox State in Blood Cells of Alzheimer’s Disease Patients. Relevant Role of Neutrophils in Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Carmen Vida

    2018-01-01

    Full Text Available Since aging is considered the most risk factor for sporadic Alzheimer’s Disease (AD, the age-related impairment of the immune system (immunosenescence, based on a chronic oxidative-inflammatory stress situation, could play a key role in the development and progression of AD. Although AD is accompanied by systemic disturbance, reflecting the damage in the brain, the changes in immune response and redox-state in different types of blood cells in AD patients have been scarcely studied. The aim was to analyze the variations in several immune functions and oxidative-inflammatory stress and damage parameters in both isolated peripheral neutrophils and mononuclear blood cells, as well as in whole blood cells, from patients diagnosed with mild (mAD and severe AD, and of age-matched controls (elderly healthy subjects as well as of adult controls. The cognitive decline of all subjects was determined by Mini-Mental State Examination (MMSE test (mAD stage was established at 20 ≤ MMSE ≤ 23 score; AD stage at <18 MMSE; elderly subjects >27 MMSE. The results showed an impairment of the immune functions of human peripheral blood neutrophils and mononuclear cells of mAD and AD patients in relation to healthy elderly subjects, who showed the typical immunosenescence in comparison with the adult individuals. However, several alterations were only observed in severe AD patients (lower chemotaxis, lipopolysaccharide lymphoproliferation, and interleukin (IL-10 release; higher basal proliferation, tumor necrosis factor (TNF-α release, and IL-10/TNF-α ratio, others only in mAD subjects (higher adherence, meanwhile others appeared in both mAD and AD patients (lower phytohemaglutinin lymphoproliferation and higher IL-6 release. This impairment of immune functions could be mediated by: (1 the higher oxidative stress and damage also observed in blood cells from mAD and AD patients and in isolated neutrophils [lower glutathione (GSH levels, high oxidized

  12. Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

    Science.gov (United States)

    Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

  13. DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression

    Directory of Open Access Journals (Sweden)

    Maceler Aldrovandi

    2017-04-01

    Full Text Available Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3. Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108 and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX, protease-activated receptors (PAR 1 and 4, cytosolic phospholipase A2 (cPLA2, phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

  14. Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram

    NARCIS (Netherlands)

    Dijkhuizen, B; deMonchy, JGR; Dubois, AEJ; Gerritsen, J; Kauffman, HF

    We quantitatively determined whether the selective phosphodiesterase (PDE) inhibitor, rolipram, inhibits changes in the adhesion molecules CD11b and L-selectin on platelet-activating factor (PAF)-stimulated human neutrophils and eosinophils in vitro. Incubations were performed in human whole blood

  15. Formation of neutrophil extracellular traps under low oxygen level

    Directory of Open Access Journals (Sweden)

    Katja Branitzki-Heinemann

    2016-11-01

    Full Text Available Since their discovery, neutrophil extracellular traps (NETs have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia inducible factor 1α (HIF-1α by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21% oxygen level and compared to hypoxic (1% conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA or S. aureus wildtype and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was

  16. Hydrogen sulfide reduces neutrophil recruitment in hind-limb ischemia-reperfusion injury in an L-selectin and ADAM-17 dependent manner

    Science.gov (United States)

    Ball, Carissa J.; Reiffel, Alyssa J.; Chintalapani, Sathvika; Kim, Minsoo; Spector, Jason A.; King, Michael R.

    2012-01-01

    Background Reperfusion following ischemia leads to neutrophil recruitment injured tissue. Selectins and β2 integrins regulate neutrophil interaction with the endothelium during neutrophil rolling and firm adhesion. Excessive neutrophil infiltration into tissue is thought to contribute to IRI damage. NaHS mitigates the damage caused by ischemia-reperfusion injury (IRI). This study's objective was to determine the effect of hydrogen sulfide (NaHS) on neutrophil adhesion receptor expression. Methods Human neutrophils were either left untreated or incubated in 20 μM NaHS, and/or 50 μg/mL pharmacological ADAM-17 inhibitor TAPI-0; activated by IL-8, fMLP, or TNF-α; and labeled against PSGL-1, LFA-1, Mac-1 α, L-selectin and β2 integrin epitopes CBRM1/5 or KIM127 for flow cytometry. Cohorts of 3 C57BL/6 mice received an intravenous dose of saline vehicle, or 20 μM NaHS with or without 50 μg/mL TAPI-0 before unilateral tourniquet induced hind-limb ischemia for 3 hours followed by 3 hours of reperfusion. Bilateral gastrocnemius muscles were processed for histology before neutrophil infiltration quantification. Results NaHS treatment significantly increased L-selectin shedding from human neutrophils following activation by fMLP and IL-8 in an ADAM-17 dependent manner. Mice treated with NaHS to raise bloodstream concentration by 20 μM prior to ischemia or reperfusion showed a significant reduction in neutrophil recruitment into skeletal muscle tissue following tourniquet-induced hindlimb IRI. Conclusions NaHS administration results in the downregulation of L-selectin expression in activated human neutrophils. This leads to a reduction in neutrophil extravasation and tissue infiltration and may partially account for the protective effects of NaHS seen in the setting of IRI. PMID:23446563

  17. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils

    DEFF Research Database (Denmark)

    Volck, B; Price, P A; Johansen, J S

    1998-01-01

    YKL-40, also called human cartilage glycoprotein-39 (HC gp-39), is a member of family 18 glycosyl hydrolases. YKL-40 is secreted by chondrocytes, synovial cells, and macrophages, and recently it has been reported that YKL-40 has a role as an autoantigen in rheumatoid arthritis (RA). The function...... of patients with RA, and the cells are assumed to play a role in joint destruction in that disorder. Therefore, we examined whether neutrophils are a source of YKL-40. YKL-40 was found to colocalize and comobilize with lactoferrin (the most abundant protein of specific granules) but not with gelatinase...... YKL-40 at the myelocyte-metamyelocyte stage, the stage of maturation at which other specific granule proteins are formed. Assuming that YKL-40 has a role as an autoantigen in RA by inducing T cell-mediated autoimmune response, YKL-40 released from neutrophils in the inflamed joint could be essential...

  18. Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation

    NARCIS (Netherlands)

    Wachtfogel, Y.T.; Hack, C.E.; Nuijens, J.H; Kettner, C.; Reilly, T.M.; Knabb, R.M.; Bischoff, Rainer; Tschesche, H.; Wenzel, H.; Kucich, U.

    1995-01-01

    Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during

  19. Phagocytosis and killing of Candida albicans by human neutrophils after exposure to structurally different lipid emulsions.

    NARCIS (Netherlands)

    Wanten, G.J.A.; Curfs, J.H.A.J.; Meis, J.F.G.M.; Naber, A.H.J.

    2001-01-01

    BACKGROUND: To test the hypothesis that structurally different lipid emulsions have distinct immune-modulating properties, we analyzed the elimination of Candida albicans by neutrophils after exposure to various emulsions. METHODS: Neutrophils from 8 volunteers were incubated in physiologic 5 mmol/L

  20. Neutrophil Extracellular Traps in Ulcerative Colitis

    DEFF Research Database (Denmark)

    Bjerg Bennike, Tue; Carlsen, Thomas Gelsing; Ellingsen, Torkell

    2015-01-01

    microscopy and confocal microscopy. RESULTS: We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did...... not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were...... validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. CONCLUSIONS: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate...

  1. Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

    International Nuclear Information System (INIS)

    Thoelke, M.S.; Casper, J.M.; Ordal, G.W.

    1990-01-01

    The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it

  2. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

    Science.gov (United States)

    Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes

    2015-01-15

    Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. CD147 is a signaling receptor for cyclophilin B.

    Science.gov (United States)

    Yurchenko, V; O'Connor, M; Dai, W W; Guo, H; Toole, B; Sherry, B; Bukrinsky, M

    2001-11-09

    Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins. Copyright 2001 Academic Press.

  4. Neutrophils: potential therapeutic targets in tularemia?

    Directory of Open Access Journals (Sweden)

    Lee-Ann H Allen

    2013-12-01

    Full Text Available The central role of neutrophils in innate immunity and host defense has long been recognized, and the ability of these cells to efficiently engulf and kill invading bacteria has been extensively studied, as has the role of neutrophil apoptosis in resolution of the inflammatory response. In the past few years additional immunoregulatory properties of neutrophils were discovered, and it is now clear that these cells play a much greater role in control of the immune response than was previously appreciated. In this regard, it is noteworthy that Francisella tularensis is one of relatively few pathogens that can successfully parasitize neutrophils as well as macrophages, DC and epithelial cells. Herein we will review the mechanisms used by F. tularensis to evade elimination by neutrophils. We will also reprise effects of this pathogen on neutrophil migration and lifespan as compared with other infectious and inflammatory disease states. In addition, we will discuss the evidence which suggests that neutrophils contribute to disease progression rather than effective defense during tularemia, and consider whether manipulation of neutrophil migration or turnover may be suitable adjunctive therapeutic strategies.

  5. Fluid flow and particle dynamics inside an evaporating droplet containing live bacteria displaying chemotaxis.

    Science.gov (United States)

    Thokchom, Ashish Kumar; Swaminathan, Rajaram; Singh, Anugrah

    2014-10-21

    Evaporation-induced particle deposition patterns like coffee rings provide easy visual identification that is beneficial for developing inexpensive and simple diagnostic devices for detecting pathogens. In this study, the effect of chemotaxis on such pattern formation has been realized experimentally in drying droplets of bacterial suspensions. We have investigated the velocity field, concentration profile, and deposition pattern in the evaporating droplet of Escherichia coli suspension in the presence and absence of nutrients. Flow visualization experiments using particle image velocimetry (PIV) were carried out with E. coli bacteria as biological tracer particles. Experiments were conducted for suspensions of motile (live) as well as nonmotile (dead) bacteria. In the absence of any nutrient gradient like sugar on the substrate, both types of bacterial suspension showed two symmetric convection cells and a ring like deposition of particles after complete evaporation. Interestingly, the droplet containing live bacterial suspension showed a different velocity field when the sugar was placed at the base of the droplet. This can be attributed to the chemoattractant nature of the sugar, which induced chemotaxis among live bacteria targeted toward the nutrient site. Deposition of the suspended bacteria was also displaced toward the nutrient site as the evaporation proceeded. Our experiments demonstrate that both velocity fields and concentration patterns can be altered by chemotaxis to modify the pattern formation in evaporating droplet containing live bacteria. These results highlight the role of bacterial chemotaxis in modifying coffee ring patterns.

  6. Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Dusenbery, D B; Sheridan, R E; Russell, R L

    1975-06-01

    The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.

  7. Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury

    DEFF Research Database (Denmark)

    Bless, N M; Smith, D; Charlton, J

    1997-01-01

    Neutrophils play an important part in the development of acute inflammatory injury. Human neutrophils contain high levels of the serine protease elastase, which is stored in azurophilic granules and is secreted in response to inflammatory stimuli. Elastase is capable of degrading many components...... of extracellular matrix [1-4] and has cytotoxic effects on endothelial cells [5-7] and airway epithelial cells. Three types of endogenous protease inhibitors control the activity of neutrophil elastase, including alpha-1 protease inhibitor (alpha-1PI), alpha-2 macroglobulin and secreted leukoproteinase inhibitor...... (SLPI) [8-10]. A disturbed balance between neutrophil elastase and these inhibitors has been found in various acute clinical conditions (such as adult respiratory syndrome and ischemia-reperfusion injury) and in chronic diseases. We investigated the effect of NX21909, a selected oligonucleotide (aptamer...

  8. PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

    Directory of Open Access Journals (Sweden)

    S. V. Zubova

    2012-01-01

    Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

  9. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Jacobsen, Christian; Strong, Roland K

    2005-01-01

    Neutrophil-gelatinase-associated lipocalin (NGAL) is a prominent protein of specific granules of human neutrophils also synthesized by epithelial cells during inflammation. NGAL binds bacterial siderophores preventing bacteria from retrieving iron from this source. Also, NGAL may be important in ...... by surface plasmon resonance analysis. Furthermore, a rat yolk sac cell line known to express high levels of megalin, endocytosed NGAL by a mechanism completely blocked by an antibody against megalin.......Neutrophil-gelatinase-associated lipocalin (NGAL) is a prominent protein of specific granules of human neutrophils also synthesized by epithelial cells during inflammation. NGAL binds bacterial siderophores preventing bacteria from retrieving iron from this source. Also, NGAL may be important...

  10. Neutrophil programming dynamics and its disease relevance.

    Science.gov (United States)

    Ran, Taojing; Geng, Shuo; Li, Liwu

    2017-11-01

    Neutrophils are traditionally considered as first responders to infection and provide antimicrobial host defense. However, recent advances indicate that neutrophils are also critically involved in the modulation of host immune environments by dynamically adopting distinct functional states. Functionally diverse neutrophil subsets are increasingly recognized as critical components mediating host pathophysiology. Despite its emerging significance, molecular mechanisms as well as functional relevance of dynamically programmed neutrophils remain to be better defined. The increasing complexity of neutrophil functions may require integrative studies that address programming dynamics of neutrophils and their pathophysiological relevance. This review aims to provide an update on the emerging topics of neutrophil programming dynamics as well as their functional relevance in diseases.

  11. Activated prostaglandin D2 receptors on macrophages enhance neutrophil recruitment into the lung

    Science.gov (United States)

    Jandl, Katharina; Stacher, Elvira; Bálint, Zoltán; Sturm, Eva Maria; Maric, Jovana; Peinhaupt, Miriam; Luschnig, Petra; Aringer, Ida; Fauland, Alexander; Konya, Viktoria; Dahlen, Sven-Erik; Wheelock, Craig E.; Kratky, Dagmar; Olschewski, Andrea; Marsche, Gunther; Schuligoi, Rufina; Heinemann, Akos

    2016-01-01

    Background Prostaglandin (PG) D2 is an early-phase mediator in inflammation, but its action and the roles of the 2 D-type prostanoid receptors (DPs) DP1 and DP2 (also called chemoattractant receptor–homologous molecule expressed on TH2 cells) in regulating macrophages have not been elucidated to date. Objective We investigated the role of PGD2 receptors on primary human macrophages, as well as primary murine lung macrophages, and their ability to influence neutrophil action in vitro and in vivo. Methods In vitro studies, including migration, Ca2+ flux, and cytokine secretion, were conducted with primary human monocyte-derived macrophages and neutrophils and freshly isolated murine alveolar and pulmonary interstitial macrophages. In vivo pulmonary inflammation was assessed in male BALB/c mice. Results Activation of DP1, DP2, or both receptors on human macrophages induced strong intracellular Ca2+ flux, cytokine release, and migration of macrophages. In a murine model of LPS-induced pulmonary inflammation, activation of each PGD2 receptor resulted in aggravated airway neutrophilia, tissue myeloperoxidase activity, cytokine contents, and decreased lung compliance. Selective depletion of alveolar macrophages abolished the PGD2-enhanced inflammatory response. Activation of PGD2 receptors on human macrophages enhanced the migratory capacity and prolonged the survival of neutrophils in vitro. In human lung tissue specimens both DP1 and DP2 receptors were located on alveolar macrophages along with hematopoietic PGD synthase, the rate-limiting enzyme of PGD2 synthesis. Conclusion For the first time, our results show that PGD2 markedly augments disease activity through its ability to enhance the proinflammatory actions of macrophages and subsequent neutrophil activation. PMID:26792210

  12. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  13. Characterisation of Neutropenia-Associated Neutrophil Elastase Mutations in a Murine Differentiation Model In Vitro and In Vivo.

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    Michael Wiesmeier

    Full Text Available Severe congenital neutropenia (SCN is characterised by a differentiation block in the bone marrow and low neutrophil numbers in the peripheral blood, which correlates with increased risk of bacterial infections. Several underlying gene defects have been identified in SCN patients. Mutations in the neutrophil elastase (ELANE gene are frequently found in SCN and cyclic neutropenia. Both mislocalization and misfolding of mutant neutrophil elastase protein resulting in ER stress and subsequent induction of the unfolded protein response (UPR have been proposed to be responsible for neutrophil survival and maturation defects. However, the detailed molecular mechanisms still remain unclear, in part due to the lack of appropriate in vitro and in vivo models. Here we used a system of neutrophil differentiation from immortalised progenitor lines by conditional expression of Hoxb8, permitting the generation of mature near-primary neutrophils in vitro and in vivo. NE-deficient Hoxb8 progenitors were reconstituted with murine and human forms of typical NE mutants representative of SCN and cyclic neutropenia, and differentiation of the cells was analysed in vitro and in vivo. ER stress induction by NE mutations could be recapitulated during neutrophil differentiation in all NE mutant-reconstituted Hoxb8 cells. Despite ER stress induction, no change in survival, maturation or function of differentiating cells expressing either murine or human NE mutants was observed. Further analysis of in vivo differentiation of Hoxb8 cells in a murine model of adoptive transfer did not reveal any defects in survival or differentiation in the mouse. Although the Hoxb8 system has been found to be useful for dissection of defects in neutrophil development, our findings indicate that the use of murine systems for analysis of NE-mutation-associated pathogenesis is complicated by differences between humans and mice in the physiology of granulopoiesis, which may go beyond possible

  14. Assessment of antioxidant activity of spray dried extracts of Psidium guajava leaves by DPPH and chemiluminescence inhibition in human neutrophils.

    Science.gov (United States)

    Fernandes, M R V; Azzolini, A E C S; Martinez, M L L; Souza, C R F; Lucisano-Valim, Y M; Oliveira, W P

    2014-01-01

    This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β -cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH(•) method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

  15. Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line.

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    Zaman, Sabiha N; Resek, Mary E; Robbins, Stephen M

    2008-10-01

    Chemokines play pivotal roles in regulating a wide variety of biological processes by modulating cell migration and recruitment. Deregulation of chemokine signaling can alter cell recruitment, contributing to the pathogenic states associated with autoimmune disease, inflammatory disorders, and sepsis. During chemotaxis, lipid rafts and their resident signaling molecules have been demonstrated to partition to different parts of the cell. Herein, we investigated the role of lipid raft resident Src-family kinases (SFK) in stromal cell-derived factor 1/CXCL12-mediated chemotaxis. We have shown that Lck-deficient J.CaM 1.6 cells are defective in CXCL12-mediated chemotaxis in contrast to their parental counterpart, Jurkat cells. Ectopic expression of the SFK hematopoietic cell kinase (Hck) in J.CaM 1.6 cells reconstituted CXCL12 responsiveness. The requirement of lipid raft association of SFK was assessed using both isoforms of Hck: the dually acylated p59(Hck) isoform that is targeted to lipid rafts and the monoacylated p61(Hck) isoform that is nonraft-associated. We have shown using several gain and loss of acylation alleles that dual acylation of Hck was required for CXCL12-mediated chemotaxis in J.CaM 1.6 cells. These results highlight the importance of the unique microenvironment provided by lipid rafts and their specific contribution in providing specificity to CXCL12 signaling.

  16. Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity

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    Jiehao Zhou

    2010-01-01

    Full Text Available We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE. The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP- 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P<.05. Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P<.05 and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.

  17. PsHint1, associated with the G-protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae.

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    Zhang, Xin; Zhai, Chunhua; Hua, Chenlei; Qiu, Min; Hao, Yujuan; Nie, Pingping; Ye, Wenwu; Wang, Yuanchao

    2016-02-01

    Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G-protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1-interacting proteins, including PsHint1, a histidine triad (HIT) domain-containing protein orthologous to human HIT nucleotide-binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms of PsGPA1. An analysis of the gene-silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα-independent pathway involved in the pathogenicity of P. sojae. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  18. Ursolic acid inhibits superoxide production in activated neutrophils and attenuates trauma-hemorrhage shock-induced organ injury in rats.

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    Tsong-Long Hwang

    Full Text Available Neutrophil activation is associated with the development of organ injury after trauma-hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma-hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma-hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma-hemorrhagic shock-induced organ injury in rats.

  19. Bid truncation, Bid/Bax targeting to the mitochondria, and caspase activation associated with neutrophil apoptosis are inhibited by granulocyte colony-stimulating factor

    NARCIS (Netherlands)

    Maianski, Nikolai A.; Roos, Dirk; Kuijpers, Taco W.

    2004-01-01

    Neutrophil apoptosis constitutes a way of managing neutrophil-mediated reactions. It allows coping with infections, but avoiding overt bystander tissue damage. Using digitonin-based subcellular fractionation and Western blotting, we found that spontaneous apoptosis of human neutrophils (after

  20. Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells.

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    Christian Schwarzer

    Full Text Available Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11 with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells. PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins and massively (10-80 fold increase, termed "swarming", but transiently (random swimming after 15 mins, to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii PA use pili to bind to epithelial cells near wounds.

  1. Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

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    Michail S Lionakis

    Full Text Available Invasive candidiasis is the 4(th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo to Ccr1(high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1(+/+ and Ccr1(-/- donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

  2. Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

    Science.gov (United States)

    Colom, Bartomeu; Bodkin, Jennifer V; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A; Nourshargh, Sussan

    2015-06-16

    Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. [Experimental study of the effects of impulse-electric discharge on chemotaxis and cytoadhesion of urinary infection pathogens].

    Science.gov (United States)

    Kuderinov, S K; Azizov, I S; Turgunov, E M; Shambilova, N A

    2006-01-01

    The aim of the experimental study was to evaluate effects of impulse-electric discharge in liquid on chemotaxis and cytoadhesion of urinary infection pathogens. Chemotaxis was determined in respect to the lung, liver, spleen, kidney, ureter, urinary bladder, urethra of white mice by S. Likholetov's modified method. Cytoadhesion was assessed by V. Brilis. The experiments show that the impulse-electric discharge holds promise for urological practice.

  4. Compound C inhibits macrophage chemotaxis through an AMPK-independent mechanism

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    Lee, Youngyi [College of Pharmacy, Woosuk University, Wanju, Jeonbuk 55338 (Korea, Republic of); Department of Biochemistry, Chonbuk National University Medical School, Jeonju, Jeonbuk 54896 (Korea, Republic of); Park, Byung-Hyun, E-mail: bhpark@jbnu.ac.kr [Department of Biochemistry, Chonbuk National University Medical School, Jeonju, Jeonbuk 54896 (Korea, Republic of); Bae, Eun Ju, E-mail: ejbae@woosuk.ac.kr [College of Pharmacy, Woosuk University, Wanju, Jeonbuk 55338 (Korea, Republic of)

    2016-01-15

    Macrophage infiltration in adipose tissue is a well-established cause of obesity-linked insulin resistance. AMP-activated protein kinase (AMPK) activation in peripheral tissues such as adipose tissue has beneficial effects on the protection against obesity-induced insulin resistance, which is mainly mediated by prevention of adipose tissue macrophage infiltration and inflammation. In examining the role of AMPK on adipose tissue inflammation, we unexpectedly found that compound C (CC), despite its inhibition of AMPK, robustly inhibited macrophage chemotaxis in RAW 264.7 cells when adipocyte conditioned medium (CM) was used as a chemoattractant. Here, we report that CC inhibition of macrophage migration occurred independently of AMPK. Mechanistically, this inhibitory effect of cell migration by CC was mediated by inhibition of the focal adhesion kinase, AKT, nuclear factor κB pathways. Moreover, the expression of chemokine monocyte chemoattractant protein-1 and pro-inflammatory genes such as tumor necrosis factor α and inducible nitric oxide synthase were prevented by CC treatment in RAW 264.7 cells stimulated with either adipocyte CM or lipopolysaccharide. Lastly, in accord with the findings of the anti-inflammatory effect of CC, we demonstrated that CC functioned as a repressor of macrophage CM-mediated insulin resistance in adipocytes. Taken together, our results suggest that CC serves as a useful inhibitory molecule against macrophage chemotaxis into adipose tissue and thus might have therapeutic potential for the treatment of obesity-linked adipose inflammation. - Highlights: • Compound C (CC) inhibits macrophage chemotaxis regardless of AMPK suppression. • CC enhances insulin sensitivity in adipocytes. • CC inhibits focal adhesion kinase, AKT, and NF-κB signaling in RAW 264.7 cells.

  5. [Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation].

    Science.gov (United States)

    Li, Jinlong; Zhang, Yidan; Zhou, Xin; Ji, Wenjie; Zhao, Jihong; Wei, Luqing; Li, Yuming

    2014-09-01

    To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis. Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen. HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees. This work established an efficient method for in vitro generation and degradation of human NETs.

  6. Mitochondria in neutrophil apoptosis

    NARCIS (Netherlands)

    van Raam, B. J.; Verhoeven, A. J.; Kuijpers, T. W.

    2006-01-01

    Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis

  7. Neutrophil Responses to Sterile Implant Materials.

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    Siddharth Jhunjhunwala

    Full Text Available In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30-500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices.

  8. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  9. Osthol attenuates neutrophilic oxidative stress and hemorrhagic shock-induced lung injury via inhibition of phosphodiesterase 4.

    Science.gov (United States)

    Tsai, Yung-Fong; Yu, Huang-Ping; Chung, Pei-Jen; Leu, Yann-Lii; Kuo, Liang-Mou; Chen, Chun-Yu; Hwang, Tsong-Long

    2015-12-01

    Oxidative stress caused by neutrophils is an important pathogenic factor in trauma/hemorrhagic (T/H)-induced acute lung injury (ALI). Osthol, a natural coumarin found in traditional medicinal plants, has therapeutic potential in various diseases. However, the pharmacological effects of osthol in human neutrophils and its molecular mechanism of action remain elusive. In this study, our data showed that osthol potently inhibited the production of superoxide anion (O2(•-)) and reactive oxidants derived therefrom as well as expression of CD11b in N-formylmethionylleucylphenylalanine (FMLP)-activated human neutrophils. However, osthol inhibited neutrophil degranulation only slightly and it failed to inhibit the activity of subcellular NADPH oxidase. FMLP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) was inhibited by osthol. Notably, osthol increased the cAMP concentration and protein kinase A (PKA) activity in activated neutrophils. PKA inhibitors reversed the inhibitory effects of osthol, suggesting that these are mediated through cAMP/PKA-dependent inhibition of ERK and Akt activation. Furthermore, the activity of cAMP-specific phosphodiesterase (PDE) 4, but not PDE3 or PDE7, was significantly reduced by osthol. In addition, osthol reduced myeloperoxidase activity and pulmonary edema in rats subjected to T/H shock. In conclusion, our data suggest that osthol has effective anti-inflammatory activity in human neutrophils through the suppression of PDE4 and protects significantly against T/H shock-induced ALI in rats. Osthol may have potential for future clinical application as a novel adjunct therapy to treat lung inflammation caused by adverse circulatory conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Neutrophil migration under normal and sepsis conditions.

    Science.gov (United States)

    Lerman, Yelena V; Kim, Minsoo

    2015-01-01

    Neutrophil migration is critical for pathogen clearance and host survival during severe sepsis. Interaction of neutrophil adhesion receptors with ligands on endothelial cells results in firm adhesion of the circulating neutrophils, followed by neutrophil activation and directed migration to sites of infection through the basement membrane and interstitial extracellular matrix. Proteolytic enzymes and reactive oxygen species are produced and released by neutrophils in response to a variety of inflammatory stimuli. Although these mediators are important for host defense, they also promote tissue damage. Excessive neutrophil migration during the early stages of sepsis may lead to an exaggerated inflammatory response with associated tissue damage and subsequent organ dysfunction. On the other hand, dysregulation of migration and insufficient migratory response that occurs during the latter stages of severe sepsis contributes to neutrophils' inability to contain and control infection and impaired wound healing. This review discusses the major steps and associated molecules involved in the balance of neutrophil trafficking, the precise regulation of which during sepsis spells life or death for the host.

  11. Inactivation of transferrin iron binding capacity by the neutrophil myeloperoxidase system

    International Nuclear Information System (INIS)

    Clark, R.A.; Pearson, D.W.

    1989-01-01

    Human serum apotransferrin was exposed to the isolated myeloperoxidase-H2O2-halide system or to phorbol ester-activated human neutrophils. Such treatment resulted in a marked loss in transferrin iron binding capacity as well as concomitant iodination of transferrin. Each component of the cell-free system (myeloperoxidase, H2O2, iodide) or neutrophil system (neutrophils, phorbol ester, iodide) was required in order to observe these changes. In the cell-free system, the H2O2 requirement was fulfilled by either reagent H2O2 or the peroxide-generating system glucose oxidase plus glucose. Both loss of iron binding capacity and transferrin iodination by either the myeloperoxidase system or activated neutrophils were blocked by azide or catalase. The isolated peroxidase system had an acidic pH optimum, whereas the intact cell system was more efficient at neutral pH. The kinetics of changes in iron binding capacity and iodination closely paralleled one another, exhibiting t1/2 values of less than 1 min for the myeloperoxidase-H2O2 system, 3-4 min for the myeloperoxidase-glucose oxidase system, and 8 min for the neutrophil system. That the occupied binding site is protected from the myeloperoxidase system was suggested by (1) a failure to mobilize iron from iron-loaded transferrin, (2) an inverse correlation between initial iron saturation and myeloperoxidase-mediated loss of iron binding capacity, and (3) decreased myeloperoxidase-mediated iodination of iron-loaded versus apotransferrin. Since as little as 1 atom of iodide bound per molecule of transferrin was associated with substantial losses in iron binding capacity, there appears to be a high specificity of myeloperoxidase-catalyzed iodination for residues at or near the iron binding sites. Amino acid analysis of iodinated transferrin (approximately 2 atoms/molecule) demonstrated that iodotyrosine was the predominant iodinated species

  12. CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera

    Directory of Open Access Journals (Sweden)

    Bettinotti Maria

    2004-03-01

    Full Text Available Abstract Genes in the Leukocyte Antigen 6 (Ly-6 superfamily encode glycosyl-phosphatidylinositol (GPI anchored glycoproteins (gp with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58–64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

  13. Chemotaxis in the cellular slime molds : I. The effect of temperature

    NARCIS (Netherlands)

    Konijn, Theo M.

    1965-01-01

    The effect of temperature on chemotaxis in the cellular slime mold Dictyostelium discoideum has been studied by incubating small populations of washed myxamoebae at different temperatures. Droplets containing a cell suspension of known density were deposited on a hydrophobic agar surface. The

  14. Ensemble models of neutrophil trafficking in severe sepsis.

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    Sang Ok Song

    Full Text Available A hallmark of severe sepsis is systemic inflammation which activates leukocytes and can result in their misdirection. This leads to both impaired migration to the locus of infection and increased infiltration into healthy tissues. In order to better understand the pathophysiologic mechanisms involved, we developed a coarse-grained phenomenological model of the acute inflammatory response in CLP (cecal ligation and puncture-induced sepsis in rats. This model incorporates distinct neutrophil kinetic responses to the inflammatory stimulus and the dynamic interactions between components of a compartmentalized inflammatory response. Ensembles of model parameter sets consistent with experimental observations were statistically generated using a Markov-Chain Monte Carlo sampling. Prediction uncertainty in the model states was quantified over the resulting ensemble parameter sets. Forward simulation of the parameter ensembles successfully captured experimental features and predicted that systemically activated circulating neutrophils display impaired migration to the tissue and neutrophil sequestration in the lung, consequently contributing to tissue damage and mortality. Principal component and multiple regression analyses of the parameter ensembles estimated from survivor and non-survivor cohorts provide insight into pathologic mechanisms dictating outcome in sepsis. Furthermore, the model was extended to incorporate hypothetical mechanisms by which immune modulation using extracorporeal blood purification results in improved outcome in septic rats. Simulations identified a sub-population (about 18% of the treated population that benefited from blood purification. Survivors displayed enhanced neutrophil migration to tissue and reduced sequestration of lung neutrophils, contributing to improved outcome. The model ensemble presented herein provides a platform for generating and testing hypotheses in silico, as well as motivating further experimental

  15. Neutrophil Reverse Migration Becomes Transparent with Zebrafish

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    Taylor W. Starnes

    2012-01-01

    Full Text Available The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo.

  16. Chemokine Receptor Ccr1 Drives Neutrophil-Mediated Kidney Immunopathology and Mortality in Invasive Candidiasis

    Science.gov (United States)

    Lionakis, Michail S.; Swamydas, Muthulekha; Wan, Wuzhou; Richard Lee, Chyi-Chia; Cohen, Jeffrey I.; Scheinberg, Phillip; Gao, Ji-Liang; Murphy, Philip M.

    2012-01-01

    Invasive candidiasis is the 4th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1lo to Ccr1high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1+/+ and Ccr1−/− donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ. PMID:22916017

  17. Assessment of Antioxidant Activity of Spray Dried Extracts of Psidium guajava Leaves by DPPH and Chemiluminescence Inhibition in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    M. R. V. Fernandes

    2014-01-01

    Full Text Available This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β-cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL produced by neutrophils stimulated with phorbol myristate acetate (PMA and the DPPH radical scavenging (DPPH* method. In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH• method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

  18. Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

    Directory of Open Access Journals (Sweden)

    Rong Jin

    Full Text Available Recent work has revealed an essential involvement of soluble CD40L (sCD40L in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40, i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better

  19. Congenital CMV with LAD type 1 and NK cell deficiency.

    Science.gov (United States)

    Rai, Narendra; Thakur, Neha

    2013-08-01

    We report a rare case of congenital cytomegalovirus (CMV) in a patient who was subsequently diagnosed as leukocyte adhesion defect type 1 with natural killer cell deficiency. The clinical course was complicated by severe CMV pneumonitis during the newborn period. Thereafter the infant suffered from recurrent skin infections without pus formation, otitis media, and bronchopneumonia since 3 months of age. The patient had congenital CMV infection as urine and blood plasma was positive for CMV from day 12 onward. Neutrophil chemotaxis studies showed a decrease in directed chemotaxis. Neutrophils were dyspoetic and nonfunctional lacking HLA DR, CD11c, and CD18. Lymphocytes were polyclonal but lacked CD56, CD16, and surface membrane immunoglobulin.

  20. Immunosenescence of Polymorphonuclear Neutrophils

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    Inga Wessels

    2010-01-01

    Full Text Available All immune cells are affected by aging, contributing to the high susceptibility to infections and increased mortality observed in the elderly. The effect of aging on cells of the adaptive immune system is well documented. In contrast, knowledge concerning age-related defects of polymorphonuclear neutrophils (PMN is limited. During the past decade, it has become evident that in addition to their traditional role as phagocytes, neutrophils are able to secrete a wide array of immunomodulating molecules. Their importance is underlined by the finding that genetic defects that lead to neutropenia increase susceptibility to infections. Whereas there is consistence about the constant circulating number of PMN throughout aging, the abilities of tissue infiltration, phagocytosis, and oxidative burst of PMN from aged donors are discussed controversially. Furthermore, there are numerous discrepancies between in vivo and in vitro results, as well as between results for murine and human PMN. Most of the reported functional changes can be explained by defective signaling pathways, but further research is required to get a detailed insight into the underlying molecular mechanisms. This could form the basis for drug development in order to prevent or treat age-related diseases, and thus to unburden the public health systems.

  1. Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

    Directory of Open Access Journals (Sweden)

    André Holdfeldt

    2016-09-01

    Full Text Available The data described here is related to the research article titled (Gabl et al., 2016 [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8 and CXCR4 (ATI-2341, respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe6-NH2 (both are FPR2 specific inhibitors, but not to the FPR1 specific inhibitor cyclosporine H.

  2. Targeting Neutrophilic Inflammation using Polymersome-Mediated Cellular Delivery

    OpenAIRE

    Robertson, J.D.; Ward, J.R.; Avila-Olias, M.; Battaglia, G.; Renshaw, S.A.

    2017-01-01

    Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In ...

  3. Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo

    Science.gov (United States)

    Colom, Bartomeu; Bodkin, Jennifer V.; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A.; Nourshargh, Sussan

    2015-01-01

    Summary Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic. PMID:26047922

  4. Cxcl8b and Cxcr2 Regulate Neutrophil Migration through Bloodstream in Zebrafish

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    Constanza Zuñiga-Traslaviña

    2017-01-01

    Full Text Available Neutrophils play an essential role during an inflammatory response, which is dependent on their rapid recruitment from the bone marrow to the vasculature. However, there is no information about the molecular signals that regulate neutrophil entry to circulation during an inflammatory process in humans. This is mainly due to the lack of a suitable model of study that contains similar set of molecules and that allows in vivo analyses. In this study, we used the zebrafish to assess the role of Cxcl8a, Cxcl8b, and Cxcr2 in neutrophil migration to blood circulation after injury. Using Tg(BACmpx:GFPi114 transgenic embryos and two damage models (severe and mild, we developed in vivo lack of function assays. We found that the transcription levels of cxcl8a, cxcl8b, and cxcr2 were upregulated in the severe damage model. In contrast, only cxcr2 and cxcl8a mRNA levels were increased during mild damage. After knocking down Cxcl8a, neutrophil quantity decreased at the injury site, while Cxcl8b decreased neutrophils in circulation. When inhibiting Cxcr2, we observed a decrease in neutrophil entry to the bloodstream. In conclusion, we identified different functions for both Cxcl8 paralogues, being the Cxcl8b/Cxcr2 axis that regulates neutrophil entry to the bloodstream, while Cxcl8a/Cxcr2 regulates the migration to the affected area.

  5. Equol Effectively Inhibits Toxic Activity of Human Neutrophils without Influencing Their Viability

    Czech Academy of Sciences Publication Activity Database

    Pažoureková, S.; Lucová, M.; Nosál, R.; Drábiková, K.; Harmatha, Juraj; Šmidrkal, J.; Jančinová, V.

    2016-01-01

    Roč. 97, 3/4 (2016), s. 138-145 ISSN 0031-7012 Institutional support: RVO:61388963 Keywords : neutrophils * equol * chemiluminescence * reactive oxygen species * p40(phox) * apoptosis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.442, year: 2016

  6. Neutrophil labeling with [99mTc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide

    International Nuclear Information System (INIS)

    Gallagher, Hayley; Ramsay, Stuart C.; Barnes, Jodie; Maggs, Jacqueline; Cassidy, Nathan; Ketheesan, Natkunam

    2006-01-01

    Introduction: [ 99m Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. Methods: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. Results: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. Conclusions: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection

  7. A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

    Science.gov (United States)

    Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

    2002-10-25

    Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

  8. Infection and cellular defense dynamics in a novel 17β-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation.

    Science.gov (United States)

    Carey, Alison J; Tan, Chee Keong; Mirza, Shaper; Irving-Rodgers, Helen; Webb, Richard I; Lam, Alfred; Ulett, Glen C

    2014-02-15

    Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (10(6)-10(7) CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

  9. Leukotriene D4 induces chemotaxis in human eosinophilc cell line, EoL-1 cells via CysLT1 receptor activation.

    Science.gov (United States)

    Shirasaki, Hideaki; Kanaizumi, Etsuko; Himi, Tetsuo

    2017-11-01

    Numerous reports have shown that cysteinyl leukotrienes (CysLTs) contribute to tissue accumulation of eosinophils in allergic airway inflammation. To date, only a few studies have reported that CysLTs promote chemotactic activity of human eosinophils in vitro. The purpose of this study was to investigate whether CysLTs promote chemotaxis in the human eosinophilic cell line, EoL-1. EoL-1 cells were induced to differentiate into mature eosinophil-like cells via incubation with butyric acid and cytokines (IL-3, IL-5 and GM-CSF). The chemotactic activity of the differentiated EoL-1 cells was assessed using the commercial cell migration assay kit. LTD 4 elicited dose-related chemotactic activity in the differntiated EoL-1 cells in the range of 1-100 nM. A typical bell-shaped dose-response curve was observed with optimal activity at 10 nM. The chemotactic activity elicited by LTD 4 (10 nM) was significantly inhibited by montelukast (control, 345 ± 19.2 × 10 3 RFU; LTD 4 10 nM alone, 511 ± 39.2 × 10 3 RFU; LTD 4 10 nM plus montelukast 100 nM, 387 ± 28.2 × 10 3 RFU). LTD 4 induces migration in eosinophilic cells via activation of CysLT1 receptor. The present in vitro model may be useful for elucidation of the mechanism underlying CysLT-induced tissue eosinophilia.

  10. Increase in neutrophil Fc gamma receptor I expression following interferon gamma treatment in rheumatoid arthritis.

    Science.gov (United States)

    Goulding, N J; Knight, S M; Godolphin, J L; Guyre, P M

    1992-04-01

    The therapeutic potential of interferon gamma (IFN gamma) in a number of disease states is still being explored, but progress is hampered by the lack of a suitable measure of in vivo biological activity. To assess the in vivo biological effects of recombinant human IFN gamma (rhIFN gamma), 14 patients were studied in a randomised, prospective, double blind, placebo controlled trial of this cytokine for the treatment of rheumatoid arthritis. The levels of Fc gamma receptors on peripheral blood neutrophils were measured at baseline and after 21 days of once daily, subcutaneous injections of rhIFN gamma or placebo. An induction of neutrophil Fc gamma receptor type I (Fc gamma RI) was seen in the group of patients receiving recombinant human rhIFN gamma but not in those receiving placebo. No change in the expression of Fc gamma RII or Fc gamma RIII was detected. The amount of induction of Fc gamma RI detected on the neutrophils of patients receiving rhIFN gamma did not correlate with clinical measures of response at either 21 days or at the end of the study (24 weeks). No significant clinical responses were observed in the rhIFN gamma group at these times. These data confirm that the reported in vitro effect of IFN gamma on human neutrophil Fc receptor expression can be reproduced in vivo.

  11. Sexy again: the renaissance of neutrophils in psoriasis.

    Science.gov (United States)

    Schön, Michael P; Broekaert, Sigrid M C; Erpenbeck, Luise

    2017-04-01

    Notwithstanding their prominent presence in psoriatic skin, the functional role of neutrophilic granulocytes still remains somewhat enigmatic. Sparked by exciting scientific discoveries regarding neutrophil functions within the last years, the interest in these short-lived cells of the innate immune system has been boosted recently. While it had been known for some time that neutrophils produce and respond to a number of inflammatory mediators, recent research has linked neutrophils with the pathogenic functions of IL-17, possibly in conjunction with the formation of NETs (neutrophil extracellular traps). Antipsoriatic therapies exert their effects, at least in part, through interference with neutrophils. Neutrophils also appear to connect psoriasis with comorbid diseases. However, directly tampering with neutrophil functions is not trivial as evinced by the failure of therapeutic approaches targeting redundantly regulated cellular communication networks. It has also become apparent that neutrophils link important pathogenic functions of the innate and the adaptive immune system and that they are intricately involved in regulatory networks underlying the pathophysiology of psoriasis. In order to advocate intensified research into the role of this interesting cell population, we here highlight some features of neutrophils and put them into perspective with our current view of the pathophysiology of psoriasis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Hyperglycemia Impairs Neutrophil-Mediated Bacterial Clearance in Mice Infected with the Lyme Disease Pathogen.

    Directory of Open Access Journals (Sweden)

    Ashkan Javid

    Full Text Available Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutrophils, which are key mediators of innate immune responses to microbes as well as pathological inflammatory processes. Neutrophils are central to immune responses to the Lyme pathogen Borrelia burgdorferi. The effect of hyperglycemia on host susceptibility to and outcomes of B. burgdorferi infection has not been examined. The present study investigated the impact of sustained obesity-independent hyperglycemia in mice on bacterial clearance, inflammatory pathology and neutrophil responses to B. burgdorferi. Hyperglycemia was associated with reduced arthritis incidence but more widespread tissue colonization and reduced clearance of bacterial DNA in multiple tissues including brain, heart, liver, lung and knee joint. B. burgdorferi uptake and killing were impaired in neutrophils isolated from hyperglycemic mice. Thus, attenuated neutrophil function in insulin-insufficient hyperglycemia was associated with reduced B. burgdorferi clearance in target organs. These data suggest that investigating the effects of comorbid conditions such as diabetes on outcomes of B. burgdorferi infections in humans may be warranted.

  13. Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

    Directory of Open Access Journals (Sweden)

    Nicolette Prevost

    2011-12-01

    Full Text Available Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing. Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze. A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration.

  14. ADAM9 Is a Novel Product of Polymorphonuclear Neutrophils

    DEFF Research Database (Denmark)

    Roychaudhuri, Robin; Hergrueter, Anja H; Polverino, Francesca

    2014-01-01

    A disintegrin and a metalloproteinase domain (ADAM) 9 is known to be expressed by monocytes and macrophages. In this study, we report that ADAM9 is also a product of human and murine polymorphonuclear neutrophils (PMNs). ADAM9 is not synthesized de novo by circulating PMNs. Rather, ADAM9 protein...

  15. Unsaturated long-chain fatty acids induce the respiratory burst of human neutrophils and monocytes in whole blood

    Directory of Open Access Journals (Sweden)

    Osthaus Wilhelm A

    2008-07-01

    Full Text Available Abstract Background It is increasingly recognized that infectious complications in patients treated with total parenteral nutrition (TPN may be caused by altered immune responses. Neutrophils and monocytes are the first line of defence against bacterial and fungal infection through superoxide anion production during the respiratory burst. To characterize the impact of three different types of lipid solutions that are applied as part of TPN formulations, we investigated the unstimulated respiratory burst activation of neutrophils and monocytes in whole blood. Methods Whole blood samples were incubated with LCT (Intralipid®, LCT/MCT (Lipofundin® and LCT-MUFA (ClinOleic® in three concentrations (0.06, 0.3 and 0.6 mg ml-1 for time periods up to one hour. Hydrogen peroxide production during the respiratory burst of neutrophils and monocytes was measured by flow cytometry. Results LCT and LCT-MUFA induced a hydrogen peroxide production in neutrophils and monocytes without presence of a physiological stimulus in contrast to LCT/MCT. Conclusion We concluded that parenteral nutrition containing unsaturated oleic (C18:1 and linoleic (C18:2 acid can induce respiratory burst of neutrophils and monocytes, resulting in an elevated risk of tissue damage by the uncontrolled production of reactive oxygen species. Contradictory observations reported in previous studies may in part be the result of different methods used to determine hydrogen peroxide production.

  16. The effect of the anaesthetic agent isoflurane on the rate of neutrophil apoptosis in vitro.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND: Volatile anaesthetic agents influence neutrophil function, and potentially, the inflammatory response to surgery. AIM: The objective of this study was to determine the effect of isoflurane (1-4%) on human polymorphonuclear neutrophil apoptosis in vitro. METHODS: Venous blood from 12 healthy volunteers was exposed to 0, 1, and 4% isoflurane delivered via a 14G Wallace flexihub internal jugular cannula, at a fresh gas flow of 0.51\\/min for 5 minutes. Isolated neutrophils were assessed for apoptosis at 1, 12, and 24 hours in culture using dual staining with annexin V-FITC and propidium iodide (Annexin-V FITC assay). Data were analysed using paired, one-tailed Student\\'s t-tests. p<0.05 was considered significant. RESULTS: At 1 hour apoptosis was inhibited in the 1% (5.1 [6.8]%; p=0.017) and 4% (4.8 [4.5]%; p=0.008) isoflurane groups compared to control (11.3 [6.9]%). At 12 and 24 hours, a dose-dependent inhibition of apoptosis was demonstrated, i.e. 4% > 1% > 0%. CONCLUSION: Human neutrophil apoptosis is inhibited in a concentration-dependent manner in vitro by isoflurane in clinical concentrations.

  17. Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines

    Directory of Open Access Journals (Sweden)

    Conor M. Henry

    2016-02-01

    Full Text Available Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ∼500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis.

  18. Neutrophils in Cancer: Two Sides of the Same Coin.

    Science.gov (United States)

    Uribe-Querol, Eileen; Rosales, Carlos

    2015-01-01

    Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

  19. Identification of glutathione adducts of α-chlorofatty aldehydes produced in activated neutrophils

    Science.gov (United States)

    Duerr, Mark A.; Aurora, Rajeev; Ford, David A.

    2015-01-01

    α-Chlorofatty aldehydes (α-ClFALDs) are produced by hypochlorous acid targeting plasmalogens during neutrophil activation. This study investigated the reaction of the α-chlorinated carbon of α-ClFALD with the nucleophile, GSH. Utilizing ESI/MS/MS, the reaction product of GSH and the 16-carbon α-ClFALD, 2-chlorohexadecanal (2-ClHDA), was characterized. The resulting conjugate of 2-ClHDA and GSH (HDA-GSH) has an intact free aldehyde, and the chlorine at the α-carbon is ejected. Stable isotope-labeled [d4]HDA-GSH was synthesized, which further confirmed the structure, and was used to quantify natural α-ClFALD conjugates of GSH (FALD-GSH) using reverse-phase LC with detection by ESI/MS/MS using selected reaction monitoring. HDA-GSH is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2-ClHDA. Furthermore, PMA-treated primary human neutrophils have elevated levels of HDA-GSH and the conjugate of 2-chlorooctadecanal (2-ClODA) and GSH (ODA-GSH), as well as elevated levels of 2-ClHDA and 2-ClODA. Production of both conjugates in PMA-stimulated neutrophils was reduced by 3-aminotriazole pretreatment, which also blocks endogenous α-ClFALD production. Additionally, plasma FALD-GSH levels were elevated in the K/BxN mouse arthritis model. Taken together, these studies demonstrate novel peptidoaldehydes derived from GSH and α-ClFALD in activated human neutrophils and in vivo in K/BxN mice. PMID:25814023

  20. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    van Raam, Bram J.; Sluiter, Wim; de Wit, Elly; Roos, Dirk; Verhoeven, Arthur J.; Kuijpers, Taco W.

    2008-01-01

    BACKGROUND: Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Deltapsi(m)), which is usually generated by the respiratory chain complexes. We investigated the source of Deltapsi(m) in neutrophils, as compared to peripheral blood

  1. Akirin1 (Mighty), a novel promyogenic factor regulates muscle regeneration and cell chemotaxis

    Energy Technology Data Exchange (ETDEWEB)

    Salerno, Monica Senna; Dyer, Kelly; Bracegirdle, Jeremy; Platt, Leanne; Thomas, Mark; Siriett, Victoria [Functional Muscle Genomics, AgResearch, Hamilton (New Zealand); Kambadur, Ravi [Functional Muscle Genomics, AgResearch, Hamilton (New Zealand); School of Biological Sciences, Nanyang Technological University, Singapore (Singapore); Sharma, Mridula, E-mail: bchmridu@nus.edu.sg [Functional Muscle Genomics, AgResearch, Hamilton (New Zealand)

    2009-07-15

    Akirin1 (Mighty) is a downstream target gene of myostatin and has been shown to be a promyogenic factor. Although expressed in many tissues, akirin1 is negatively regulated by myostatin specifically in skeletal muscle tissue. In this manuscript we have characterized the possible function of akirin1 in postnatal muscle growth. Molecular and immunohistological analyses indicated that while low levels of akirin1 are associated with quiescent satellite cells (SC), higher levels of akirin1 are detected in activated proliferating SC indicating that akirin1 could be associated with satellite cell activation. In addition to SC, macrophages also express akirin1, and increased expression of akirin1 resulted in more efficient chemotaxis of both macrophages and myoblasts. Akirin1 appears to regulate chemotaxis of both macrophages and myoblasts by reorganising actin cytoskeleton, leading to more efficient lamellipodia formation via a PI3 kinase dependent pathway. Expression analysis during muscle regeneration also indicated that akirin1 expression is detected very early (day 2) in regenerating muscle, and expression gradually peaks to coincide the nascent myotube formation stage of muscle regeneration. Based on these results we propose that akirin1 could be acting as a transducer of early signals of muscle regeneration. Thus, we speculate that myostatin regulates key steps of muscle regeneration including chemotaxis of inflammatory cells, SC activation and migration through akirin1.

  2. Akirin1 (Mighty), a novel promyogenic factor regulates muscle regeneration and cell chemotaxis

    International Nuclear Information System (INIS)

    Salerno, Monica Senna; Dyer, Kelly; Bracegirdle, Jeremy; Platt, Leanne; Thomas, Mark; Siriett, Victoria; Kambadur, Ravi; Sharma, Mridula

    2009-01-01

    Akirin1 (Mighty) is a downstream target gene of myostatin and has been shown to be a promyogenic factor. Although expressed in many tissues, akirin1 is negatively regulated by myostatin specifically in skeletal muscle tissue. In this manuscript we have characterized the possible function of akirin1 in postnatal muscle growth. Molecular and immunohistological analyses indicated that while low levels of akirin1 are associated with quiescent satellite cells (SC), higher levels of akirin1 are detected in activated proliferating SC indicating that akirin1 could be associated with satellite cell activation. In addition to SC, macrophages also express akirin1, and increased expression of akirin1 resulted in more efficient chemotaxis of both macrophages and myoblasts. Akirin1 appears to regulate chemotaxis of both macrophages and myoblasts by reorganising actin cytoskeleton, leading to more efficient lamellipodia formation via a PI3 kinase dependent pathway. Expression analysis during muscle regeneration also indicated that akirin1 expression is detected very early (day 2) in regenerating muscle, and expression gradually peaks to coincide the nascent myotube formation stage of muscle regeneration. Based on these results we propose that akirin1 could be acting as a transducer of early signals of muscle regeneration. Thus, we speculate that myostatin regulates key steps of muscle regeneration including chemotaxis of inflammatory cells, SC activation and migration through akirin1.

  3. Neutrophils in Cancer: Two Sides of the Same Coin

    Directory of Open Access Journals (Sweden)

    Eileen Uribe-Querol

    2015-01-01

    Full Text Available Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

  4. The impact of odor–reward memory on chemotaxis in larval Drosophila

    Science.gov (United States)

    Schleyer, Michael; Reid, Samuel F.; Pamir, Evren; Saumweber, Timo; Paisios, Emmanouil; Davies, Alexander

    2015-01-01

    How do animals adaptively integrate innate with learned behavioral tendencies? We tackle this question using chemotaxis as a paradigm. Chemotaxis in the Drosophila larva largely results from a sequence of runs and oriented turns. Thus, the larvae minimally need to determine (i) how fast to run, (ii) when to initiate a turn, and (iii) where to direct a turn. We first report how odor-source intensities modulate these decisions to bring about higher levels of chemotactic performance for higher odor-source intensities during innate chemotaxis. We then examine whether the same modulations are responsible for alterations of chemotactic performance by learned odor “valence” (understood throughout as level of attractiveness). We find that run speed (i) is neither modulated by the innate nor by the learned valence of an odor. Turn rate (ii), however, is modulated by both: the higher the innate or learned valence of the odor, the less often larvae turn whenever heading toward the odor source, and the more often they turn when heading away. Likewise, turning direction (iii) is modulated concordantly by innate and learned valence: turning is biased more strongly toward the odor source when either innate or learned valence is high. Using numerical simulations, we show that a modulation of both turn rate and of turning direction is sufficient to account for the empirically found differences in preference scores across experimental conditions. Our results suggest that innate and learned valence organize adaptive olfactory search behavior by their summed effects on turn rate and turning direction, but not on run speed. This work should aid studies into the neural mechanisms by which memory impacts specific aspects of behavior. PMID:25887280

  5. NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.

    Science.gov (United States)

    Prince, Lynne R; Prosseda, Svenja D; Higgins, Kathryn; Carlring, Jennifer; Prestwich, Elizabeth C; Ogryzko, Nikolay V; Rahman, Atiqur; Basran, Alexander; Falciani, Francesco; Taylor, Philip; Renshaw, Stephen A; Whyte, Moira K B; Sabroe, Ian

    2017-08-24

    The lifespan of neutrophils is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis-related genes, the most highly regulated of these being NR4A2 and NR4A3 Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA (8-AHA-cAMP and N6-MB-cAMP) and treatment with endogenous activators of PKA, including adenosine and prostaglandin E2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA-dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and small interfering RNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologs in zebrafish larvae significantly reduces the absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis. © 2017 by The American Society of Hematology.

  6. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

    Directory of Open Access Journals (Sweden)

    Heike L Rittner

    2009-04-01

    Full Text Available In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR and/or toll like receptor (TLR agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively. Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim

  7. Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils.

    Science.gov (United States)

    Vega, Antonio; Chacón, Pedro; Alba, Gonzalo; El Bekay, Rajaa; Martín-Nieto, José; Sobrino, Francisco

    2006-07-01

    Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE(2) and thromboxane A(2) release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-kappaB, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-kappaB pathway, such as N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe(2)(+)/Cu(2)(+) ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-kappaB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions.

  8. Radiation-induced muscositis and neutrophil granulocytes in oral mucosa; Strahleninduzierte Mukositis und neutrophile Granulozyten in der Mundschleimhaut

    Energy Technology Data Exchange (ETDEWEB)

    Schmidberger, H.; Rave-Fraenk, M.; Kim, S.; Hille, A.; Pradier, O.; Hess, C.F. [Klinik fuer Strahlentherapie und Radioonkologie, Univ. Goettingen (Germany)

    2003-10-01

    Background: Chemotherapy-induced mucositis can be related to a decrease in oral neutrophils. We tested the relationship between radiation-induced mucositis and oral neutrophil counts. Patients and Methods: Oral neutrophil counts were obtained for ten patients with head and neck cancer who received radiotherapy of the pharynx and oral cavity. Four patients received additional chemotherapy (5-FU, Mitomycin). Counts were obtained before and during treatment; four healthy volunteers were included in the study as well. For evaluation, a quantitative mouth rinse assay, including neutrophil-staining with acridin-orange, was applied. Results: We observed large inter-individual variations with respect to neutrophil counts for patients and control persons (Table 1). During treatment (irradiation or chemoirradiation), large intra-individual variations were seen additionally (Figure 1). We found a correlation between neutrophil counts and clinical reaction grade. Neutrophil counts increased with increasing mucositis (Figure 2). This increase was more pronounced for patients treated with chemoirradiation compared to radiation alone. Treatment breaks at weekends had no clear influence on neutrophil counts. Conclusions: We observed a weak correlation between neutrophil counts and clinical reaction grade. However, the variations in neutrophil counts are too large to utilize this parameter as a surrogate for clinical mucositis grading. The assumption that a decrease in oral neutrophils is associated with radiation-induced mucositis was clearly negated. (orig.) [German] Hintergrund: Die chemotherapieinduzierte Mukositis kann mit einer Verarmung der Mundschleimhaut an neutrophilen Granulozyten vergesellschaftet sein. Wir ueberprueften den Zusammenhang zwischen der radiogenen Mukositis und der Anzahl neutrophiler Granulozyten. Patienten und Methoden: Bei zehn Patienten mit Tumoren der Kopf-Hals-Region, die sich einer Strahlentherapie unterzogen, wurde die Anzahl enoraler neutrophiler

  9. Neutrophil labeling with [{sup 99m}Tc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Gallagher, Hayley [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Ramsay, Stuart C. [School of Medicine, James Cook University, Townsville, Queensland (Australia) and Townsville Nuclear Medicine, Mater Hospital, Townsville, Queensland 4812 (Australia)]. E-mail: stuart.ramsey@jcu.edu.au; Barnes, Jodie [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Maggs, Jacqueline [Department of Nuclear Medicine, Townsville Hospital, Townsville, Queensland 4814 (Australia); Cassidy, Nathan [Townsville Nuclear Medicine, Mater Hospital, Townsville, Queensland 4812 (Australia); Ketheesan, Natkunam [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); School of Medicine, James Cook University, Townsville, Queensland (Australia)

    2006-04-15

    Introduction: [{sup 99m}Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. Methods: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. Results: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. Conclusions: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection.

  10. Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts

    Science.gov (United States)

    Schäfer, Edith; Tarantola, Marco; Polo, Elena; Westendorf, Christian; Oikawa, Noriko; Bodenschatz, Eberhard; Geil, Burkhard; Janshoff, Andreas

    2013-01-01

    Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells. PMID:23349816

  11. Correlation between the neutrophil-lymphocyte count ratio and bacterial infection in patient with human immunodeficiency virus

    Science.gov (United States)

    Kusnadi, D.; Liwang, M. N. I.; Katu, S.; Mubin, A. H.; Halim, R.

    2018-03-01

    Parameters for starting antibiotic therapy such as CRP andleukocytosis are considered non-specific. Previous studies have shown the Neutrophil-Lymphocyte Count Ratio (NLCR) can serve as the basis of bacterial infection, the level of infection, and the basis of antibiotic therapy. Compared with the Procalcitonin parameter, this NLCR is rapid, an inexpensive and requires no additional sampling. To determine the correlation between The Neutrophil-LymphocyteCount Ratio to bacterial infection in HIV patients. This study was a cross-sectional observational approach to HIV subject at Wahidin Sudirohusodo and Hasanuddin University Hospital. The subjects performed routine blood, microbiology test,and blood Procalcitonin levels tests. Then performed NLCR calculations based on routine blood results. The subjects then grouped the presence or absence of bacterial infection.In 146 study subjects, there were 78 (53.4%) with bacterial infections and 68 (46.6%) without bacterial infection as controls. Subjects with bacterial infections had higher total neutrophils (84.83) compared with non-bacterial infections. Subjects with bacterial infections had total lymphocytes with an average of 8.51 lower than non-bacterial infections. Subjects with bacterial infections had higher NLCR values with an average of 12.80. The Neutrophil-Lymphocyte Count Ratio can become a marker of bacterial infection in HIV patients.

  12. Neutrophils in Tuberculosis: Heterogeneity Shapes the Way?

    Science.gov (United States)

    2017-01-01

    Infection with M. tuberculosis remains one of the most common infections in the world. The outcome of the infection depends on host ability to mount effective protection and balance inflammatory responses. Neutrophils are innate immune cells implicated in both processes. Accordingly, during M. tuberculosis infection, they play a dual role. Particularly, they contribute to the generation of effector T cells, participate in the formation of granuloma, and are directly involved in tissue necrosis, destruction, and infection dissemination. Neutrophils have a high bactericidal potential. However, data on their ability to eliminate M. tuberculosis are controversial, and the results of neutrophil depletion experiments are not uniform. Thus, the overall roles of neutrophils during M. tuberculosis infection and factors that determine these roles are not fully understood. This review analyzes data on neutrophil defensive and pathological functions during tuberculosis and considers hypotheses explaining the dualism of neutrophils during M. tuberculosis infection and tuberculosis disease. PMID:28626346

  13. Evidence for bacterial chemotaxis to cyanobacteria from a radioassay technique

    International Nuclear Information System (INIS)

    Kangatharalingam, N.; Wang, Lizhu; Priscu, J.C.

    1991-01-01

    Lyngbya birgei and Aphanizomenon flos-aquae elicited a significant chemotactic attraction of Aeromonas hydrophila compared with controls lacking cyanobacteria. There was a positive exponential relationship between biomass (chlorophyll a) of L. birgei and A. flos-aquae and chemotactic attraction of A. hydrophila. The assay equipment was simple and reliable and could be used to study bacterial chemotaxis in other species in situ

  14. Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira

    Science.gov (United States)

    Scharrig, Emilia; Carestia, Agostina; Ferrer, María F.; Cédola, Maia; Pretre, Gabriela; Drut, Ricardo; Picardeau, Mathieu; Schattner, Mirta; Gómez, Ricardo M.

    2015-01-01

    NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden. PMID:26161745

  15. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    B.J. van Raam (Bram); W.J. Sluiter (Wim); F.R.C. de Wit (Frank); D. Roos (Dirk); A.J. Verhoeven (Arthur); T.W. Kuijpers (Taco W.)

    2008-01-01

    textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory chain complexes. We investigated the source of ΔΨm in neutrophils, as compared to peripheral blood

  16. Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis

    Directory of Open Access Journals (Sweden)

    Tahtouh Muriel

    2012-02-01

    Full Text Available Abstract Background In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR, which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand. Methods Recombinant HmC1q (rHmC1q was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques. Results rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia. Conclusions A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal

  17. Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis.

    Science.gov (United States)

    Tahtouh, Muriel; Garçon-Bocquet, Annelise; Croq, Françoise; Vizioli, Jacopo; Sautière, Pierre-Eric; Van Camp, Christelle; Salzet, Michel; Nagnan-le Meillour, Patricia; Pestel, Joël; Lefebvre, Christophe

    2012-02-22

    In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand. Recombinant HmC1q (rHmC1q) was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques. rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia. A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech.

  18. Rapid Sequestration of Leishmania mexicana by Neutrophils Contributes to the Development of Chronic Lesion.

    Directory of Open Access Journals (Sweden)

    Benjamin P Hurrell

    2015-05-01

    Full Text Available The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing.

  19. Semi-Automatic Rating Method for Neutrophil Alkaline Phosphatase Activity.

    Science.gov (United States)

    Sugano, Kanae; Hashi, Kotomi; Goto, Misaki; Nishi, Kiyotaka; Maeda, Rie; Kono, Keigo; Yamamoto, Mai; Okada, Kazunori; Kaga, Sanae; Miwa, Keiko; Mikami, Taisei; Masauzi, Nobuo

    2017-01-01

    The neutrophil alkaline phosphatase (NAP) score is a valuable test for the diagnosis of myeloproliferative neoplasms, but it has still manually rated. Therefore, we developed a semi-automatic rating method using Photoshop ® and Image-J, called NAP-PS-IJ. Neutrophil alkaline phosphatase staining was conducted with Tomonaga's method to films of peripheral blood taken from three healthy volunteers. At least 30 neutrophils with NAP scores from 0 to 5+ were observed and taken their images. From which the outer part of neutrophil was removed away with Image-J. These were binarized with two different procedures (P1 and P2) using Photoshop ® . NAP-positive area (NAP-PA) and granule (NAP-PGC) were measured and counted with Image-J. The NAP-PA in images binarized with P1 significantly (P < 0.05) differed between images with NAP scores from 0 to 3+ (group 1) and those from 4+ to 5+ (group 2). The original images in group 1 were binarized with P2. NAP-PGC of them significantly (P < 0.05) differed among all four NAP score groups. The mean NAP-PGC with NAP-PS-IJ indicated a good correlation (r = 0.92, P < 0.001) to results by human examiners. The sensitivity and specificity of NAP-PS-IJ were 60% and 92%, which might be considered as a prototypic method for the full-automatic rating NAP score. © 2016 Wiley Periodicals, Inc.

  20. Chemotaxis of C. elegans in 3D media: a model for navigation of undulatory microswimmers

    Science.gov (United States)

    Patel, Amar; Bilbao, Alejandro; Rahman, Mizanur; Vanapalli, Siva; Blawzdziewicz, Jerzy

    2017-11-01

    While the natural environment of C. elegans consists of complex 3D media (e.g., decomposing organic matter and water), most studies of chemotactic behavior of this nematode are limited to 2D. We present a 3D chemotaxis model that combines a realistic geometrical representation of body movements associated with 3D maneuvers, an analysis of mechanical interactions of the nematode body with the surrounding medium to determine nematode trajectories, and a simple memory-function description of chemosensory apparatus that controls the frequency, magnitude, and timing of turning maneuvers. We show that two main chemotaxis strategies of C. elegans moving in 2D, i.e., the biased random walk and gradual turn, are effective also in 3D, provided that 2D turns are supplemented by the roll maneuvers that enable 3D reorientation. Optimal choices of chemosensing and gait-control parameters are discussed; we show that the nematode can maintain efficient chemotaxis in burrowing and swimming by adjusting the undulation frequency alone, without changing the chemotactic component of the body control. Understanding how C. elegans efficiently navigates in 3D media may help in developing self-navigating artificial microswimmers. Supported by NSF Grant No. CBET 1603627.

  1. 18F-fluoro-2-deoxyglucose PET informs neutrophil accumulation and activation in lipopolysaccharide-induced acute lung injury.

    Science.gov (United States)

    Rodrigues, Rosana S; Bozza, Fernando A; Hanrahan, Christopher J; Wang, Li-Ming; Wu, Qi; Hoffman, John M; Zimmerman, Guy A; Morton, Kathryn A

    2017-05-01

    Molecular imaging of the earliest events related to the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) could facilitate therapeutic development and patient management. We previously reported that 18 F-fluoro-2-deoxyglucose ( 18 F-FDG) PET identifies ALI/ARDS prior to radiographic abnormalities. The purpose of this study was to establish the time courses of 18 F-FDG uptake, edema and neutrophil recruitment in an endotoxin-induced acute lung injury model and to examine molecular events required for 14 C-2DG uptake in activated neutrophils. Lung uptake of 18 F-FDG was measured by PET in control male Sprague Dawley rats and at 2, 6 and 24h following the intraperitoneal injection of 10mg/kg LPS. Lung edema (attenuation) was measured by microCT. Neutrophil influx into the lungs was measured by myeloperoxidase assay. Control and activated human donor neutrophils were compared for uptake of 14 C-2DG, transcription and content of hexokinase and GLUT isoforms and for hexokinase (HK) activity. Significant uptake of 18 F-FDG occurred by 2h following LPS, and progressively increased to 24h. Lung uptake of 18 F-FDG preceded increased CT attenuation (lung edema). Myeloperoxidase activity in the lungs, supporting neutrophil influx, paralleled 18 F-FDG uptake. Activation of isolated human neutrophils resulted in increased uptake of 14 C-2DG, expression of GLUT 3 and GLUT 4 and expression and increased HK1 activity. Systemic endotoxin-induced ALI results in very early and progressive uptake of 18 F-FDG, parallels neutrophil accumulation and occurs earlier than lung injury edema. Activated neutrophils show increased uptake of 14 C-2DG, expression of specific GLUT3, GLUT4 and HK1 protein and HK activity. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: 18 F-FDG pulmonary uptake is an early biomarker of neutrophil recruitment in ALI and is associated with specific molecular events that mediate 14 C-2DG uptake in activated neutrophils. 18 F

  2. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    O’Day, Danton H.; Huber, Robert J.; Suarez, Andres

    2012-01-01

    Highlights: ► Extracellular calmodulin is present throughout growth and development in Dictyostelium. ► Extracellular calmodulin localizes within the ECM during development. ► Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. ► Extracellular calmodulin exists in eukaryotic microbes. ► Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca 2+ /CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  3. Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

    International Nuclear Information System (INIS)

    Ai, Shingo; Cheng Xianwu; Inoue, Aiko; Nakamura, Kae; Okumura, Kenji; Iguchi, Akihisa; Murohara, Toyoaki; Kuzuya, Masafumi

    2007-01-01

    Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation

  4. Interleukin-17A and Neutrophils in a Murine Model of Bird-Related Hypersensitivity Pneumonitis.

    Directory of Open Access Journals (Sweden)

    Masahiro Ishizuka

    Full Text Available Hypersensitivity pneumonitis (HP is an immune mediated lung disease induced by the repeated inhalation of a wide variety of antigens. Bird-related hypersensitivity pneumonitis (BRHP is one of the most common forms of HP in human and results from the inhalation of avian antigens. The findings of a recent clinical analysis suggest that in addition to Th1 factors, the levels of interleukin(IL-17 and IL-17-associated transcripts are increased in the setting of HP, and that both IL-17A and neutrophils are crucial for the development of pulmonary inflammation in murine models of HP. Our objectives were to investigate the roles of IL-17A and neutrophils in granuloma-forming inflammation in an acute HP model. We developed a mouse model of acute BRHP using pigeon dropping extract. We evaluated the process of granuloma formation and the roles of both IL-17A and neutrophils in a model. We found that the neutralization of IL-17A by the antibody attenuated granuloma formation and the recruitment of neutrophils, and also decreased the expression level of chemokine(C-X-C motif ligand 5 (CXCL5 in the acute HP model. We confirmed that most of the neutrophils in the acute HP model exhibited immunoreactivity to the anti-IL-17 antibody. We have identified the central roles of both IL-17A and neutrophils in the pathogenesis of granuloma formation in acute HP. We have also assumed that neutrophils are an important source of IL-17A in an acute HP model, and that the IL-17A-CXCL5 pathway may be responsible for the recruitment of neutrophils.

  5. The complex interplay between neutrophils and cancer.

    Science.gov (United States)

    Rakic, Andrea; Beaudry, Paul; Mahoney, Douglas J

    2018-03-01

    Neutrophils are the most abundant type of white blood cell, and are an essential component of the innate immune system. They characteristically arrive rapidly at sites of infection and injury, and release a variety of cytokines and toxic molecules to eliminate pathogens and elicit an acute inflammatory response. Research into the function of neutrophils in cancer suggest they have divergent roles. Indeed, while most studies have found neutrophils to be associated with cancer progression, others have also documented anticancer effects. In this review, we describe the investigations into neutrophil populations that have been implicated in promoting tumor growth and metastasis as well those demonstrating antitumor functions. The collective research suggests a complex role for neutrophils in cancer biology, which raises the prospect of their targeting for the treatment of cancer.

  6. Identification of glutathione adducts of α-chlorofatty aldehydes produced in activated neutrophils.

    Science.gov (United States)

    Duerr, Mark A; Aurora, Rajeev; Ford, David A

    2015-05-01

    α-Chlorofatty aldehydes (α-ClFALDs) are produced by hypochlorous acid targeting plasmalogens during neutrophil activation. This study investigated the reaction of the α-chlorinated carbon of α-ClFALD with the nucleophile, GSH. Utilizing ESI/MS/MS, the reaction product of GSH and the 16-carbon α-ClFALD, 2-chlorohexadecanal (2-ClHDA), was characterized. The resulting conjugate of 2-ClHDA and GSH (HDA-GSH) has an intact free aldehyde, and the chlorine at the α-carbon is ejected. Stable isotope-labeled [d4]HDA-GSH was synthesized, which further confirmed the structure, and was used to quantify natural α-ClFALD conjugates of GSH (FALD-GSH) using reverse-phase LC with detection by ESI/MS/MS using selected reaction monitoring. HDA-GSH is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2-ClHDA. Furthermore, PMA-treated primary human neutrophils have elevated levels of HDA-GSH and the conjugate of 2-chlorooctadecanal (2-ClODA) and GSH (ODA-GSH), as well as elevated levels of 2-ClHDA and 2-ClODA. Production of both conjugates in PMA-stimulated neutrophils was reduced by 3-aminotriazole pretreatment, which also blocks endogenous α-ClFALD production. Additionally, plasma FALD-GSH levels were elevated in the K/BxN mouse arthritis model. Taken together, these studies demonstrate novel peptidoaldehydes derived from GSH and α-ClFALD in activated human neutrophils and in vivo in K/BxN mice. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  7. Spontaneous neutrophil migration patterns during sepsis after major burns.

    Science.gov (United States)

    Jones, Caroline N; Moore, Molly; Dimisko, Laurie; Alexander, Andrew; Ibrahim, Amir; Hassell, Bryan A; Warren, H Shaw; Tompkins, Ronald G; Fagan, Shawn P; Irimia, Daniel

    2014-01-01

    Finely tuned to respond quickly to infections, neutrophils have amazing abilities to migrate fast and efficiently towards sites of infection and inflammation. Although neutrophils ability to migrate is perturbed in patients after major burns, no correlations have yet been demonstrated between altered migration and higher rate of infections and sepsis in these patients when compared to healthy individuals. To probe if such correlations exist, we designed microfluidic devices to quantify the neutrophil migration phenotype with high precision. Inside these devices, moving neutrophils are confined in channels smaller than the neutrophils and forced to make directional decisions at bifurcations and around posts. We employed these devices to quantify neutrophil migration across 18 independent parameters in 74 blood samples from 13 patients with major burns and 3 healthy subjects. Blinded, retrospective analysis of clinical data and neutrophil migration parameters revealed that neutrophils isolated from blood samples collected during sepsis migrate spontaneously inside the microfluidic channels. The spontaneous neutrophil migration is a unique phenotype, typical for patients with major burns during sepsis and often observed one or two days before the diagnosis of sepsis is confirmed. The spontaneous neutrophil migration phenotype is rare in patients with major burns in the absence of sepsis, and is not encountered in healthy individuals. Our findings warrant further studies of neutrophils and their utility for early diagnosing and monitoring sepsis in patients after major burns.

  8. The impact of odor-reward memory on chemotaxis in larval Drosophila.

    Science.gov (United States)

    Schleyer, Michael; Reid, Samuel F; Pamir, Evren; Saumweber, Timo; Paisios, Emmanouil; Davies, Alexander; Gerber, Bertram; Louis, Matthieu

    2015-05-01

    How do animals adaptively integrate innate with learned behavioral tendencies? We tackle this question using chemotaxis as a paradigm. Chemotaxis in the Drosophila larva largely results from a sequence of runs and oriented turns. Thus, the larvae minimally need to determine (i) how fast to run, (ii) when to initiate a turn, and (iii) where to direct a turn. We first report how odor-source intensities modulate these decisions to bring about higher levels of chemotactic performance for higher odor-source intensities during innate chemotaxis. We then examine whether the same modulations are responsible for alterations of chemotactic performance by learned odor "valence" (understood throughout as level of attractiveness). We find that run speed (i) is neither modulated by the innate nor by the learned valence of an odor. Turn rate (ii), however, is modulated by both: the higher the innate or learned valence of the odor, the less often larvae turn whenever heading toward the odor source, and the more often they turn when heading away. Likewise, turning direction (iii) is modulated concordantly by innate and learned valence: turning is biased more strongly toward the odor source when either innate or learned valence is high. Using numerical simulations, we show that a modulation of both turn rate and of turning direction is sufficient to account for the empirically found differences in preference scores across experimental conditions. Our results suggest that innate and learned valence organize adaptive olfactory search behavior by their summed effects on turn rate and turning direction, but not on run speed. This work should aid studies into the neural mechanisms by which memory impacts specific aspects of behavior. © 2015 Schleyer et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Pathophysiology of neutrophil-mediated extracellular redox reactions.

    Science.gov (United States)

    Jaganjac, Morana; Cipak, Ana; Schaur, Rudolf Joerg; Zarkovic, Neven

    2016-01-01

    Neutrophil granulocyte leukocytes (neutrophils) play fundamental role in the innate immune response. In the presence of adequate stimuli, neutrophils release excessive amount of reactive oxygen species (ROS) that may induce cell and tissue injury. Oxidative burst of neutrophils acts as a double-edged sword. It may contribute to the pathology of atherosclerosis and brain injury but is also necessary in resolving infections. Moreover, neutrophil-derived ROS may also have both a tumor promoting and tumor suppressing role. ROS have a specific activities and diffusion distance, which is related to their short lifetime. Therefore, the manner in which ROS will act depends on the cells targeted and the intra- and extracellular levels of individual ROS, which can further cause production of reactive aldehydes like 4-hydroxynonenal (HNE) that act as a second messengers of ROS. In this review we discuss the influence of neutrophil mediated extracellular redox reactions in ischemia reperfusion injury, transplant rejection and chronic diseases (atherosclerosis, inflammatory bowel diseases and cancer). At the end a brief overview of cellular mechanisms to maintain ROS homeostasis is given.

  10. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence...... and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced...

  11. Chemotaxis and Actin Oscillations

    Science.gov (United States)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  12. The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping.

    Science.gov (United States)

    Bible, Amber; Russell, Matthew H; Alexandre, Gladys

    2012-07-01

    The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche.

  13. Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

    Science.gov (United States)

    Henry, Conor M; Sullivan, Graeme P; Clancy, Danielle M; Afonina, Inna S; Kulms, Dagmar; Martin, Seamus J

    2016-02-02

    Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ~500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  14. IFN-γ alters the expression of diverse immunity related genes in a cell culture model designed to represent maturing neutrophils.

    Directory of Open Access Journals (Sweden)

    Michael A Ellison

    Full Text Available The cytokine interferon-γ (IFN-γ is approved as a drug to treat chronic granulomatous disease (CGD and osteopetrosis and is also used in hyperimmunoglobulin E syndromes. Patients with CGD have defects in proteins of the NOX2 NADPH oxidase system. This leads to reduced production of microbicidal ROS by PMNs and recurrent life threatening infections. The goal of this study was to better understand how IFN-γ might support phagocyte function in these diseases, and to obtain information that might expand potential uses for IFN-γ. Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death with a half-life of only 5-10 hours. Therefore we reasoned that IFN-γ might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells, a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we investigated transcription and protein expression in PLB-985 cells undergoing maturation in the presence or absence of IFN-γ. We observed IFN-γ induced differences in expression of genes known to be involved in classical aspects of neutrophil function (transmigration, chemotaxis, phagocytosis, killing and pattern recognition as well as genes involved in apoptosis and other mechanisms that regulating neutrophil number. We also observed differences for genes involved in the major histocompatibility complex I (MHCI and MHCII systems whose involvement in neutrophil function is controversial and not well defined. Finally, we observed significant changes in expression of genes encoding guanylate binding proteins (Gbps that are known to have roles in immunity but which have not as yet been linked to neutrophil function. We propose that changes in the

  15. Age is the work of art? Impact of neutrophil and organism age on neutrophil extracellular trap formation.

    Science.gov (United States)

    Ortmann, Weronika; Kolaczkowska, Elzbieta

    2018-03-01

    Neutrophil extracellular traps or NETs are released by highly activated neutrophils in response to infectious agents, sterile inflammation, autoimmune stimuli and cancer. In the cells, the nuclear envelop disintegrates and decondensation of chromatin occurs that depends on peptidylarginine deiminase 4 (PAD4) and neutrophil elastase (NE). Subsequently, proteins from neutrophil granules (e.g., NE, lactoferrin and myeloperoxidase) and the nucleus (histones) bind to decondensed DNA and the whole structure is ejected from the cell. The DNA decorated with potent antimicrobials and proteases can act to contain dissemination of infection and in sterile inflammation NETs were shown to degrade cytokines and chemokines via serine proteases. On the other hand, overproduction of NETs, or their inadequate removal and prolonged presence in vasculature or tissues, can lead to bystander damage or even initiation of diseases. Considering the pros and cons of NET formation, it is of relevance if the stage of neutrophil maturation (immature, mature and senescent cells) affects the capacity to produce NETs as the cells of different age-related phenotypes dominate in given (pathological) conditions. Moreover, the immune system of neonates and elderly individuals is weaker than in adulthood. Is the same pattern followed when it comes to NETs? The overall importance of individual and neutrophil age on the capacity to release NETs is reviewed in detail and the significance of these facts is discussed.

  16. Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

    Science.gov (United States)

    Pazos, Michael A; Lanter, Bernard B; Yonker, Lael M; Eaton, Alex D; Pirzai, Waheed; Gronert, Karsten; Bonventre, Joseph V; Hurley, Bryan P

    2017-08-01

    Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

  17. Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

    Directory of Open Access Journals (Sweden)

    Michael A Pazos

    2017-08-01

    Full Text Available Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3, initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4. We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2 activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

  18. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine

    NARCIS (Netherlands)

    Peschel, A.; Jack, R.W.; Otto, M.; Collins, L.V.; Staubitz, P.; Nicholson, G.; Kalbacher, H.; Nieuwenhuizen, W.F.; Jung, G.; Tarkowski, A.; Kessel, K.P.M. van; Strijp, J.A.G. van

    2001-01-01

    Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance

  19. Angiotensin-(1-7 Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

    Directory of Open Access Journals (Sweden)

    Lívia C. Barroso

    2017-11-01

    Full Text Available Defective resolution of inflammation may be crucial for the initiation and development of chronic inflammatory diseases, such as arthritis. Therefore, it has been suggested that therapeutic strategies based on molecules that facilitate inflammation resolution present great potential for the treatment of chronic inflammatory diseases. In this study, we investigated the effects and role of angiotensin-(1-7 [Ang-(1-7] in driving resolution of neutrophilic inflammation in a model of arthritis. For this purpose, male C57BL/6 mice were subjected to antigen-induced arthritis and treated with Ang-(1-7 at the peak of the inflammatory process. Analysis of the number of inflammatory cells, apoptosis, and immunofluorescence for NF-κB was performed in the exudate collected from the knee cavity. Neutrophil accumulation in periarticular tissue was measured by assaying myeloperoxidase activity. Apoptosis of human neutrophil after treatment with Ang-(1-7 was evaluated morphologically and by flow cytometry, and NF-κB phosphorylation by immunofluorescence. Efferocytosis was evaluated in vivo. Therapeutic treatment with Ang-(1-7 at the peak of inflammation promoted resolution, an effect associated with caspase-dependent neutrophils apoptosis and NF-κB inhibition. Importantly, Ang-(1-7 was also able to induce apoptosis of human neutrophils, an effect associated with NF-κB inhibition. The pro-resolving effects of Ang-(1-7 were inhibited by the Mas receptor antagonist A779. Finally, we showed that Ang-(1-7 increased the efferocytic ability of murine macrophages. Our results clearly demonstrate that Ang-(1-7 resolves neutrophilic inflammation in vivo acting in two key step of resolution: apoptosis of neutrophils and their removal by efferocytosis. Ang-(1-7 is a novel mediator of resolution of inflammation.

  20. Mkit: A Cell Migration Assay Based on Microfluidic Device and Smartphone

    Science.gov (United States)

    Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

    2017-01-01

    Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications, we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. PMID:28772229

  1. Chemotaxis of Dictyostelium discoideum: collective oscillation of cellular contacts.

    Directory of Open Access Journals (Sweden)

    Edith Schäfer

    Full Text Available Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.

  2. Role of oncogene 24p3 neutrophil gelatinase-associated lipocalin (NGAL) in digestive system cancers.

    Science.gov (United States)

    Michalak, Łukasz; Bulska, Magdalena; Kudłacz, Katarzyna; Szcześniak, Piotr

    2016-01-04

    Neutrophil gelatinase-associated lipocalin, known also as 24p3 lipocalin, lipocalin-2 or uterocalin (in mouse), is a small secretory protein binding small molecular weight ligands which takes part in numerous processes including apoptosis induction in leukocytes, iron transport, smell, and prostaglandins and retinol transport [19]. It was discovered in activated neutrophils as a covalent peptide associated with human gelatinase neutrophils [7]. Neutrophil lipocalin is secreted physiologically in the digestive system, respiratory tract, renal tubular cells, liver or immunity system. Systematic (circulated in plasma) neutrophil gelatinase come from multiple sources; it may be synthesized in the liver, secreted from activated neutrophils or macrophages, or derive from atherosclerosis or inflammatory endothelial cells [17]. NGAL is stored secondarily in granulates with lactoferrin, calprotectin or MAC-1, which take part in neutrophils' action and migration [13,19]. NGAL participates in acute and chronic inflammation (production of NGAL is indicated by factors conducive to cancer progression) [13,21]. NGAL levels increase in inflammatory or endothelial damage. NGAL level is measured in blood or urine. It is known as a kidney failure factor [7,20]. NGAL is therefore one of the most promising new generation biomarkers in clinical nephrology [6]. The role of NGAL in digestive system neoplasms has not been explored in detail. However, overexpression of this marker was proved in neoplasms such as esophageal carcinoma, stomach cancer, pancreatic cancer or colon cancer, which may indicate an association between concentration and neoplasm [3].

  3. Cryptococcus neoformans modulates extracellular killing by neutrophils

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    Asfia eQureshi

    2011-09-01

    Full Text Available We recently established a key role for host sphingomyelin synthase (SMS in the regulation of the killing activity of neutrophils against Cryptococcus neoformans. In this work, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and NK cells (Tgε26 mice. To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike C. albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. Next, we monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the medium and found that pre-incubation with live but not heat-killed fungal cells significantly inhibits further killing activity of the medium. We next studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption-ionization (MALDI tissue imaging in infected lung we found that similarly to previous observations in the isogenic wild type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells.

  4. Bacterial lipoprotein delays apoptosis in human neutrophils through inhibition of caspase-3 activity: regulatory roles for CD14 and TLR-2.

    LENUS (Irish Health Repository)

    Power, Colm P

    2012-02-03

    The human sepsis syndrome resulting from bacterial infection continues to account for a significant proportion of hospital mortality. Neutralizing strategies aimed at individual bacterial wall products (such as LPS) have enjoyed limited success in this arena. Bacterial lipoprotein (BLP) is a major constituent of the wall of diverse bacterial forms and profoundly influences cellular function in vivo and in vitro, and has been implicated in the etiology of human sepsis. Delayed polymorphonuclear cell (PMN) apoptosis is a characteristic feature of human sepsis arising from Gram-negative or Gram-positive bacterial infection. Bacterial wall product ligation and subsequent receptor-mediated events upstream of caspase inhibition in neutrophils remain incompletely understood. BLP has been shown to exert its cellular effects primarily through TLR-2, and it is now widely accepted that lateral associations with the TLRs represent the means by which CD14 communicates intracellular messages. In this study, we demonstrate that BLP inhibits neutrophil mitochondrial membrane depolarization with a subsequent reduction in caspase-3 processing, ultimately leading to a significant delay in PMN apoptosis. Pretreatment of PMNs with an anti-TLR-2 mAb or anti-CD14 mAb prevented BLP from delaying PMN apoptosis to such a marked degree. Combination blockade using both mAbs completely prevented the effects of BLP (in 1 and 10 ng\\/ml concentrations) on PMN apoptosis. At higher concentrations of BLP, the antiapoptotic effects were observed, but were not as pronounced. Our findings therefore provide the first evidence of a crucial role for both CD14 and TLR-2 in delayed PMN apoptosis arising from bacterial infection.

  5. Contribution of neutrophils to acute lung injury.

    Science.gov (United States)

    Grommes, Jochen; Soehnlein, Oliver

    2011-01-01

    Treatment of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), remain unsolved problems of intensive care medicine. ALI/ARDS are characterized by lung edema due to increased permeability of the alveolar-capillary barrier and subsequent impairment of arterial oxygenation. Lung edema, endothelial and epithelial injury are accompanied by an influx of neutrophils into the interstitium and broncheoalveolar space. Hence, activation and recruitment of neutrophils are regarded to play a key role in progression of ALI/ARDS. Neutrophils are the first cells to be recruited to the site of inflammation and have a potent antimicrobial armour that includes oxidants, proteinases and cationic peptides. Under pathological circumstances, however, unregulated release of these microbicidal compounds into the extracellular space paradoxically can damage host tissues. This review focuses on the mechanisms of neutrophil recruitment into the lung and on the contribution of neutrophils to tissue damage in ALI.

  6. Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

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    Jitka Y. Sagiv

    2015-02-01

    Full Text Available Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs. LDNs consist of both immature myeloid-derived suppressor cells (MDSCs and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

  7. A cheZ-Like Gene in Azorhizobium caulinodans Is a Key Gene in the Control of Chemotaxis and Colonization of the Host Plant.

    Science.gov (United States)

    Liu, Xiaolin; Liu, Wei; Sun, Yu; Xia, Chunlei; Elmerich, Claudine; Xie, Zhihong

    2018-02-01

    Chemotaxis can provide bacteria with competitive advantages for survival in complex environments. The CheZ chemotaxis protein is a phosphatase, affecting the flagellar motor in Escherichia coli by dephosphorylating the response regulator phosphorylated CheY protein (CheY∼P) responsible for clockwise rotation. A cheZ gene has been found in Azorhizobium caulinodans ORS571, in contrast to other rhizobial species studied so far. The CheZ protein in strain ORS571 has a conserved motif similar to that corresponding to the phosphatase active site in E. coli The construction of a cheZ deletion mutant strain and of cheZ mutant strains carrying a mutation in residues of the putative phosphatase active site showed that strain ORS571 participates in chemotaxis and motility, causing a hyperreversal behavior. In addition, the properties of the cheZ deletion mutant revealed that ORS571 CheZ is involved in other physiological processes, since it displayed increased flocculation, biofilm formation, exopolysaccharide (EPS) production, and host root colonization. In particular, it was observed that the expression of several exp genes, involved in EPS synthesis, was upregulated in the cheZ mutant compared to that in the wild type, suggesting that CheZ negatively controls exp gene expression through an unknown mechanism. It is proposed that CheZ influences the Azorhizobium -plant association by negatively regulating early colonization via the regulation of EPS production. This report established that CheZ in A. caulinodans plays roles in chemotaxis and the symbiotic association with the host plant. IMPORTANCE Chemotaxis allows bacteria to swim toward plant roots and is beneficial to the establishment of various plant-microbe associations. The level of CheY phosphorylation (CheY∼P) is central to the chemotaxis signal transduction. The mechanism of the signal termination of CheY∼P remains poorly characterized among Alphaproteobacteria , except for Sinorhizobium meliloti , which

  8. Macrophage migration inhibitory factor triggers chemotaxis of CD74+CXCR2+ NKT cells in chemically induced IFN-γ-mediated skin inflammation.

    Science.gov (United States)

    Hsieh, Chia-Yuan; Chen, Chia-Ling; Lin, Yee-Shin; Yeh, Trai-Ming; Tsai, Tsung-Ting; Hong, Ming-Yuan; Lin, Chiou-Feng

    2014-10-01

    IFN-γ mediates chemically induced skin inflammation; however, the mechanism by which IFN-γ-producing cells are recruited to the sites of inflammation remains undefined. Secretion of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, from damaged cells may promote immune cell recruitment. We hypothesized that MIF triggers an initial step in the chemotaxis of IFN-γ-producing cells in chemically induced skin inflammation. Using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears, MIF expression was examined, and its role in this process was investigated pharmacologically. The cell populations targeted by MIF, their receptor expression patterns, and the effects of MIF on cell migration were examined. TPA directly caused cytotoxicity accompanied by MIF release in mouse ear epidermal keratinocytes, as well as in human keratinocytic HaCaT cells. Treatment with the MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester considerably attenuated TPA-induced ear swelling, leukocyte infiltration, epidermal cell proliferation, and dermal angiogenesis. Inhibition of MIF greatly diminished the dermal infiltration of IFN-γ(+) NKT cells, whereas the addition of exogenous TPA and MIF to NKT cells promoted their IFN-γ production and migration, respectively. MIF specifically triggered the chemotaxis of NKT cells via CD74 and CXCR2, and the resulting depletion of NKT cells abolished TPA-induced skin inflammation. In TPA-induced skin inflammation, MIF is released from damaged keratinocytes and then triggers the chemotaxis of CD74(+)CXCR2(+) NKT cells for IFN-γ production. Copyright © 2014 by The American Association of Immunologists, Inc.

  9. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

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    Ledderose Carola

    2011-10-01

    Full Text Available Abstract Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR. This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells, 7 (neutrophils or 8 (unselected leukocytes potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

  10. Hidden truth of circulating neutrophils (polymorphonuclear neutrophil function in periodontally healthy smoker subjects

    Directory of Open Access Journals (Sweden)

    Chitra Agarwal

    2016-01-01

    Full Text Available Context: Tobacco smoking is considered to be a major risk factor associated with periodontal disease. Smoking exerts a major effect on the protective elements of the immune response, resulting in an increase in the extent and severity of periodontal destruction. Aims: The aim of the present study was to assess viability and phagocytic function of neutrophils in circulating blood of the smokers and nonsmokers who are periodontally healthy. Settings and Design: Two hundred subjects in the mean range of 20–30 years of age were included in the study population. It was a retrospective study carried out for 6 months. Materials and Methods: Two hundred subjects were divided into four groups: 50 nonsmokers, 50 light smokers (15 cigarettes/day. Full mouth plaque index, sulcus bleeding index, and probing depths were measured. Percentage viability of circulating neutrophils and average number of phagocytosed Candida albicans were recorded. Statistical Analysis Used: Means and standard deviations were calculated from data obtained within the groups. Comparison between the smokers and nonsmokers was performed by Kruskal–Wallis ANOVA analysis. Comparison between smoker groups was performed using Mann–Whitney–Wilcoxon test. Results: Percentage viability of neutrophils was significantly less in heavy smokers (66.9 ± 4.0, moderate (76.6 ± 4.2, light smokers (83.1 ± 2.5 as compared to nonsmokers (92.3 ± 2.6 (P < 0.01. The ability of neutrophils to phagocytose, i.e., mean particle number was significantly less in light smokers (3.5 ± 0.5, moderate smokers (2.3 ± 0.5, and heavy smokers (1.4 ± 0.5 compared to nonsmokers (4.9 ± 0.7 (P < 0.01 with evidence of dose-response effect. Conclusions: Smoking significantly affects neutrophils viability and phagocytic function in periodontally healthy population.

  11. Suppression of neutrophil accumulation in mice by cutaneous application of geranium essential oil

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    Oshima Haruyuki

    2005-02-01

    Full Text Available Abstract Background Previous studies suggested that essential oils suppressed the adherence response of human neutrophils in vitro and that intraperitoneal application of geranium oil suppressed the neutrophil accumulation into peritoneal cavity in vivo. Usually, essential oils are applied through skin in aromatherapy in inflammatory symptoms. The purpose of this study is to assess the effects of cutaneous application of essential oils on the accumulation of neutrophils in inflammatory sites in skin of mice. Methods Inflammation with accumulation of inflammatory cells was induced by injection of curdlan, a (1→3-β-D-glucan in skin or peritoneal cavity of mice. Essential oils were applied cutaneously to the mice immediately and 3 hr after intradermal injection of curdlan. The skin with inflammatory lesion was cut off 6 hr after injection of curdlan, and the homogenates were used for myeloperoxidase (MPO: a marker enzyme of neutrophil granule assay. Results The MPO activity of the skin lesion induced by curdlan was suppressed dose-dependently by cutaneous application of geranium oil. Other oils such as lavender, eucalyptus and tea tree oils also suppressed the activity, but their activities seemed weaker than geranium. Juniper oil didn't suppress the activity Conclusion Cutaneous application of essential oils, especially geranium oil, can suppress the inflammatory symptoms with neutrophil accumulation and edema.

  12. Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway.

    Science.gov (United States)

    Takeda, Kosuke; Shao, Danying; Adler, Micha; Charest, Pascale G; Loomis, William F; Levine, Herbert; Groisman, Alex; Rappel, Wouter-Jan; Firtel, Richard A

    2012-01-03

    Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein-coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase-activating protein acting as a global inhibitor.

  13. Advanced Role of Neutrophils in Common Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Jinping Liu

    2017-01-01

    Full Text Available Respiratory diseases, always being a threat towards the health of people all over the world, are most tightly associated with immune system. Neutrophils serve as an important component of immune defense barrier linking innate and adaptive immunity. They participate in the clearance of exogenous pathogens and endogenous cell debris and play an essential role in the pathogenesis of many respiratory diseases. However, the pathological mechanism of neutrophils remains complex and obscure. The traditional roles of neutrophils in severe asthma, chronic obstructive pulmonary diseases (COPD, pneumonia, lung cancer, pulmonary fibrosis, bronchitis, and bronchiolitis had already been reviewed. With the development of scientific research, the involvement of neutrophils in respiratory diseases is being brought to light with emerging data on neutrophil subsets, trafficking, and cell death mechanism (e.g., NETosis, apoptosis in diseases. We reviewed all these recent studies here to provide you with the latest advances about the role of neutrophils in respiratory diseases.

  14. β-Glucan induces reactive oxygen species production in human neutrophils to improve the killing of Candida albicans and Candida glabrata isolates from vulvovaginal candidiasis.

    Directory of Open Access Journals (Sweden)

    Patricia de Souza Bonfim-Mendonça

    Full Text Available Vulvovaginal candidiasis (VVC is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent. Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO activity, and the release of tumor necrosis factor α (TNF-α, interleukin-8 (IL-8, IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.

  15. Neutrophil extracellular trap formation in supragingival biofilms.

    Science.gov (United States)

    Hirschfeld, Josefine; Dommisch, Henrik; Skora, Philipp; Horvath, Gabor; Latz, Eicke; Hoerauf, Achim; Waller, Tobias; Kawai, Toshihisa; Jepsen, Søren; Deschner, James; Bekeredjian-Ding, Isabelle

    2015-01-01

    Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins. Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation. Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation. Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter

  16. The peptidomimetic Lau-(Lys-βNSpe)6-NH2 antagonizes formyl peptide receptor 2 expressed in mouse neutrophils

    DEFF Research Database (Denmark)

    Skovbakke, Sarah Line; Winther, Malene; Gabl, Michael

    2016-01-01

    /differences between the human and murine FPR family members is required. Compared to FPR1 and FPR2 expressed by human neutrophils, very little is known about agonist/antagonist recognition patterns for their murine orthologues, but now we have identified two potent and selective formylated peptide agonists (f...... to be devoid of effect on their murine orthologues as determined by their inability to inhibit superoxide release from murine neutrophils upon stimulation with receptor-specific agonists. The Boc-FLFLF peptide was found to be a selective antagonist for Fpr1, whereas the lipidated peptidomimetic Lau...

  17. Neutrophil Microvesicles from Healthy Control and Rheumatoid Arthritis Patients Prevent the Inflammatory Activation of Macrophages

    Directory of Open Access Journals (Sweden)

    Hefin I. Rhys

    2018-03-01

    Full Text Available Microvesicles (MVs are emerging as a novel means to enact cell-to-cell communication in inflammation. Here, we aimed to ascertain the ability of neutrophil-derived MVs to modulate target cell behaviour, the focus being the macrophage.MVs were generated in response to tumour necrosis factor-α, from healthy control neutrophils or those from rheumatoid arthritis patients. MVs were used to stimulate human monocyte-derived macrophages in vitro, or administered intra-articularly in the K/BxN mouse model of arthritis. A macrophage/fibroblast-like synoviocyte co-culture system was used to study the effects of vesicles on the crosstalk between these cells.We demonstrate a direct role for phosphatidylserine and annexin-A1 exposed by the MVs to counteract classical activation of the macrophages, and promote the release of transforming growth factor-β, respectively. Classically-activated macrophages exposed to neutrophil MVs no longer activated fibroblast-like synoviocytes in subsequent co-culture settings. Finally, intra-articular administration of neutrophil MVs from rheumatoid arthritis patients in arthritic mice affected the phenotype of joint macrophages.Altogether these data, with the identification of specific MV determinants, open new opportunities to modulate on-going inflammation in the synovia – mainly by affecting macrophage polarization and potentially also fibroblast-like synoviocytes - through the delivery of autologous or heterologous MVs produced from neutrophils. Keywords: Neutrophils, Macrophages, Vesicles, Rheumatoid arthritis

  18. A computational model for how cells choose temporal or spatial sensing during chemotaxis.

    Science.gov (United States)

    Tan, Rui Zhen; Chiam, Keng-Hwee

    2018-03-01

    Cell size is thought to play an important role in choosing between temporal and spatial sensing in chemotaxis. Large cells are thought to use spatial sensing due to large chemical difference at its ends whereas small cells are incapable of spatial sensing due to rapid homogenization of proteins within the cell. However, small cells have been found to polarize and large cells like sperm cells undergo temporal sensing. Thus, it remains an open question what exactly governs spatial versus temporal sensing. Here, we identify the factors that determines sensing choices through mathematical modeling of chemotactic circuits. Comprehensive computational search of three-node signaling circuits has identified the negative integral feedback (NFB) and incoherent feedforward (IFF) circuits as capable of adaptation, an important property for chemotaxis. Cells are modeled as one-dimensional circular system consisting of diffusible activator, inactivator and output proteins, traveling across a chemical gradient. From our simulations, we find that sensing outcomes are similar for NFB or IFF circuits. Rather than cell size, the relevant parameters are the 1) ratio of cell speed to the product of cell diameter and rate of signaling, 2) diffusivity of the output protein and 3) ratio of the diffusivities of the activator to inactivator protein. Spatial sensing is favored when all three parameters are low. This corresponds to a cell moving slower than the time it takes for signaling to propagate across the cell diameter, has an output protein that is polarizable and has a local-excitation global-inhibition system to amplify the chemical gradient. Temporal sensing is favored otherwise. We also find that temporal sensing is more robust to noise. By performing extensive literature search, we find that our prediction agrees with observation in a wide range of species and cell types ranging from E. coli to human Fibroblast cells and propose that our result is universally applicable.

  19. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

    Science.gov (United States)

    Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B.

    2014-03-01

    Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks `outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

  20. A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

    DEFF Research Database (Denmark)

    Dang, Pham My-Chan; Stensballe, Allan; Boussetta, Tarek

    2006-01-01

    mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM......Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem...

  1. Hypoxia upregulates neutrophil degranulation and potential for tissue injury

    Science.gov (United States)

    Hoenderdos, Kim; Lodge, Katharine M; Hirst, Robert A; Chen, Cheng; Palazzo, Stefano G C; Emerenciana, Annette; Summers, Charlotte; Angyal, Adri; Porter, Linsey; Juss, Jatinder K; O'Callaghan, Christopher; Chilvers, Edwin R

    2016-01-01

    Background The inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown. Methods and results Following exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release. Conclusion Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion. PMID:27581620

  2. 18F-fluoro-2-deoxyglucose PET informs neutrophil accumulation and activation in lipopolysaccharide-induced acute lung injury genetic algorithm

    International Nuclear Information System (INIS)

    Rodrigues, Rosana S.; Bozza, Fernando A.; Hanrahan, Christopher J.; Wang, Li-Ming; Wu, Qi; Hoffman, John M.; Zimmerman, Guy A.; Morton, Kathryn A.

    2017-01-01

    Introduction: Molecular imaging of the earliest events related to the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) could facilitate therapeutic development and patient management. We previously reported that 18 F-fluoro-2-deoxyglucose ( 18 F-FDG) PET identifies ALI/ARDS prior to radiographic abnormalities. The purpose of this study was to establish the time courses of 18 F-FDG uptake, edema and neutrophil recruitment in an endotoxin-induced acute lung injury model and to examine molecular events required for 14 C-2DG uptake in activated neutrophils. Methods: Lung uptake of 18 F-FDG was measured by PET in control male Sprague Dawley rats and at 2, 6 and 24 h following the intraperitoneal injection of 10 mg/kg LPS. Lung edema (attenuation) was measured by microCT. Neutrophil influx into the lungs was measured by myeloperoxidase assay. Control and activated human donor neutrophils were compared for uptake of 14 C-2DG, transcription and content of hexokinase and GLUT isoforms and for hexokinase (HK) activity. Results: Significant uptake of 18 F-FDG occurred by 2 h following LPS, and progressively increased to 24 h. Lung uptake of 18 F-FDG preceded increased CT attenuation (lung edema). Myeloperoxidase activity in the lungs, supporting neutrophil influx, paralleled 18 F-FDG uptake. Activation of isolated human neutrophils resulted in increased uptake of 14 C-2DG, expression of GLUT 3 and GLUT 4 and expression and increased HK1 activity. Conclusion: Systemic endotoxin-induced ALI results in very early and progressive uptake of 18 F-FDG, parallels neutrophil accumulation and occurs earlier than lung injury edema. Activated neutrophils show increased uptake of 14 C-2DG, expression of specific GLUT3, GLUT4 and HK1 protein and HK activity. Advances in knowledge and implications for patient care: 18 F-FDG pulmonary uptake is an early biomarker of neutrophil recruitment in ALI and is associated with specific molecular events that mediate 14

  3. Sinking, merging and stationary plumes in a coupled chemotaxis-fluid model: a high-resolution numerical approach

    KAUST Repository

    Chertock, A.

    2012-02-02

    Aquatic bacteria like Bacillus subtilis are heavier than water yet they are able to swim up an oxygen gradient and concentrate in a layer below the water surface, which will undergo Rayleigh-Taylor-type instabilities for sufficiently high concentrations. In the literature, a simplified chemotaxis-fluid system has been proposed as a model for bio-convection in modestly diluted cell suspensions. It couples a convective chemotaxis system for the oxygen-consuming and oxytactic bacteria with the incompressible Navier-Stokes equations subject to a gravitational force proportional to the relative surplus of the cell density compared to the water density. In this paper, we derive a high-resolution vorticity-based hybrid finite-volume finite-difference scheme, which allows us to investigate the nonlinear dynamics of a two-dimensional chemotaxis-fluid system with boundary conditions matching an experiment of Hillesdon et al. (Bull. Math. Biol., vol. 57, 1995, pp. 299-344). We present selected numerical examples, which illustrate (i) the formation of sinking plumes, (ii) the possible merging of neighbouring plumes and (iii) the convergence towards numerically stable stationary plumes. The examples with stable stationary plumes show how the surface-directed oxytaxis continuously feeds cells into a high-concentration layer near the surface, from where the fluid flow (recurring upwards in the space between the plumes) transports the cells into the plumes, where then gravity makes the cells sink and constitutes the driving force in maintaining the fluid convection and, thus, in shaping the plumes into (numerically) stable stationary states. Our numerical method is fully capable of solving the coupled chemotaxis-fluid system and enabling a full exploration of its dynamics, which cannot be done in a linearised framework. © 2012 Cambridge University Press.

  4. Neutrophilic dermatoses in a patient with collagenous colitis

    Directory of Open Access Journals (Sweden)

    Didac Barco

    2010-01-01

    Full Text Available We report the case of a 75-year old woman with collagenous colitis who presented with erythematous and edematous plaques on the periorbital and eyelid regions, accompanied by oral ulcers. Histopathology showed a dermal neutrophilic infiltrate plus mild septal and lobular panniculitis with lymphocytes, neutrophils and eosinophils. Five years earlier she had presented a flare of papules and vesicles on the trunk, together with oral ulcers; a skin biopsy revealed a neutrophilic dermal infiltrate and Sweet’s syndrome was diagnosed. Both the neutrophilic panniculitis and the Sweet’s syndrome were accompanied by fever, malaise and diarrhea. Cutaneous and intestinal symptoms disappeared with corticoid therapy. The two types of neutrophilic dermatoses that appeared in periods of colitis activity suggest that intestinal and cutaneous manifestations may be related.

  5. Association of microparticles and neutrophil activation with decompression sickness.

    Science.gov (United States)

    Thom, Stephen R; Bennett, Michael; Banham, Neil D; Chin, Walter; Blake, Denise F; Rosen, Anders; Pollock, Neal W; Madden, Dennis; Barak, Otto; Marroni, Alessandro; Balestra, Costantino; Germonpre, Peter; Pieri, Massimo; Cialoni, Danilo; Le, Phi-Nga Jeannie; Logue, Christopher; Lambert, David; Hardy, Kevin R; Sward, Douglas; Yang, Ming; Bhopale, Veena B; Dujic, Zeljko

    2015-09-01

    Decompression sickness (DCS) is a systemic disorder, assumed due to gas bubbles, but additional factors are likely to play a role. Circulating microparticles (MPs)--vesicular structures with diameters of 0.1-1.0 μm--have been implicated, but data in human divers have been lacking. We hypothesized that the number of blood-borne, Annexin V-positive MPs and neutrophil activation, assessed as surface MPO staining, would differ between self-contained underwater breathing-apparatus divers suffering from DCS vs. asymptomatic divers. Blood was analyzed from 280 divers who had been exposed to maximum depths from 7 to 105 meters; 185 were control/asymptomatic divers, and 90 were diagnosed with DCS. Elevations of MPs and neutrophil activation occurred in all divers but normalized within 24 h in those who were asymptomatic. MPs, bearing the following proteins: CD66b, CD41, CD31, CD142, CD235, and von Willebrand factor, were between 2.4- and 11.7-fold higher in blood from divers with DCS vs. asymptomatic divers, matched for time of sample acquisition, maximum diving depth, and breathing gas. Multiple logistic regression analysis documented significant associations (P < 0.001) between DCS and MPs and for neutrophil MPO staining. Effect estimates were not altered by gender, body mass index, use of nonsteroidal anti-inflammatory agents, or emergency oxygen treatment and were modestly influenced by divers' age, choice of breathing gas during diving, maximum diving depth, and whether repetitive diving had been performed. There were no significant associations between DCS and number of MPs without surface proteins listed above. We conclude that MP production and neutrophil activation exhibit strong associations with DCS. Copyright © 2015 the American Physiological Society.

  6. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    Directory of Open Access Journals (Sweden)

    Mark A Little

    Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  7. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    LENUS (Irish Health Repository)

    Little, Mark A

    2012-01-01

    Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  8. Laminar flow assisted anisotropic bacteria absorption for chemotaxis delivery of bacteria-attached microparticle

    Science.gov (United States)

    Huh, Keon; Oh, Darong; Son, Seok Young; Yoo, Hyung Jung; Song, Byeonghwa; Cho, Dong-il Dan; Seo, Jong-Mo; Kim, Sung Jae

    2016-12-01

    The concepts of microrobots has been drawn significant attentions recently since its unprecedented applicability in nanotechnology and biomedical field. Bacteria attached microparticles presented in this work are one of pioneering microrobot technology for self-propulsion or producing kinetic energy from ambient for their motions. Microfluidic device, especially utilizing laminar flow characteristics, were employed for anisotropic attachment of Salmonella typhimurium flagellated chemotactic bacteria to 30 um × 30 um and 50 um × 50 um microparticles that made of biodegradable polymer. Any toxic chemicals or harmful treatments were excluded during the attachment process and it finished within 100 s for the anisotropic attachment. The attachments were directly confirmed by fluorescent intensity changes and SEM visualization. Chemotaxis motions were tracked using aspartate and the maximum velocity of the bacteria-attached microrobot was measured to be 5 um/s which is comparable to prior state of art technologies. This reusable and scalable method could play a key role in chemotaxis delivery of functional microparticles such as drug delivery system.

  9. Effect of Isolation Techniques on Viability of Bovine Blood Neutrophils

    Directory of Open Access Journals (Sweden)

    P. Sláma

    2006-01-01

    Full Text Available The effect of selected isolation methods on the viability of neutrophil granulocytes (neutrophils from the blood of healthy Holstein x Bohemian Red Pied crossbred heifers was evaluated. Two methods of neutrophil isolation were used: a neutrophil isolation on the basis of hypotonic erythrocyte lysis (in two variants: after the erythrocyte lysis proper, the cells were centrifuged at either 200 g or 1000 g, and b neutrophil isolation with FACS Lysing Solution as the lysing agent. The viability of the isolated neutrophils was evaluated on the basis of apoptosis and necrosis. The results obtained with flow cytometry (FCM suggest that, from the isolation techniques used, the method based on FACS Lysing Solution impaired the neutrophil viability least. After the application of this method, 5.36 ± 2.15% of neutrophils were apoptotic and 0.51 ± 0.12% were necrotic. In contrast, when the hypotonic erythrocyte lysis was used, the proportion of apoptotic neutrophils amounted to 42.14 ± 7.12% and 49.00 ± 14.70%, respectively, and 41.12 ± 5.55% and 36.91 ± 24.38% respectively of necrotic neutrophils (P < 0.01. This was also confirmed by the light microscopy. After the isolation with FASC Lysing Solution, 1.92 ± 1.74% of neutrophils were apoptotic and 1.05 ± 0.76% were necrotic, as distinct from after the hypotonic erythrocyte lysis where 9.43 ± 3.69% of neutrophils were apoptotic and 12.67 ± 4.74% of necrotic after centrifugation at 200 g, while 12.60 ± 4.35 were apoptotic and 14.96 ± 12.64% were necrotic after centrifugation at 1000 g. It follows from the above-mentioned data that hypotonic lysis is not a suitable method for the isolation of neutrophils, as the method itself markedly affects cell viability.

  10. d(− Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

    Directory of Open Access Journals (Sweden)

    Pablo Alarcón

    2017-08-01

    Full Text Available Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(− lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(− lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(− lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET production (NETosis in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(− lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(− lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1. d(− lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(− lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(− lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

  11. Enhanced {sup 18}F-FDG uptake in activated neutrophils is unaffected by respiratory burst inhibition with RGD

    Energy Technology Data Exchange (ETDEWEB)

    Paik, J. Y.; Lee, K. H.; Go, B. H.; Jeong, K. H.; Kim, H. K.; Choi, J. S.; Choi, Y.; Kim, P. T [Samsung Medical Center, Seoul (Korea, Republic of)

    2004-07-01

    Respiratory burst generation is an important response of activated neutrophils and is associated with enhanced glucose metabolism. Since such activation in dependent on adhesion through integrins, we investigated how integrin occupation with RGD influences respiratory burst response and {sup 18}F-FDG uptake in neutrophils. Human neutrophils separated from healthy volunteers were incubated in RPMI media. For RGD peptide inhibitory experiments, neutrophils were preincubated with 200 {mu} g/ml of cRGD peptides ad 37.deg. for 2 hr prior. Respiratory burst generation and uptake of {sup 18}F-FDG was then measured with or without PMA stimulation. Cellular total hexokinase levels were assayed with a colorimetric method. Treatment with RGD in the basal state resulted in a significant but relatively small increase in neutrophil superoxide release to 1.5{+-}0.25 fold o control levels (p<0.005). Whereas PMA stimulation caused a marked increase in superoxide generation, pretreatment with RGD caused a significant attenuation of this response to 35.6{+-}0.2% (p<0.005). PMA stimulation resulted in a significant increase in {sup 18}F-FDG uptake. However, unlike the attenution of superoxide generation, neutrophils pretreated with RGD before PMA stimulation showed an identical magnitude of enhanced {sup 18}F-FDG uptake (201.8{+-}20.5 of controls, p=0.0001). In addition, hexokinase levels were increased to comparable levels of approximately 1.5 fold for PMA stimulated neutrophils irrespective of RGD pretreatment. In conclusion, soluble RGD blocks stimulation of respiratory burst activation in neutrophils but does not inhibit stimulation of cellular glucose metabolism. This dissociation may contribute to maximally enhanced neutrophil FDG uptake in inflammatory lesions regardless of the occupancy of their integrin receptors.

  12. Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

    Science.gov (United States)

    Ko, Hyun-Kyung; Guo, Li-wu; Su, Bing; Gao, Lingqiu; Gelman, Irwin H

    2014-01-01

    Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the

  13. The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

    LENUS (Irish Health Repository)

    Hayes, Elaine

    2012-02-01

    Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

  14. The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

    LENUS (Irish Health Repository)

    Hayes, Elaine

    2011-04-01

    Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

  15. Different innate neutrophil responses in controlled and uncontrolled asthma

    NARCIS (Netherlands)

    Tang, Francesca; Foxley, Gloria; Gibson, Peter; Burgess, Janette; Baines, Katherine; Oliver, Brian

    2015-01-01

    Introduction: Respiratory viruses are a major cause of asthma exacerbations. Neutrophilic inflammation occurs during infections and is associated with difficult to treat asthma. The role of neutrophils in viral infections and whether neutrophil dysfunction contributes to exacerbation pathogenesis

  16. Gasdermin D Exerts Anti-inflammatory Effects by Promoting Neutrophil Death

    Directory of Open Access Journals (Sweden)

    Hiroto Kambara

    2018-03-01

    Full Text Available Summary: Gasdermin D (GSDMD is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE, released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies. : Kambara et al. find that GSDMD deficiency augments host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, establishing GSDMD as a negative regulator of innate immunity. GSDMD cleavage and activation in neutrophils is mediated by ELANE, released from cytoplasmic granules into the cytosol in aging neutrophils. Keywords: GSDMD, neutrophil death, neutrophil elastase, innate immunity, host defense

  17. High glucose impairs superoxide production from isolated blood neutrophils

    DEFF Research Database (Denmark)

    Perner, A; Nielsen, S E; Rask-Madsen, J

    2003-01-01

    Superoxide (O(2)(-)), a key antimicrobial agent in phagocytes, is produced by the activity of NADPH oxidase. High glucose concentrations may, however, impair the production of O(2)(-) through inhibition of glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the formation of NADPH. This study...... measured the acute effects of high glucose or the G6PD inhibitor dehydroepiandrosterone (DHEA) on the production of O(2)(-) from isolated human neutrophils....

  18. Neutrophils, a candidate biomarker and target for radiation therapy?

    Science.gov (United States)

    Schernberg, Antoine; Blanchard, Pierre; Chargari, Cyrus; Deutsch, Eric

    2017-11-01

    Neutrophils are the most abundant blood-circulating white blood cells, continuously generated in the bone marrow. Growing evidence suggests they regulate the innate and adaptive immune system during tumor evolution. This review will first summarize the recent findings on neutrophils as a key player in cancer evolution, then as a potential biomarker, and finally as therapeutic targets, with respective focuses on the interplay with radiation therapy. A complex interplay: Neutrophils have been associated with tumor progression through multiple pathways. Ionizing radiation has cytotoxic effects on cancer cells, but the sensitivity to radiation therapy in vivo differ from isolated cancer cells in vitro, partially due to the tumor microenvironment. Different microenvironmental states, whether baseline or induced, can modulate or even attenuate the effects of radiation, with consequences for therapeutic efficacy. Inflammatory biomarkers: Inflammation-based scores have been widely studied as prognostic biomarkers in cancer patients. We have performed a large retrospective cohort of patients undergoing radiation therapy (1233 patients), with robust relationship between baseline blood neutrophil count and 3-year's patient's overall survival in patients with different cancer histologies. (Pearson's correlation test: p = .001, r = -.93). Therapeutic approaches: Neutrophil-targeting agents are being developed for the treatment of inflammatory and autoimmune diseases. Neutrophils either can exert antitumoral (N1 phenotype) or protumoral (N2 phenotype) activity, depending on the Tumor Micro Environment. Tumor associated N2 neutrophils are characterized by high expression of CXCR4, VEGF, and gelatinase B/MMP9. TGF-β within the tumor microenvironment induces a population of TAN with a protumor N2 phenotype. TGF-β blockade slows tumor growth through activation of CD8 + T cells, macrophages, and tumor associated neutrophils with an antitumor N1 phenotype. This supports

  19. Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    2016-01-01

    Full Text Available Distinct tumor microenvironment forms in each progression step of cancer and has diverse capacities to induce both adverse and beneficial consequences for tumorigenesis. It is now known that immune cells can be activated to favor tumor growth and progression, most probably influenced by the tumor microenvironment. Tumor-associated macrophages and tumor-associated neutrophils can exert protumoral functions, enhancing tumor cell invasion and metastasis, angiogenesis, and extracellular matrix remodeling, while inhibiting the antitumoral immune surveillance. Considering that neutrophils in inflammatory environments recruit macrophages and that recruited macrophages affect neutrophil functions, there may be various degrees of interaction between tumor-associated macrophages and tumor-associated neutrophils. Platelets also play an important role in the recruitment and regulation of monocytic and granulocytic cells in the tumor tissues, suggesting that platelet function may be essential for generation of tumor-associated macrophages and tumor-associated neutrophils. In this review, we will explore the biology of tumor-associated macrophages and tumor-associated neutrophils and their possible interactions in the tumor microenvironment. Special attention will be given to the recruitment and activation of these tumor-associated cells and to the roles they play in maintenance of the tumor microenvironment and progression of tumors.

  20. Granule protein processing and regulated secretion in neutrophils

    Directory of Open Access Journals (Sweden)

    Avinash eSheshechalam

    2014-09-01

    Full Text Available Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

  1. Mkit: A cell migration assay based on microfluidic device and smartphone.

    Science.gov (United States)

    Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

    2018-01-15

    Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS 2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS 2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS 2 -based cell functional assay for testing cell migration (the M kit ). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the M kit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the M kit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the M kit . In addition to research applications, we demonstrated the effective use of the M kit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed M kit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    International Nuclear Information System (INIS)

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-01-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10 -7 to 10 -4 M), naloxone inhibited (p 2 - ) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O 2 - released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of 3 H FMLP to HN. Using 3 H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10 -5 ) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED 50 for naloxone inhibition of O 2 - (1 x 10 -5 M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D 2 or E 2 . Conclusions: (1) naloxone inhibits FMLP-stimulated O 2 but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN

  3. Effect of moderate exercise on peritoneal neutrophils from juvenile rats.

    Science.gov (United States)

    Braz, Glauber Ruda; Ferreira, Diorginis Soares; Pedroza, Anderson Apolonio; da Silva, Aline Isabel; Sousa, Shirley Maria; Pithon-Curi, Tania Cristina; Lagranha, Claudia

    2015-09-01

    Previous studies showed that moderate exercise in adult rats enhances neutrophil function, although no studies were performed in juvenile rats. We evaluated the effects of moderate exercise on the neutrophil function in juvenile rats. Viability and neutrophils function were evaluated. Moderate exercise did not impair the viability and mitochondrial transmembrane potential of neutrophils, whereas there was greater reactive oxygen species production (164%; p < 0.001) and phagocytic capacity (29%; p < 0.05). Our results suggest that moderate exercise in juvenile rats improves neutrophil function, similar to adults.

  4. Intracellular accumulation of potent amiloride analogues by human neutrophils

    International Nuclear Information System (INIS)

    Simchowitz, L.; Woltersdorf, O.W. Jr.; Cragoe, E.J. Jr.

    1987-01-01

    The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using [ 14 C]amiloride and the 14 C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids

  5. Yersinia pestis subverts the dermal neutrophil response in a mouse model of bubonic plague.

    Science.gov (United States)

    Shannon, Jeffrey G; Hasenkrug, Aaron M; Dorward, David W; Nair, Vinod; Carmody, Aaron B; Hinnebusch, B Joseph

    2013-08-27

    The majority of human Yersinia pestis infections result from introduction of bacteria into the skin by the bite of an infected flea. Once in the dermis, Y. pestis can evade the host's innate immune response and subsequently disseminate to the draining lymph node (dLN). There, the pathogen replicates to large numbers, causing the pathognomonic bubo of bubonic plague. In this study, several cytometric and microscopic techniques were used to characterize the early host response to intradermal (i.d.) Y. pestis infection. Mice were infected i.d. with fully virulent or attenuated strains of dsRed-expressing Y. pestis, and tissues were analyzed by flow cytometry. By 4 h postinfection, there were large numbers of neutrophils in the infected dermis and the majority of cell-associated bacteria were associated with neutrophils. We observed a significant effect of the virulence plasmid (pCD1) on bacterial survival and neutrophil activation in the dermis. Intravital microscopy of i.d. Y. pestis infection revealed dynamic interactions between recruited neutrophils and bacteria. In contrast, very few bacteria interacted with dendritic cells (DCs), indicating that this cell type may not play a major role early in Y. pestis infection. Experiments using neutrophil depletion and a CCR7 knockout mouse suggest that dissemination of Y. pestis from the dermis to the dLN is not dependent on neutrophils or DCs. Taken together, the results of this study show a very rapid, robust neutrophil response to Y. pestis in the dermis and that the virulence plasmid pCD1 is important for the evasion of this response. Yersinia pestis remains a public health concern today because of sporadic plague outbreaks that occur throughout the world and the potential for its illegitimate use as a bioterrorism weapon. Since bubonic plague pathogenesis is initiated by the introduction of Y. pestis into the skin, we sought to characterize the response of the host's innate immune cells to bacteria early after

  6. Demodex-associated bacterial proteins induce neutrophil activation.

    LENUS (Irish Health Repository)

    2012-02-01

    Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea thus suggesting a possible role for bacterial proteins in the etiology of this condition. Objectives: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. Methods: Bacterial cells were lysed and proteins were partially purified by AKTA-FPLC. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. Results: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of MMP-9, an enzyme known to degrade collagen and cathelicidin, an antimicrobial peptide. In addition neutrophils exposed to the bacterial proteins demonstrated elevated rates of Il-8 and TNF-alpha production. Conclusions: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

  7. Neutrophils are resistant to Yersinia YopJ/P-induced apoptosis and are protected from ROS-mediated cell death by the type III secretion system.

    Directory of Open Access Journals (Sweden)

    Justin L Spinner

    2010-02-01

    Full Text Available The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis and YopP (Y. enterocolitica rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS and cell death. PMN reactive oxygen species (ROS production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

  8. Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice: Implications for Superficial Erosion.

    Science.gov (United States)

    Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K; Swirski, Filip K; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J; Libby, Peter

    2017-06-23

    Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis ( P <0.05). These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era. © 2017 American Heart Association, Inc.

  9. Interval and continuous exercise regimens suppress neutrophil-derived microparticle formation and neutrophil-promoted thrombin generation under hypoxic stress.

    Science.gov (United States)

    Chen, Yi-Ching; Ho, Ching-Wen; Tsai, Hsing-Hua; Wang, Jong-Shyan

    2015-04-01

    Acute hypoxic exposure increases vascular thrombotic risk. The release of procoagulant-rich microparticles from neutrophils accelerates the pathogenesis of inflammatory thrombosis. The present study explicates the manner in which interval and continuous exercise regimens affect neutrophil-derived microparticle (NDMP) formation and neutrophil/NDMP-mediated thrombin generation (TG) under hypoxic condition. A total of 60 sedentary males were randomized to perform either aerobic interval training [AIT; 3-min intervals at 40% and 80% V̇O2max (maximal O2 consumption)] or moderate continuous training (MCT; sustained 60% V̇O2max) for 30 min/day, 5 days/week for 5 weeks, or to a control (CTL) group who did not receive any form of training. At rest and immediately after hypoxic exercise test (HE, 100 W under 12% O2 for 30 min), the NDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. Before the intervention, HE (i) elevated coagulant factor VIII/fibrinogen concentrations and shortened activated partial thromboplastin time (aPTT), (ii) increased total and tissue factor (TF)-rich/phosphatidylserine (PS)-exposed NDMP counts and (iii) enhanced the peak height and rate of TG promoted by neutrophils/NDMPs. Following the 5-week intervention, AIT exhibited higher enhancement of V̇O2max than did MCT. Notably, both MCT and AIT attenuated the extents of HE-induced coagulant factor VIII/fibrinogen elevations and aPTT shortening. Furthermore, the two exercise regimens significantly decreased TF-rich/PS-exposed NDMP formation and depressed neutrophil/NDMP-mediated dynamic TG at rest and following HE. Hence, we conclude that AIT is superior to MCT for enhancing aerobic capacity. Moreover, either AIT or MCT effectively ameliorates neutrophil/NDMP-promoted TG by down-regulating expression of procoagulant factors during HE, which may reduce thrombotic risk evoked by hypoxia. Moreover, either AIT or MCT effectively ameliorates neutrophil

  10. Neutrophils, dendritic cells and Toxoplasma.

    Science.gov (United States)

    Denkers, Eric Y; Butcher, Barbara A; Del Rio, Laura; Bennouna, Soumaya

    2004-03-09

    Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.

  11. Neutrophil heterogeneity: implications for homeostasis and pathogenesis

    NARCIS (Netherlands)

    Silvestre-Roig, Carlos; Hidalgo, Andres; Soehnlein, Oliver

    2016-01-01

    Neutrophils are polymorphonuclear leukocytes of the phagocytic system that act as first line of host defense against invading pathogens but are also important mediators of inflammation-induced injury. In contrast to other members of the innate immune system, neutrophils are classically considered a

  12. Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils

    International Nuclear Information System (INIS)

    Della Bianca, V.; Grzeskowiak, M.; Lissandrini, D.; Rossi, F.

    1991-01-01

    The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: (1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of [3H]alkyl-phosphatidic acid [( 3H]alkyl-PA) and [3H]alkyl-diglyceride [( 3H]alkyl-DG) in cells labeled with [3H]alkyl-lyso-phosphatidylcholine; (2) in the presence of ethanol [3H]alkyl-phosphatidylethanol was formed, and accumulation of [3H]alkyl-PA and [3H]alkyl-DG was depressed; (3) propranolol, by inhibiting the dephosphorylation of [3H]alkyl-PA, completely inhibited the accumulation of [3H]alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: (a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; (b) these two processes were insensitive to staurosporine

  13. Neutrophils in Homeostasis, Immunity, and Cancer.

    Science.gov (United States)

    Nicolás-Ávila, José Ángel; Adrover, José M; Hidalgo, Andrés

    2017-01-17

    Neutrophils were among the first leukocytes described and visualized by early immunologists. Prominent effector functions during infection and sterile inflammation classically placed them low in the immune tree as rapid, mindless aggressors with poor regulatory functions. This view is currently under reassessment as we uncover new aspects of their life cycle and identify transcriptional and phenotypic diversity that endows them with regulatory properties that extend beyond their lifetime in the circulation. These properties are revealing unanticipated roles for neutrophils in supporting homeostasis, as well as complex disease states such as cancer. We focus this review on these emerging functions in order to define the true roles of neutrophils in homeostasis, immunity, and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Neutrophilic dermatoses in a patient with collagenous colitis

    OpenAIRE

    Didac Barco; Maria A. Barnadas; Esther Roé; Francisco J. Sancho; Elena Ricart; Agustín Alomar

    2010-01-01

    We report the case of a 75-year old woman with collagenous colitis who presented with erythematous and edematous plaques on the periorbital and eyelid regions, accompanied by oral ulcers. Histopathology showed a dermal neutrophilic infiltrate plus mild septal and lobular panniculitis with lymphocytes, neutrophils and eosinophils. Five years earlier she had presented a flare of papules and vesicles on the trunk, together with oral ulcers; a skin biopsy revealed a neutrophilic dermal infiltrate...

  15. Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; De Carli, Geraldo Attilio; Bonan, Carla Denise; Tasca, Tiana

    2012-03-01

    Trichomonas vaginalis is a parasite from the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. The neutrophil infiltration has been considered to be primarily responsible for cytological changes observed at infection site, and the chemoattractants can play an important role in this leukocytic recruitment. Nitric oxide (NO) is one of the most widespread mediator compounds, and it is implicated in modulation of immunological mechanisms. Extracellular nucleotides and nucleosides are signaling molecules involved in several processes, including immune responses and control of leukocyte trafficking. Ectonucleoside triphosphate diphosphohydrolase members, ecto-5'-nucleotidase, and adenosine deaminase (ectoADA) have been characterized in T. vaginalis. Herein, we investigated the effects of purinergic system on NO production by neutrophils stimulated with T. vaginalis. The trophozoites were able to induce a high NO synthesis by neutrophils through iNOS pathway. The extracellular nucleotides ATP, ADP, and ATPγS (a non-hydrolyzable ATP analog) showed no significant change in NO secretion. In contrast, adenosine and its degradation product, inosine, promoted a low production of the compound. The immunosuppressive effect of adenosine upon NO release by neutrophils occurred due to adenosine A(2A) receptor activation. The ecto-5'-nucleotidase activity displayed by T. vaginalis was shown to be important in adenosine generation, indicating the efficiency of purinergic cascade. Our data suggest the influence of purinergic signaling, specifically adenosinergic system, on NO production by neutrophils in T. vaginalis infection, contributing to the immunological aspects of disease.

  16. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

    Science.gov (United States)

    von Brühl, Marie-Luise; Stark, Konstantin; Steinhart, Alexander; Chandraratne, Sue; Konrad, Ildiko; Lorenz, Michael; Khandoga, Alexander; Tirniceriu, Anca; Coletti, Raffaele; Köllnberger, Maria; Byrne, Robert A.; Laitinen, Iina; Walch, Axel; Brill, Alexander; Pfeiler, Susanne; Manukyan, Davit; Braun, Siegmund; Lange, Philipp; Riegger, Julia; Ware, Jerry; Eckart, Annekathrin; Haidari, Selgai; Rudelius, Martina; Schulz, Christian; Echtler, Katrin; Brinkmann, Volker; Schwaiger, Markus; Preissner, Klaus T.; Wagner, Denisa D.; Mackman, Nigel; Engelmann, Bernd

    2012-01-01

    Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features. PMID:22451716

  17. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

    Directory of Open Access Journals (Sweden)

    Robert C. Allen

    2015-01-01

    Full Text Available Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2 facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (O2*1 is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism.

  18. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Serena Zilio

    2016-09-01

    Full Text Available Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer.

  19. Systems biology of neutrophil differentiation and immune response

    DEFF Research Database (Denmark)

    Theilgaard-Mönch, Kim; Porse, Bo T; Borregaard, Niels

    2005-01-01

    Systems biology has emerged as a new scientific field, which aims at investigating biological processes at the genomic and proteomic levels. Recent studies have unravelled aspects of neutrophil differentiation and immune responses at the systems level using high-throughput technologies. These stu......Systems biology has emerged as a new scientific field, which aims at investigating biological processes at the genomic and proteomic levels. Recent studies have unravelled aspects of neutrophil differentiation and immune responses at the systems level using high-throughput technologies....... These studies have identified a plethora of novel effector proteins stored in the granules of neutrophils. In addition, these studies provide evidence that neutrophil differentiation and immune response are governed by a highly coordinated transcriptional programme that regulates cellular fate and function...

  20. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

    Directory of Open Access Journals (Sweden)

    María Laura Chiribao

    2014-01-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response, a great number of transcription factors (including the majority of NFκB family members, and host metabolism (cholesterol, fatty acids, and phospholipids. These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

  1. Diverse novel functions of neutrophils in immunity, inflammation, and beyond.

    Science.gov (United States)

    Mócsai, Attila

    2013-07-01

    Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10-20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellular pathogens such as viruses and mycobacteria. They have been shown to intimately shape the adaptive immune response at various levels, including marginal zone B cells, plasmacytoid dendritic cells and T cell populations, and even to control NK cell homeostasis. Neutrophils have been shown to mediate an alternative pathway of systemic anaphylaxis and to participate in allergic skin reactions. Finally, neutrophils were found to be involved in physiological and pathological processes beyond the immune system, such as diabetes, atherosclerosis, and thrombus formation. Many of those functions appear to be related to their unique ability to release neutrophil extracellular traps even in the absence of pathogens. This review summarizes those novel findings on versatile functions of neutrophils and how they change our view of neutrophil biology in health and disease.

  2. Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins in Sinorhizobium meliloti.

    Science.gov (United States)

    Zatakia, Hardik M; Arapov, Timofey D; Meier, Veronika M; Scharf, Birgit E

    2018-03-15

    The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli , the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilis IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better

  3. [The significances of peripheral neutrophils CD(55) and myeloperoxidase expression in patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody associated vasculitis].

    Science.gov (United States)

    Zhou, X L; Zheng, M J; Shuai, Z W; Zhang, L; Zhang, M M; Chen, S Y

    2017-06-01

    Objective: To investigate the expression of CD(55) and myeloperoxidase (MPO) on neutrophils in patients with MPO-specific anti-neutrophil cytoplasmic antibody associated vasculitis(MPO-AAV), and analyze the relationship between the expression and clinical manifestation. Methods: Forty untreated patients with active MPO-AAV (patient group) and 30 healthy volunteers (control group) were enrolled in this study. The CD(55) on neutrophils and both membrane and cytoplasmic MPO were detected by flow cytometry. Serum fragment-from the activated complement factor B(Ba) and MPO were measured by ELISA. The clinical activity of vasculitis was valued by Birmingham vasculitis activity score-version 3(BVAS-V3). The significance of laboratory data was evaluated by Spearman correlation test and multivariate linear regression analysis. Results: (1)The mean fluorescence intensity(MFI) of CD(55) expressed on neutrophils was significantly higher than that in control group[4 068.6±2 306.0 vs 2 999.5±1 504.9, P =0.033]. Similar results of serum MPO and Ba in patient group were found compared to controls [500.0(381.0, 612.7) IU/L vs 286.9(225.5, 329.1) IU/L, P <0.001; 35.2(25.2, 79.5) ng/L vs 18.0(15.0, 28.0) ng/L, P <0.001], respectively. However, MIF of cytoplasmic MPO in patients was significantly lower than that of control group(1 577.1±1 175.9 vs 3 105.3±2 323.0, P =0.003) . (2) In patient group, cytoplasmic intensity of MPO was negatively associated with the serum levels of MPO( r =-0.710, P <0.001) and Ba ( r =-0.589, P =0.001). Moreover, serum MPO was positively associated with serum Ba( r =0.691, P <0.001). Membrane intensity of CD(55) on neutrophils was positively correlated with patient age ( r =0.514, P =0.001), C reactive protein ( r =0.376, P =0.018), peripheral neutrophils count ( r =0.485, P =0.001) and BVAS-V3 ( r =0.484, P =0.002), whereas negative correlation between membrane CD(55) and disease duration was seen ( r =-0.403, P =0.01). (3) The result of multiple

  4. Antiphospholipid Antibodies Promote the Release of Neutrophil Extracellular Traps: A New Mechanism of Thrombosis in the Antiphospholipid Syndrome

    Science.gov (United States)

    Yalavarthi, Srilakshmi; Gould, Travis J.; Rao, Ashish N.; Mazza, Levi F.; Morris, Alexandra E.; Núñez-Álvarez, Carlos; Hernández-Ramírez, Diego; Bockenstedt, Paula L.; Liaw, Patricia C.; Cabral, Antonio R.; Knight, Jason S.

    2015-01-01

    Objective Antiphospholipid antibodies (aPL), especially those targeting beta-2-glycoprotein I (β2GPI), are well known to activate endothelial cells, monocytes, and platelets, with prothrombotic implications. In contrast, the interaction of aPL with neutrophils has not been extensively studied. Neutrophil extracellular traps (NETs) have recently been recognized as an important activator of the coagulation cascade, as well as an integral component of arterial and venous thrombi. Here, we hypothesized that aPL might activate neutrophils to release NETs, thereby predisposing to the arterial and venous thrombosis inherent to the antiphospholipid syndrome (APS). Methods Neutrophils, sera, and plasma were prepared and characterized from patients with primary APS (n=52), or from healthy volunteers. No patient carried a concomitant diagnosis of systemic lupus erythematosus. Results Sera and plasma from patients with primary APS have elevated levels of both cell-free DNA and NETs, as compared to healthy volunteers. Freshly-isolated APS neutrophils are predisposed to high levels of spontaneous NET release. Further, APS-patient sera, as well as IgG purified from APS patients, stimulate NET release from control neutrophils. Human aPL monoclonals, especially those targeting β2GPI, also enhance NET release. The induction of APS NETs can be abrogated with inhibitors of reactive oxygen species formation and toll-like receptor 4 signaling. Highlighting the potential clinical relevance of these findings, APS NETs promote thrombin generation. Conclusion Neutrophil NET release warrants further investigation as a novel therapeutic target in APS. PMID:26097119

  5. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

    Directory of Open Access Journals (Sweden)

    Prajna Jena

    Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

  6. Neutrophils in traumatic brain injury (TBI): friend or foe?

    Science.gov (United States)

    Liu, Yang-Wuyue; Li, Song; Dai, Shuang-Shuang

    2018-05-17

    Our knowledge of the pathophysiology about traumatic brain injury (TBI) is still limited. Neutrophils, as the most abundant leukocytes in circulation and the first-line transmigrated immune cells at the sites of injury, are highly involved in the initiation, development, and recovery of TBI. Nonetheless, our understanding about neutrophils in TBI is obsolete, and mounting evidences from recent studies have challenged the conventional views. This review summarizes what is known about the relationships between neutrophils and pathophysiology of TBI. In addition, discussions are made on the complex roles as well as the controversial views of neutrophils in TBI.

  7. Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

    Science.gov (United States)

    Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K.; Swirski, Filip K.; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J.; Libby, Peter

    2017-01-01

    Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective This study tested in vivo the involvement of disturbed flow, and of neutrophils, hyaluronan, and TLR2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results : In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era. PMID:28428204

  8. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-03-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10/sup -7/ to 10/sup -4/M), naloxone inhibited (p < .001) the release of superoxide (O/sub 2//sup -/) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O/sub 2//sup -/ released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of /sup 3/H FMLP to HN. Using /sup 3/H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10/sup -5/) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED/sub 50/ for naloxone inhibition of O/sub 2//sup -/ (1 x 10/sup -5/M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D/sub 2/ or E/sub 2/. Conclusions: (1) naloxone inhibits FMLP-stimulated O/sub 2/ but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN.

  9. Inhibition of neutrophil migration by aggregated immunoglobulin attached to micropore membranes.

    Science.gov (United States)

    Kemp, A S; Brown, S

    1980-01-01

    The effect of substrate-bound immunoglobulin on neutrophil migration was examined. Immunoglobulin aggregates bound to micropore membranes inhibited the neutrophil response to a chemotactic stimulus. This inhibition was reversed by the presence of aggregates in suspension suggesting competition between substrate-bound and free aggregates for neutrophil surface binding sites. The immobilization of neutrophils by substrate-bound aggregated immunoglobulin suggests a mechanism for the accumulation of neutrophils at sites of immune complex deposition and tissue-bound antibodies in vivo. PMID:7380477

  10. Characterization of Pharmacologic and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study.

    Science.gov (United States)

    Bekker, Pirow; Dairaghi, Daniel; Seitz, Lisa; Leleti, Manmohan; Wang, Yu; Ertl, Linda; Baumgart, Trageen; Shugarts, Sarah; Lohr, Lisa; Dang, Ton; Miao, Shichang; Zeng, Yibin; Fan, Pingchen; Zhang, Penglie; Johnson, Daniel; Powers, Jay; Jaen, Juan; Charo, Israel; Schall, Thomas J

    2016-01-01

    The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773.

  11. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

    Science.gov (United States)

    Ricci-Azevedo, Rafael; Oliveira, Aline Ferreira; Conrado, Marina C A V; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina

    2016-04-01

    ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an

  12. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

    Directory of Open Access Journals (Sweden)

    Rafael Ricci-Azevedo

    2016-04-01

    Full Text Available ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1 immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS, form neutrophil extracellular traps (NETs and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF and interleukin-1beta (IL-1β release; otherwise, transforming growth factor-beta (TGF-β production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be

  13. The effect of midazolam on neutrophil mitogen-activated protein kinase.

    LENUS (Irish Health Repository)

    Ghori, Kamran

    2010-06-01

    Neutrophil p38 mitogen-activated protein kinase (MAPK) is a key enzyme in the intracellular signalling pathway that is responsible for many neutrophil functions, which are important in neutrophil-endothelial interaction. The imidazole compounds are inhibitors of this enzyme system. The objectives of this in-vitro investigation were to examine the effect of midazolam on neutrophil p38 MAPK activation (phosphorylation) following in-vitro ischaemia-reperfusion injury, and the expression of adhesion molecule CD11b\\/CD18.

  14. Down-regulated resistin level in consequence of decreased neutrophil counts in untreated Grave's disease.

    Science.gov (United States)

    Peng, Ying; Qi, Yicheng; Huang, Fengjiao; Chen, Xinxin; Zhou, Yulin; Ye, Lei; Wang, Weiqing; Ning, Guang; Wang, Shu

    2016-11-29

    Resistin, belongs to cysteine-rich secretory protein, is mainly produced by circulating leukocytes, such as neutrophils monocytes and macrophages in humans. To date, few but controversial studies have reported about resistin concentrations in hyperthyroid patients, especially in Graves' disease (GD). We undertaked a controlled, prospective study to explore the serum resistin concentration in GD patients before and after -MMI treatment. In addition, we also investigated the main influencing factor on serum resistin level and discuessed the potential role of serum resistin plays in GD patients. 39 untreated GD (uGD) patients, including 8 males and 31 females, were enrolled in our investigation. All of these patients were prescribed with MMI treatment, in addition to 25 healthy controls. Anthropometric parameters and hormone assessment were measured. Enzyme-linked immunosorbent assay was used to detect serum resistin concentration in different stages of GD patients. Furthermore, neutrophil cell line NB4 with or without T3 treatment to detect the effect of thyroid hormones on resistin expression. The serum resistin level and neutrophil counts in untreated GD patients were significantly declined. And all of these parameters were recovered to normal after MMI treatment in ethyroid GD (eGD) and TRAb-negative conversion (nGD) patients. Resistin concentration exhibited a negative correlation with FT3 and FT4, but a positive correlation with absolute number of neutrophiles in uGD patients, whereas did not correlate with thyroid autoimmune antibodies and BMI. Neutrophile cell line, NB4, produced decreased expression of resistin when stimulated with T3. Our study showed a decrease of serum resistin level in GD patients and we suggested that the serum resistin might primarily secreted from circulating neutrophils and down-regulated by excessive thyroid hormones in GD patients.

  15. Effects of Wharton's jelly-derived mesenchymal stem cells on neonatal neutrophils

    Directory of Open Access Journals (Sweden)

    Khan I

    2014-12-01

    Full Text Available Imteyaz Khan,1 Liying Zhang,2 Moiz Mohammed,1 Faith E Archer,1 Jehan Abukharmah,1 Zengrong Yuan,2 S Saif Rizvi,1 Michael G Melek,1 Arnold B Rabson,1,2 Yufang Shi,2 Barry Weinberger,1 Anna M Vetrano1,21Department of Pediatrics, Division of Neonatology, Rutgers Robert Wood Johnson Medical School, 2Rutgers Child Health Institute of New Jersey, New Brunswick, NJ, USABackground: Mesenchymal stem cells (MSCs have been proposed as autologous therapy for inflammatory diseases in neonates. MSCs from umbilical cord Wharton's jelly (WJ-MSCs are accessible, with high proliferative capacity. The effects of WJ-MSCs on neutrophil activity in neonates are not known. We compared the effects of WJ-MSCs on apoptosis and the expression of inflammatory, oxidant, and antioxidant mediators in adult and neonatal neutrophils.Methods: WJ-MSCs were isolated, and their purity and function were confirmed by flow cytometry. Neutrophils were isolated from cord and adult blood by density centrifugation. The effects of neutrophil/WJ-MSC co-culture on apoptosis and gene and protein expression were measured.Results: WJ-MSCs suppressed neutrophil apoptosis in a dose-dependent manner. WJ-MSCs decreased gene expression of NADPH oxidase-1 in both adult and neonatal neutrophils, but decreased heme oxygenase-1 and vascular endothelial growth factor and increased catalase and cyclooxygenase-2 in the presence of lipopolysaccharide only in adult cells. Similarly, generation of interleukin-8 was suppressed in adult but not neonatal neutrophils. Thus, WJ-MSCs dampened oxidative, vascular, and inflammatory activity by adult neutrophils, but neonatal neutrophils were less responsive. Conversely, Toll-like receptor-4, and cyclooxygenase-2 were upregulated in WJ-MSCs only in the presence of adult neutrophils, suggesting an inflammatory MSC phenotype that is not induced by neonatal neutrophils.Conclusion: Whereas WJ-MSCs altered gene expression in adult neutrophils in ways suggesting anti

  16. Suppressed neutrophil function in children with acute lymphoblastic leukemia.

    Science.gov (United States)

    Tanaka, Fumiko; Goto, Hiroaki; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Naruto, Takuya; Nishimaki, Shigeru; Yokota, Shumpei

    2009-10-01

    Infection is a major obstacle in cancer chemotherapy. Neutropenia has been considered to be the most important risk factor for severe infection; however, other factors, such as impaired neutrophil function, may be involved in susceptibility to infection in patients undergoing chemotherapy. In this study, we analyzed neutrophil function in children with acute lymphoblastic leukemia (ALL). Whole blood samples were obtained from 16 children with ALL at diagnosis, after induction chemotherapy, and after consolidation chemotherapy. Oxidative burst and phagocytic activity of neutrophils were analyzed by flow cytometry. Oxidative burst of neutrophils was impaired in ALL patients. The percentage of neutrophils with normal oxidative burst after PMA stimulation was 59.0 +/- 13.2 or 70.0 +/- 21.0% at diagnosis or after induction chemotherapy, respectively, which was significantly lower compared with 93.8 +/- 6.1% in healthy control subjects (P = 0.00004, or 0.002, respectively); however, this value was normal after consolidation chemotherapy. No significant differences were noted in phagocytic activity in children with ALL compared with healthy control subjects. Impaired oxidative burst of neutrophils may be one risk factor for infections in children with ALL, especially in the initial periods of treatment.

  17. Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

    Directory of Open Access Journals (Sweden)

    Ajantha Nadesalingam

    2018-03-01

    Full Text Available Tonicity of saline (NaCl is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA, Gram-negative bacterial cell wall component lipopolysaccharide (LPS, calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus, and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus. We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12, and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol. This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

  18. Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

    Science.gov (United States)

    Shamloo, Amir

    2014-09-01

    This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

  19. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Vlasova, Irina I., E-mail: irina.vlasova@yahoo.com [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Vakhrusheva, Tatyana V. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Sokolov, Alexey V.; Kostevich, Valeria A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Research Institute for Experimental Medicine, Russian Academy of Medical Science, Saint Petersburg (Russian Federation); Gusev, Alexandr A.; Gusev, Sergey A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Melnikova, Viktoriya I. [Institute of Developmental Biology, Russian Academy of Science, Moscow (Russian Federation); Lobach, Anatolii S. [Institute of Problems of Chemical Physics, Russian Academy of Science, Chernogolovka (Russian Federation)

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H{sub 2}O{sub 2} system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. -- Highlights: ► Myeloperoxidase (MPO) product hypochlorous acid is able to degrade CNTs. ► PEGylated SWCNTs stimulate isolated neutrophils to produce hypochlorous acid. ► SWCNTs are capable of activating neutrophils in blood samples. ► Activation of

  20. Changes in Neutrophil Functions in Astronauts

    Science.gov (United States)

    Kaur, Indreshpal; Simons, Elizabeth R.; Castro, Victoria; Pierson, Duane L.

    2002-01-01

    Neutrophil functions (phagocytosis, oxidative burst, degranulation) and expression of surface markers involved in these functions were studied in 25 astronauts before and after 4 space shuttle missions. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch (preflight or L-10), immediately after landing (landing or R+0), and again at 3 days after landing (postflight or R+3). Blood samples were also collected from 9 healthy low-stressed subjects at 3 time points simulating a 10-day shuttle mission. The number of neutrophils increased at landing by 85 percent when compared to the preflight numbers. Neutrophil functions were studied in whole blood using flow cytometric methods. Phagocytosis of E.coli-FITC and oxidative burst capacity of the neutrophils following the 9 to 11 day missions were lower at all three sampling points than the mean values for control subjects. Phagocytosis and oxidative burst capacity of the astronauts was decreased even 10-days before space flight. Mission duration appears to be a factor in phagocytic and oxidative functions. In contrast, following the short-duration (5-days) mission, these functions were unchanged from control values. No consistent changes in degranulation were observed following either short or medium length space missions. The expression of CD16, CD32, CD11a, CD11b, CD11c, L-selectin and CD36 was measured and found to be variable. Specifically, CD16 and CD32 did not correlate with the changes in oxidative burst and phagocytosis. We can conclude from this study that the stresses associated with space flight can alter the important functions of neutrophils.