Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

Science.gov (United States)

Llewellyn-Jones, C G; Lomas, D A; Stockley, R A

1994-06-01

Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors

International Nuclear Information System (INIS)

Bode, W.; Meyer, E. Jr.; Powers, J.C.

1989-01-01

The serine protease family of enzymes is one of the most widely studied group of enzymes, as evidenced by the fact that more crystal structures are available for individuals of this superfamily than for any other homologous group of enzymes. These enzymes contain a conserved triad of catalytic residues including Ser-195, His-57, and Asp-102. The active-site serine is very nucleophilic, and serine proteases are inhibited by specific serine protease reagents such as diisopropyl phosphorofluoridate (DFP), phenylmethanesulfonyl fluoride, and 3,4-dichloroisocoumarin. Elastases are a group of proteases that possess the ability to cleave the important connective tissue protein elastin. Elastin has the unique property of elastic recoil, is widely distributed in vertebrate tissue, and is particularly abundant in the lungs, arteries, skin, and ligaments. Human neutrophil elastase and pancreatic elastase are two major serine proteases that cleave elastin. Neutrophil elastase is found in the dense granules of polymorphonuclear leukycytes and is essential for phagocytosis and defense against infection by invading microorganisms. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestines where it becomes activated by trypsin and then participates in digestion. Both elastases cleave substrates at peptide bonds where the P 1 residue is an amino acid residue with a small alkyl side chain

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.

In Vitro Activities against Cystic Fibrosis Pathogens of Synthetic Host Defence Propeptides Processed by Neutrophil Elastase.

LENUS (Irish Health Repository)

Desgranges, Stephane

2011-02-22

The antimicrobial and haemolytic activities of a host defence peptide can be controlled by modification as a propeptide of reduced net charge which can be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.

Epithelial-to-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma and Pancreatic Tumor Cell Lines: The Role of Neutrophils and Neutrophil-Derived Elastase

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Thomas Große-Steffen

2012-01-01

Full Text Available Pancreatic ductal adenocarcinoma (PDAC is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN and the tumor cell transition, biopsies of patients with PDAC (n=115 were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and—by implication—to tumor progression is possible.

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...

Gasdermin D Exerts Anti-inflammatory Effects by Promoting Neutrophil Death

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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

Macrophage elastase (MMP-12: a pro-inflammatory mediator?

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Soazig Nénan

2005-03-01

Full Text Available As many metalloproteinases (MMPs, macrophage elastase (MMP-12 is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstrutive pulmonary disease (COPD, have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12 in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

Role of LTB4 in the pathogenesis of elastase-induced murine pulmonary emphysema

Science.gov (United States)

Paige, Mikell; Hanna, Halim; Kim, Su H.; Burdick, Marie D.; Strieter, Robert M.

2010-01-01

Exaggerated levels of the leukotriene B4 (LTB4) frequently coexist at sites of inflammation and tissue remodeling. Therefore, we hypothesize that the LTB4 pathway plays an important role in the pathogenesis of neutrophilic inflammation that contributes to pulmonary emphysema. In this study, significant levels of LTB4 were detected in human lung tissues with emphysema compared with lungs without emphysema (9,497 ± 2,839 vs. 4,142 ± 1,173 pg/ml, n = 9 vs. 10, P = 0.04). To further determine the biological role of LTB4 in the pathogenesis of emphysema, we compared the lungs of wild-type (WT) and LTA4 hydrolase−/− mice (LTB4 deficient, LTA4H−/−) exposed to intranasal elastase or vehicle control. We found that intranasal elastase induced accumulation of LTB4 in the lungs and caused progressively worsening emphysema between 14 and 28 days after elastase exposure in WT mice but not in LTA4H−/− mice. Premortem physiology documented increased lung compliance in elastase-exposed WT mice compared with elastase-exposed LTA4H−/− mice as measured by Flexivent (0.058 ± 0.005 vs. 0.041 ± 0.002 ml/cmH2O pressure). Postmortem morphometry documented increased total lung volume and alveolar sizes in elastase-exposed WT mice compared with elastase-exposed LTA4H−/− mice as measured by volume displacement and alveolar chord length assessment. Furthermore, elastase-exposed LTA4H−/− mice were found to have significantly delayed influx of the CD45highCD11bhighLy6Ghigh leukocytes compatible with neutrophils compared with elastase-exposed WT mice. Mechanistic insights to these phenotypes were provided by demonstrating protection from elastase-induced murine emphysema with neutrophil depletion in the elastase-exposed WT mice and by demonstrating time-dependent modulation of cysteinyl leukotriene biosynthesis in the elastase-exposed LTA4H−/− mice compared with elastase-exposed WT mice. Together, these findings demonstrated that LTB4 played an important role in

Pancreatic elastase in human serum. Determination by radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Geokas, M.C. (Univ. of California, Davis); Brodrick, J.W.; Johnson, J.H.; Largman, C.

1977-01-10

This study demonstrates that a serine endopeptidase of pancreatic origin (elastase 2) circulates in human blood. A specific and highly sensitive radioimmunoassay has been developed for pancreatic elastase 2 in human serum. The inactivation of elastase 2 employed as radioiodinated tracer with an active site-specific reagent (phenylmethanesulfonyl fluoride) was necessary to prevent its binding by serum ..cap alpha../sub 1/-antitrypsin and ..cap alpha../sub 2/-macroglobulin while maintaining its immunoreactivity. The assay is based upon competition of standard human pancreatic elastase 2 with /sup 125/I-labeled phenylmethanesulfonyl elastase 2 for specific antibody binding sites, after which a second antibody precipitation step is used to separate bound from free /sup 125/I-labeled phenylmethanesulfonyl elastase 2. The minimum detectable concentration of elastase 2 was 0.9 ng/ml. The average normal fasting serum level determined was 71 ng/ml, approximately 80-fold greater than the minimum detectable amount.

Elastase-induced emphysema: retention of instilled proteinase in the rat

International Nuclear Information System (INIS)

Sandhaus, R.A.; Janoff, A.

1982-01-01

Airway instillation of proteinases with the ability to degrade elastin has been used to produce disease in the rat analogous to human pulmonary emphysema. This study examined the retention, localization, and fate of endotracheally instilled elastase using 125 I labeled enzyme and immunoperoxidase histochemistry. Porcine pancreatic elastase labeled with 125 I was detected in rat lungs through 96 h after instillation; over half of the label was still present after 7 h. Similar results were obtained when elastase was reacted with a specific, catalytic site inactivator prior to instillation. Trypsin and denatured elastase, however, were cleared much more rapidly from the lung (less than half of the label present after 30 min). When lungs were homogenized after instillation of active elastase, the soluble fraction contained elastase bound to rat alpha1-antitrypsin. In addition, a small amount of label (less than 10%) appeared bound to insoluble components for extended periods of time. Using immunoperoxidase histochemistry, it was found that exogenous elastase was rapidly contained with pulmonary alveolar macrophages, as well as associated with alveolar septums and other parenchymal structures. Similar results were obtained with elastase from both porcine pancreas and human neutrophils. These results suggest that exogenous elastase in the rat, and perhaps endogenous elastolytic enzymes in humans, may have several fates in the lungs: complex formation with endogenous inhibitors, containment within the macrophage, and/or association with connective tissue targets

Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

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Ramesh Tati

2017-02-01

Full Text Available Adequate cleavage of von Willebrand factor (VWF prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3, cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

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

Aggressive re-warming at 38.5 degrees C following deep hypothermia at 21 degrees C increases neutrophil membrane bound elastase activity and pro-inflammatory factor release

NARCIS (Netherlands)

Tang, Min; Zhao, Xiao-gang; He, Yi; Gu, Yan; Mei, Ju

2016-01-01

Background: Cardiopulmonary bypass (CPB) is often performed under hypothermic condition. The effects of hypothermia and re-warming on neutrophil activity are unclear. This study aimed to compare the effects of different hypothermia and re-warming regimens on neutrophil membrane bound elastase (MBE)

Characterization of Total Phenolic Constituents from the Stems of Spatholobus suberectus Using LC-DAD-MSn and Their Inhibitory Effect on Human Neutrophil Elastase Activity

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Yiming Li

2013-06-01

Full Text Available Spatholobus suberectus Dunn, belonging to the legume family (Fabaceae, has been used as a Traditional Chinese Medicine for the treatment of anemia, menoxenia and rheumatism. A limited number of studies report that various types of flavonoids are the main characteristic constituents of this herb. We have now found that S. suberectus contains about 2% phenolic components and characterized the major phenolic components as homogeneous B-type procyanidin conjugates using a liquid chromatography with diode-array detection-ESI mass spectrometry (LC-DAD/ESI-MS method. This is the first report on occurrence of most B-type procyanidins in this herb. Moreover, the total phenolics extract was assayed for inhibitory activity on human neutrophil elastase and its IC50 was found to be 1.33 μg/mL.

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....

The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease as alarm antiproteinases in inflammatory lung disease

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Sallenave Jean-Michel

2000-08-01

Full Text Available Abstract Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.

Eunicellin-based diterpenoids from the Formosan soft coral Klyxum molle with inhibitory activity on superoxide generation and elastase release by neutrophils.

Science.gov (United States)

Lin, Ming-Chang; Chen, Bo-Wei; Huang, Chiung-Yao; Dai, Chang-Feng; Hwang, Tsong-Long; Sheu, Jyh-Horng

2013-09-27

Eleven new eunicellin-based diterpenoids possessing a cladiellane skeleton with a C-2, C-9 ether bridge, klymollins I-S (1-11), have been isolated from the EtOAc extract of the soft coral Klyxum molle from Taiwan waters. The structures of compounds 1-11 were elucidated by extensive spectroscopic analysis, including 2D NMR spectroscopy (COSY, HSQC, HMBC, and NOESY). Compound 5 exhibited cytotoxicity toward several cancer cell lines. Compound 5 is the first eunicellin-based metabolite bearing a phenyl group and displays significant inhibition of both superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophils.

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.

Expression of IL-17A concentration and effector functions of peripheral blood neutrophils in food allergy hypersensitivity patients.

Science.gov (United States)

Żbikowska-Gotz, Magdalena; Pałgan, Krzysztof; Gawrońska-Ukleja, Ewa; Kuźmiński, Andrzej; Przybyszewski, Michał; Socha, Ewa; Bartuzi, Zbigniew

2016-03-01

Lymphocytes Th17 and other types of immune system cells produce IL17. By induction of cytokines and chemokines, the IL17 cytokine is involved in mechanisms of allergic reaction with participation of neutrophil granulocytes. It affects activation, recruitment, and migration of neutrophils to the tissues, regulating inflammatory reaction intensity. Excited neutrophils secrete inter alia elastase and reactive oxygen species (ROS)--significant mediators of inflammation process responsible for tissues damage.The aim of the study was to evaluate the concentrations of serum interleukin 17A, serum neutrophil elastase, and ROS production by neutrophils in patients with food allergy.The study included 30 patients with food allergy diagnosed based on interview, clinical symptoms, positive SPT, placebo controlled double-blind oral provocation trial, and the presence of asIgE in blood serum against selected food allergens using fluoro-immuno-enzymatic method FEIA UNICap 100. The control group consisted of 10 healthy volunteers. The concentrations of IL17A were determined in all patients using ELISA method with eBioscience kits, and elastase using BenderMed Systems kits. Chemiluminescence of non-stimulated neutrophils was evaluated using luminol-dependent kinetic method for 40 min on Luminoskan (Labsystems luminometer).The results of serum IL-17A concentrations and the values of chemiluminescence obtained by non-activated neutrophils, as well as elastase concentrations, were higher in patients with food allergic hypersensitivity compared to healthy volunteers.This study demonstrates a significance of IL-17A and activated neutrophil granulocytes in the course of diseases with food allergic hypersensitivity. © The Author(s) 2015.

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

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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

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice

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Rafael Almeida-Reis

2017-01-01

Full Text Available Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.  C57BL/6 mice received intratracheal elastase (ELA group or saline (SAL group. One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group. Controls received saline and BbCI (SALBC group. After 28 days, we evaluated respiratory mechanics, exhaled nitric oxide, and bronchoalveolar lavage fluid. In lung tissue we measured airspace enlargement, quantified neutrophils, TNFα-, MMP-9-, MMP-12-, TIMP-1-, iNOS-, and eNOS-positive cells, 8-iso-PGF2α, collagen, and elastic fibers in alveolar septa and airways. MUC-5-positive cells were quantified only in airways. Results. BbCI reduced elastase-induced changes in pulmonary mechanics, airspace enlargement and elastase-induced increases in total cells, and neutrophils in BALF. BbCI reduced macrophages and neutrophils positive cells in alveolar septa and neutrophils and TNFα-positive cells in airways. BbCI attenuated elastic and collagen fibers, MMP-9- and MMP-12-positive cells, and isoprostane and iNOS-positive cells in alveolar septa and airways. BbCI reduced MUC5ac-positive cells in airways. Conclusions. BbCI improved lung mechanics and reduced lung inflammation and airspace enlargement and increased oxidative stress levels induced by elastase. BbCI may have therapeutic potential in chronic obstructive pulmonary disease.

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.

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

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.

LENUS (Irish Health Repository)

Guyot, Nicolas

2008-11-21

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.

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

Directory of Open Access Journals (Sweden)

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.

Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling.

Science.gov (United States)

Kristensen, Jacob H; Karsdal, Morten A; Sand, Jannie Mb; Willumsen, Nicholas; Diefenbach, Claudia; Svensson, Birte; Hägglund, Per; Oersnes-Leeming, Diana J

2015-05-03

During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin. Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40). Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.

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

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.

Direct interaction between caffeic acid phenethyl ester and human neutrophil elastase inhibits the growth and migration of PANC-1 cells.

Science.gov (United States)

Duan, Jianhui; Xiaokaiti, Yilixiati; Fan, Shengjun; Pan, Yan; Li, Xin; Li, Xuejun

2017-05-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors of the digestive system, but the mechanisms of its development and progression are unclear. Inflammation is thought to be fundamental to pancreatic cancer development and caffeic acid phenethyl ester (CAPE) is an active component of honey bee resin or propolis with anti-inflammatory and anticancer activities. We investigated the inhibitory effects of CAPE on cell growth and migration induced by human neutrophil elastase (HNE) and report that HNE induced cancer cell migration at low doses and growth at higher doses. In contrast, lower CAPE doses inhibited migration and higher doses of CAPE inhibited the growth induced by HNE. HNE activity was significantly inhibited by CAPE (7.5-120 µM). Using quantitative real-time PCR and western blotting, we observed that CAPE (18-60 µM) did not affect transcription and translation of α1-antitrypsin (α1-AT), an endogenous HNE inhibitor. However, in an in silico drug target docking model, we found that CAPE directly bound to the binding pocket of HNE (25.66 kcal/mol) according to CDOCKER, and the residue of the catalytic site stabilized the interaction between CAPE and HNE as evidenced by molecular dynamic simulation. Response unit (RU) values of surface plasmon resonance (SPR) significantly increased with incremental CAPE doses (7.5-120 µM), indicating that CAPE could directly bind to HNE in a concentration-dependent manner. Thus, CAPE is an effective inhibitor of HNE via direct interaction whereby it inhibits the migration and growth of PANC-1 cells in a dose-dependent manner.

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

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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

A dietary supplement improves facial photoaging and skin sebum, hydration and tonicity modulating serum fibronectin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins.

Science.gov (United States)

Di Cerbo, Alessandro; Laurino, Carmen; Palmieri, Beniamino; Iannitti, Tommaso

2015-03-01

Excessive exposure to the sun can cause severe photoaging as early as the second decade of life resulting in a loss of physiological elastic fiber functions. We designed a first study to assess differences in facial skin pH, sebum, elasticity, hydration and tonicity and serum levels of fibronectin, elastin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins between patients affected by facial photoaging and healthy controls. In a second study we tested the hypothesis that a dietary supplement would improve facial photoaging, also promoting changes in the above mentioned skin and serum parameters. In the first study we enrolled 30 women [age: 47.5 ± 1.6 years (mean ± standard error of the mean)] affected by moderate facial photoaging (4 cm ≤ Visual Analogue Scale (VAS)Skin Tester was used to analyze differences in facial skin parameters between patients affected by facial photoaging and healthy controls. Skin Tester was also used to assess the effect of VISCODERM Pearls on facial skin parameters and compared with placebo 2 weeks after the end of treatment. Serum levels of fibronectin, elastin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins were measured by enzyme-linked immunosorbent assay in the first cohort of patients affected by facial photoaging and healthy controls and, at baseline and 2 weeks after the end of treatment, in the second cohort of patients who underwent treatment with VISCODERM Pearls and placebo. VAS photoaging score was higher in patients affected by photoaging, if compared with healthy controls (p hydration and tonicity were decreased in patients affected by photoaging, if compared with healthy controls (all p hydration and tonicity were increased in the active treatment group vs. placebo (p skin hydration, tonicity and elasticity and increased skin pH and sebum. Treatment with the dietary supplement VISCODERM Pearls significantly improved VAS photoaging score and skin hydration, sebum and tonicity 2 weeks

Elastolytic activity of human blood monocytes characterized by a new monoclonal antibody against human leucocyte elastase. Relationship to rheumatoid arthritis

DEFF Research Database (Denmark)

Jensen, H S; Christensen, L D

1990-01-01

The leucocyte elastase of human blood monocytes was investigated by applying a new monoclonal antibody which did not block the enzyme activity against elastin. In a fixed population of mononuclear cells (MNC) and using fluorescence activated cell sorting (FACS), the human leucocyte elastase (HLE...

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

Elastase-induced emphysema in guinea pigs

International Nuclear Information System (INIS)

Loscutoff, S.M.

1979-01-01

Pulmonary function changes measured in guinea pigs 4 to 5 wk following intratracheal instillation of crystalline porcine pancreatic elastase resembled comparable changes in humans with moderately severe pulmonary emphysema. Compared with saline-treated controls, elastase-treated animals had increased values for all divisions of lung volume, increased static compliance and prolonged time constants. Since humans with emphysema are especially sensitive to air pollutants, elastase-treated animals may be useful as sensitive animal models in inhalatio toxicology

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.

Oenothera paradoxa defatted seeds extract and its bioactive component penta-O-galloyl-β-D-glucose decreased production of reactive oxygen species and inhibited release of leukotriene B4, interleukin-8, elastase, and myeloperoxidase in human neutrophils.

Science.gov (United States)

Kiss, Anna K; Filipek, Agnieszka; Czerwińska, Monika; Naruszewicz, Marek

2010-09-22

In this study, we analyzed ex vivo the effect of an aqueous extract of Oenothera paradoxa defatted seeds on the formation of neutrophil-derived oxidants. For defining active compounds, we also tested lypophilic extract constituents such as gallic acid, (+)-catechin, ellagic acid, and penta-O-galloyl-β-D-glucose and a hydrophilic fraction containing polymeric procyanidins. The anti-inflammatory potential of the extract and compounds was tested by determining the release from activated neutrophils of elastase, myeloperoxidase, interleukin-8 (IL-8), and leukotriene B4 (LTB4), which are considered relevant for the pathogenesis of cardiovascular diseases. The extract of O. paradoxa defatted seeds displays potent antioxidant effects against both 4β-phorbol-12β-myristate-α13-acetate- and formyl-met-leu-phenylalanine-induced reactive oxygen species production in neutrophils with IC50 values around 0.2 μg/mL. All types of polyphenolics present in the extract contributed to the extract antioxidant activity. According to their IC50 values, penta-O-galloyl-β-D-glucose was the more potent constituent of the extract. In cell-free assays, we demonstrated that this effect is partially due to the scavenging of O2- and H2O2 oxygen species. The extract and especially penta-O-galloyl-β-D-glucose significantly inhibit elastase, myeloperoxidase IL-8, and LTB4 release with an IC50 for penta-O-galloyl-β-D-glucose of 17±1, 15±1, 6.5±2.5, and around 20 μM, respectively. The inhibition of penta-O-galloyl-β-D-glucose on reactive oxygen species and especially on O2- production, myeloperoxidase, and chemoattractant release may reduce the interaction of polymorphonuclear leukocyte with the vascular endothelium and by that potentially diminish the risk of progression of atherosclerosis development.

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

Neutrophil extracellular traps in the host defense against sepsis induced by Burkholderia pseudomallei (melioidosis)

NARCIS (Netherlands)

de Jong, Hanna K.; Koh, Gavin C. K. W.; Achouiti, Ahmed; van der Meer, Anne J.; Bulder, Ingrid; Stephan, Femke; Roelofs, Joris J. T. H.; Day, Nick P. J.; Peacock, Sharon J.; Zeerleder, Sacha; Wiersinga, W. Joost

2014-01-01

Neutrophil extracellular traps (NETs) are a central player in the host response to bacteria: neutrophils release extracellular DNA (nucleosomes) and neutrophil elastase to entrap and kill bacteria. We studied the role of NETs in Burkholderia pseudomallei infection (melioidosis), an important cause

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

Neutrophils degrade subendothelial matrices in the presence of alpha-1-proteinase inhibitor. Cooperative use of lysosomal proteinases and oxygen metabolites.

OpenAIRE

Weiss, S J; Regiani, S

1984-01-01

Triggered neutrophils rapidly degraded labeled matrices secreted by cultured, venous endothelial cells via a process dependent on elastase but not oxygen metabolites. In the presence of high concentrations of alpha-1-proteinase inhibitor, the ability of the stimulated neutrophil to solubilize the matrix was impaired. However, at lower concentrations of alpha-1-proteinase inhibitor the neutrophil could enhance the degradative potential of its released elastase by a H2O2-dependent process. Coin...

NETosis Delays Diabetic Wound Healing in Mice and Humans.

Science.gov (United States)

Fadini, Gian Paolo; Menegazzo, Lisa; Rigato, Mauro; Scattolini, Valentina; Poncina, Nicol; Bruttocao, Andrea; Ciciliot, Stefano; Mammano, Fabio; Ciubotaru, Catalin Dacian; Brocco, Enrico; Marescotti, Maria Cristina; Cappellari, Roberta; Arrigoni, Giorgio; Millioni, Renato; Vigili de Kreutzenberg, Saula; Albiero, Mattia; Avogaro, Angelo

2016-04-01

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Primary structure of human pancreatic elastase 2 determined by sequence analysis of the cloned mRNA

International Nuclear Information System (INIS)

Fletcher, T.S.; Shen, W.F.; Largman, C.

1987-01-01

A cDNA encoding elastase 2 has been cloned from a human pancreatic cDNA library. The cDNA contains a translation initiation site and a poly(A) recognition site and encodes a protein of 269 amino acids, including a proposed 16-residue signal peptide. The amino acid sequence of the deduced mature protein contains a 12-residue activation peptide containing a cysteine at residue 1 similar to that of chymotryspin. The proposed active enzyme contains all of the characteristic active-site amino acids, including His-57, Asp-102, and Ser-195. The S1 binding pocket is bounded by Gly-216 and Ser-226, making this pocket intermediate in size between chymotrypsins and elastase 1 or protease E, consistent with the substrate specificity of elastase 2 for long-chain aliphatic or aromatic amino acids. Computer modeling studies using the amino acid sequence of elastase 2 superimposed on the X-ray structure of porcine elastase 1 suggest that a change of Gln-192 in elastase 1 to Asn-192 in elastase 2 may account for the lower catalytic efficiency of the latter enzyme. Several basic residues appear to be near the ends of the extended binding pocket of elastases which might serve to anchor the enzyme to the elastin substrate. These studies indicate that elastases 2 and elastase 1 both contain an Arg-65A as well as a basic dipeptide at 223/224 which is not present in chymotrypsins. In addition, Arg-217A is present in humaan elastase 2 but absent in rat pancreatic protein which has been proposed to be an elastase 2 on the basis of sequence homology, but which was not isolated during screening of rat pancreatic tissue extracts for elastolytic activity

Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

Directory of Open Access Journals (Sweden)

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.

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

Ly6G-mediated depletion of neutrophils is dependent on macrophages.

Science.gov (United States)

Bruhn, Kevin W; Dekitani, Ken; Nielsen, Travis B; Pantapalangkoor, Paul; Spellberg, Brad

2016-01-01

Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.

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

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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

Pathophysiology of neutrophil-mediated extracellular redox reactions.

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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.

Chemokine Receptor Ccr1 Drives Neutrophil-Mediated Kidney Immunopathology and Mortality in Invasive Candidiasis

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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

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

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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.

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

Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L.

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Hernández González, Jorge Enrique; García-Fernández, Rossana; Valiente, Pedro Alberto

2015-01-01

The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, Ki = 2.35·10-8 M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (Ki = 1.3·10-9 M, and Ki = 1.2·10-8 M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor's P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases.

Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases.

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Sullivan, Graeme P; Henry, Conor M; Clancy, Danielle M; Mametnabiev, Tazhir; Belotcerkovskaya, Ekaterina; Davidovich, Pavel; Sura-Trueba, Sylvia; Garabadzhiu, Alexander V; Martin, Seamus J

2018-03-07

Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptide-based pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36β processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.

A Potential Role for Acrolein in Neutrophil-Mediated Chronic Inflammation.

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Noerager, Brett D; Xu, Xin; Davis, Virginia A; Jones, Caleb W; Okafor, Svetlana; Whitehead, Alicia; Blalock, J Edwin; Jackson, Patricia L

2015-12-01

Neutrophils (PMNs) are key mediators of inflammatory processes throughout the body. In this study, we investigated the role of acrolein, a highly reactive aldehyde that is ubiquitously present in the environment and produced endogenously at sites of inflammation, in mediating PMN-mediated degradation of collagen facilitating proline-glycine-proline (PGP) production. We treated peripheral blood neutrophils with acrolein and analyzed cell supernatants and lysates for matrix metalloproteinase-9 (MMP-9) and prolyl endopeptidase (PE), assessed their ability to break down collagen and release PGP, and assayed for the presence of leukotriene A4 hydrolase (LTA4H) and its ability to degrade PGP. Acrolein treatment induced elevated production and functionality of collagen-degrading enzymes and generation of PGP fragments. Meanwhile, LTA4H levels and triaminopeptidase activity declined with increasing concentrations of acrolein thereby sparing PGP from enzymatic destruction. These findings suggest that acrolein exacerbates the acute inflammatory response mediated by neutrophils and sets the stage for chronic pulmonary and systemic inflammation.

Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase

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Irina Kuznetsova

2017-12-01

Full Text Available Oncolytic viruses are currently established as a novel type of immunotherapy. The challenge is to safely target oncolytic viruses to tumors. Previously, we have generated influenza A viruses (IAVs containing deletions in the viral interferon antagonist. Those deletions have attenuated the virus in normal tissue but allowed replication in tumor cells. IAV entry is mediated by hemagglutinin (HA, which needs to be activated by a serine protease, for example, through trypsin. To further target the IAV to tumors, we have changed the trypsin cleavage site to an elastase cleavage site. We chose this cleavage site because elastase is expressed in the tumor microenvironment. Moreover, the exchange of the cleavage site previously has been shown to attenuate viral growth in lungs. Newly generated elastase-activated influenza viruses (AE viruses grew to similar titers in tumor cells as the trypsin-activated counterparts (AT viruses. Intratumoral injection of AE viruses into syngeneic B16f1 melanoma-derived tumors in mice reduced tumor growth similar to AT viruses and had a better therapeutic effect in heterologous human PANC-1-derived tumors. Therefore, the introduction of the attenuation marker “elastase cleavage site” in viral HA allows for safe, effective oncolytic virus therapy.

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

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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.

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.

Heightened systemic levels of neutrophil and eosinophil granular proteins in pulmonary tuberculosis and reversal following treatment.

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Moideen, Kadar; Kumar, Nathella Pavan; Nair, Dina; Banurekha, Vaithilingam V; Bethunaickan, Ramalingam; Babu, Subash

2018-04-09

Granulocytes are activated during tuberculosis (TB) infection and act as immune effector cells and granulocyte responses are implicated in TB pathogenesis. Plasma levels of neutrophil and eosinophil granular proteins provide an indirect measure of degranulation. In this study, we wanted to examine the levels of neutrophil and eosinophil granular proteins in individuals with pulmonary tuberculosis (PTB) and to compare them with the levels in latent TB (LTB) individuals. Hence, we measured the plasma levels of myeloperoxidase (MPO), neutrophil elastase, and proteinase-3; major basic protein (MBP), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) in these individuals. Finally, we also measured the levels of all of these parameters in PTB individuals following anti-tuberculosis (ATT) treatment. Our data reveal that PTB individuals are characterized by significantly higher plasma levels of MPO, elastase, human proteinase 3 as well as MBP and EDN in comparison to LTB individuals. Our data also reveal that ATT resulted in reversal of all of these changes, indicating an association with TB disease. Finally, our data show that the systemic levels of MPO and proteinase-3 can significantly discriminate PTB from LTB individuals. Thus, our data suggest that neutrophil and eosinophil granular proteins could play a potential role in the innate immune response and therefore, the pathogenesis of pulmonary TB. Copyright © 2018 American Society for Microbiology.

The role of neutrophilic mediators in acute inflammation of the gut

International Nuclear Information System (INIS)

Ritter, C. von.

1988-01-01

Activation of granulocytes within the lamina propria by luminally derived bacterial products may represent an important mechanism in the pathogenesis of inflammatory bowel disease. One objective of this thesis was to determine the effects of luminal perfusion with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a bacterial product that attracts and activates granulocytes, on mucosal permeability in different regions of the rat small intestine and colon. Mucosal permeability was measured using the blood-to-lumen clearance of 51 Cr-EDTA during luminal perfusion with FMLP dissolved in Tyrode's solution. Of the bowel segments studied, mucosal permeability was significantly increased only in the distal 10 cm of the ileum. In order to define the role of neutrophilic oxidants in FMLP-induced ileitis, we evaluated the protective effect of several free radical scavengers and antioxidant enzymes. Pretreatment with the either superoxide dismutase or catalase had no effect on the FMLP-induced increase in mucosal permeability. However, treatment with either Mn-desferrioxamine, PZ51, desferrioxamine, or dimethylsulfoxide significantly attenuated FMLP-induced mucosal damage. Non-oxidative toxins released from activated neutrophil may be another mechanism by which FMLP increases mucosal permeability. In order to investigate the role of neutrophilic proteases in FMLP-induced ileitis, the effects of the nonspecific protease inhibitor soybean trypsin inhibitor, and the elastase inhibitors MeOSuc-Ala-Ala-Pro-Val-CH 2 Cl(MAAPV) and Eglin C on the FMLP-induced increases in 51 Cr-EDTA clearance were determined

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

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

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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.

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

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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

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.

Elastase-induced pulmonary emphysema: insights from experimental models

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Mariana A. Antunes

2011-12-01

Full Text Available Several distinct stimuli can be used to reproduce histological and functional features of human emphysema, a leading cause of disability and death. Since cigarette smoke is the main cause of emphysema in humans, experimental researches have attempted to reproduce this situation. However, this is an expensive and cumbersome method of emphysema induction, and simpler, more efficacious alternatives have been sought. Among these approaches, elastolytic enzymes have been widely used to reproduce some characteristics of human cigarette smoke-induced disease, such as: augmentation of airspaces, inflammatory cell influx into the lungs, and systemic inflammation. Nevertheless, the use of elastase-induced emphysema models is still controversial, since the disease pathways involved in elastase induction may differ from those occurring in smoke-induced emphysema. This indicates that the choice of an emphysema model may impact the results of new therapies or drugs being tested. The aim of this review is to compare the mechanisms of disease induction in smoke and elastase emphysema models, to describe the differences among various elastase models, and to establish the advantages and disadvantages of elastase-induced emphysema models. More studies are required to shed light on the mechanisms of elastase-induced emphysema.Diversos estímulos podem ser utilizados para reproduzir características histológicas e funcionais do enfisema humano, uma das principais causas de incapacidade e morte. Uma vez que a fumaça de cigarro é a principal causa de enfisema em humanos, estudos experimentais têm tentado reproduzir esta situação. No entanto, esse é um método dispendioso e complicado para a indução do enfisema e, alternativas mais simples e eficazes, têm sido pesquisadas. Entre essas abordagens, enzimas elastolíticas vêm sendo amplamente utilizadas para reproduzir algumas das características do enfisema humano, tais como: aumento dos espaços a

Elastase-coupled beads as a tool for characterizing localized alveolar tissue destruction associated with the onset of emphysema

Science.gov (United States)

Craig, J. M.; Scott, A. L.

2013-01-01

Intratracheal elastase challenge of laboratory animals has long been established as a model for observing the physiological and morphological changes that result from alveolar destruction, the hallmark of emphysema. However, instillation of elastase suspended in buffer results in widespread inflammation and variable emphysematous lesions, which has made the identification of specific cellular and molecular events associated with the onset of emphysema difficult to define. Here we establish a bead-based elastase delivery system that induces localized tissue destruction, a key event in the initiation of emphysema. Elastase was coupled to bisacrylamide beads, which were shown to retain enzymatic activity prior to intratracheal administration in mice. C57BL/6 mice were given a single dose of 40,000 beads, which became distributed throughout the small airways and parenchyma of the lung. Elastase-coupled beads resulted in a quantifiable loss of alveolar tissue immediately surrounding the beads, an effect that was not observed with beads that lacked protein altogether or with beads containing elastase inactivated by an irreversible inhibitor. Furthermore, beads bound with active elastase elicited local recruitment of mononuclear cells, including macrophages, and polymorphonuclear neutrophils to the site of bead deposition, a feature consistent with the cellular infiltration observed following conventional solubilized elastase challenges. This work identifies a novel bead-based enzyme delivery system that also extends the elastase model of emphysema to permit the characterization of mechanisms that drive alveolar surface area loss following elastin degradation in focal emphysematous lesions. PMID:23558388

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

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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.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection: A New Mechanism of Host Defense.

Science.gov (United States)

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Miller, Derek; Unkel, Ronald; Shaman, Majid; Jacques, Suzanne M; Panaitescu, Bogdan; Garcia-Flores, Valeria; Hassan, Sonia S

2017-08-01

Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

Enhancement by platelets of oxygen radical responses of human neutrophils

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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.

Cutaneous expressions of interleukin-6 and neutrophil elastase as well as levels of serum IgA antibodies to gliadin nonapeptides, tissue transglutaminase and epidermal transglutaminase: implications for both autoimmunity and autoinflammation involvement in dermatitis herpetiformis.

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Gornowicz-Porowska, Justyna; Bowszyc-Dmochowska, Monika; Seraszek-Jaros, Agnieszka; Kaczmarek, Elżbieta; Pietkiewicz, Paweł; Dmochowski, Marian

2014-01-01

Dermatitis herpetiformis (DH) seems to be a chronic immune-mediated inflammatory disease of partially known origin. In light of its known biological functions and its involvement in tissue pathology in other disease states, particularly in nickel-induced allergic contact dermatitis coexisting with DH, it would appear that the central and peripheral response by neutrophils and their mediators (e.g. neutrophil elastase - NE) in DH may be partially mediated by interleukin-6 (IL-6). The aim of the study was to assess the role of IL -6 in DH lesions by examining the relationships between IL -6/NE cutaneous expression and levels of serum anti-nonapeptides of gliadin (npG) IgA, anti-tissue transglutaminase (tTG) immunoglobulin A (IgA), anti-epidermal transglutaminase (eTG) IgA in DH. In total, 24 DH patients having IgA cutaneous deposition were studied. Immunohistochemistry on paraffin-embedded sections with quantitative digital morphometry was used to measure the intensity of IL -6 and NE cutaneous expressions. Levels of serum anti-npG IgA, anti-tTG IgA and anti-eTG IgA were evaluated with ELISA. We found no statistically significant correlation between the NE and IL -6 expression intensities. Our results revealed also a lack of correlations between NE/IL -6 expressions and levels of anti-npG IgA, anti-tTG IgA, anti-eTG IgA in DH. However, the IL -6 expression level was significantly lower than that of NE. The lack of correlations suggested no substantial interactions between IL -6, NE, IgA/npG, IgA/tTG or IgA/eTG in DH. Presented results might indicate the heterogenetic nature of DH pathogenesis suggesting further that both autoimmune and autoinflammatory phenomena may be involved in DH cutaneous pathology.

Hyperglycemia Impairs Neutrophil-Mediated Bacterial Clearance in Mice Infected with the Lyme Disease Pathogen.

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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.

Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

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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.

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines

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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.

IFN-γ protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils

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Savarin Carine

2012-05-01

Full Text Available Abstract Background The interplay between IFN-γ, IL-17 and neutrophils during CNS inflammatory disease is complex due to cross-regulatory factors affecting both positive and negative feedback loops. These interactions have hindered the ability to distinguish the relative contributions of neutrophils, Th1 and Th17 cell-derived effector molecules from secondary mediators to tissue damage and morbidity. Methods Encephalitis induced by a gliatropic murine coronavirus was used as a model to assess the direct contributions of neutrophils, IFN-γ and IL-17 to virus-induced mortality. CNS inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed wild-type (WT or IFN-γ deficient (GKO memory CD4+ T cells into infected SCID mice, coupled with antibody-mediated neutrophil depletion and cytokine blockade. Results Transfer of GKO memory CD4+ T cells into infected SCID mice induced rapid mortality compared to recipients of WT memory CD4+ T cells, despite similar virus control and demyelination. In contrast to recipients of WT CD4+ T cells, extensive neutrophil infiltration and IL-17 expression within the CNS in recipients of GKO CD4+ T cells provided a model to directly assess their contribution(s to disease. Recipients of WT CD4+ T cells depleted of IFN-γ did not express IL-17 and were spared from mortality despite abundant CNS neutrophil infiltration, indicating that mortality was not mediated by excessive CNS neutrophil accumulation. By contrast, IL-17 depletion rescued recipients of GKO CD4+ T cells from rapid mortality without diminishing neutrophils or reducing GM-CSF, associated with pathogenic Th17 cells in CNS autoimmune models. Furthermore, co-transfer of WT and GKO CD4+ T cells prolonged survival in an IFN-γ dependent manner, although IL-17 transcription was not reduced. Conclusions These data demonstrate that IL-17 mediates detrimental clinical consequences in an IFN-γ-deprived environment, independent of

Does human leukocyte elastase degrade intact skin elastin?

DEFF Research Database (Denmark)

Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes

2012-01-01

This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues...... and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage......, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very...

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

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

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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.

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

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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.

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

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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.

Inhibition of NET Release Fails to Reduce Adipose Tissue Inflammation in Mice

NARCIS (Netherlands)

Braster, Quinte; Silvestre Roig, Carlos; Hartwig, Helene; Beckers, Linda; den Toom, Myrthe; Döring, Yvonne; Daemen, Mat J.; Lutgens, Esther; Soehnlein, Oliver

2016-01-01

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of

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.

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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.

Elastase production by B16-F10 melanoma cells

International Nuclear Information System (INIS)

Shrager, J.B.; Yusa, T.; Netland, P.A.; Zetter, B.R.

1986-01-01

Elastolytic activity was found in sonicates of mouse B16-F10 melanoma cells and in medium conditioned by these cells. Degradation of elastin was determined by measuring the release of soluble 3 H-peptides from labelled insoluble elastin. The activity secreted from B16-F10 cells was soluble and was not associated with membrane vesicles. The secreted activity was partially inhibited by incubation with phenymethylsulfonylfluoride (PMSF) and was abolished by incubation with the alpha-1-protease inhibitor, with pepstatin A or with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). In contrast, the activity was unaffected by incubation with elastatinal, with the plasmin inhibitor Σ-aminocaproic acid (EACA), the metalloproteinase inhibitor ethylenediamine-tetra-acetic acid (EDTA), the soybean trypsin inhibitor or the trypsin inhibitor N proportional to-p-tosyl-L-lysine chloromethyl ketone (TLCK). These results suggest that the majority of the tumor cell-derived elastolytic activity is attributable to a serine protease that differs in specificity from the well characterized elastases previously isolated from neutrophils, macrophages or from mammalian pancreas. The authors postulate that the release of elastase from lung-colonizing B16-F10 cells may facilitate their invasion of elastin-rich lung tissue

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

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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.

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

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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.

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

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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.

Cocaine/levamisole-associated autoimmune syndrome: a disease of neutrophil-mediated autoimmunity.

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Cascio, Michael J; Jen, Kuang-Yu

2018-01-01

Levamisole was previously used for its immunomodulatory properties to treat rheumatoid arthritis and some cancers. However, because of serious side-effects, it was taken off the market in the United States. Recently, levamisole has reemerged as a popular cocaine adulterant. Some individuals who consume levamisole-adulterated cocaine can develop a life-threatening autoimmune syndrome. In this review, the medical consequences of levamisole exposure and postulated mechanisms by which levamisole induces these adverse effects are discussed. Although agranulocytosis and cutaneous vasculitis are the major findings in patients who develop cocaine/levamisole-associated autoimmune syndrome (CLAAS), more recent experience indicates that other organ systems can be involved as well. Current studies point to neutrophil activation and neutrophil extracellular trap formation with subsequent antineutrophil cytoplasmic antibody-mediated tissue injury as a possible mechanism of CLAAS. In the past decade, the detrimental effects of levamisole have reemerged because of its popularity as a cocaine adulterant. Although infrequent, some individuals develop a systemic autoimmune syndrome characterized by immune-mediated agranulocytosis and antineutrophil cytoplasmic antibody-mediated vasculitis. Mechanistically, neutrophil antigens appear to be a major player in inducing CLAAS. Prompt cessation of levamisole exposure is key to treatment, although relapses are frequent because of the addictive effects of cocaine and the high prevalence of levamisole within the cocaine supply.

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.

Ozone-induced airway hyperresponsiveness in patients with asthma: role of neutrophil-derived serine proteinases.

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Hiltermann, T J; Peters, E A; Alberts, B; Kwikkers, K; Borggreven, P A; Hiemstra, P S; Dijkman, J H; van Bree, L A; Stolk, J

1998-04-01

Proteinase inhibitors may be of potential therapeutic value in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma. Our aim was to study the role of neutrophils, and neutrophil-derived serine proteinases in an acute model in patients with asthma. Exposure to ozone induces an acute neutrophilic inflammatory reaction accompanied by an increase in airway hyperresponsiveness. It is thought that these two effects of ozone are linked, and that neutrophil-derived serine proteinases (i.e. elastase) may play a role in the ozone-induced airway hyperresponsiveness. Therefore, we examined the effect of recombinant antileukoprotease (rALP), one of the major serine proteinase inhibitors in the lung, on ozone-induced changes in airway hyperresponsiveness in this model. We observed that 16 h after exposure to ozone, airway hyperresponsiveness to methacholine was increased both following placebo and rALP treatment. There was no significant difference between placebo and rALP treatment (change in area under the dose-response curve to methacholine: 117.3+/-59.0 vs 193.6+/-59.6 % fall x DD; p=.12). Moreover, the immediate decrease in FEV1 after ozone exposure was not significantly different between the two groups (placebo: -29.6+/-6.7%; rALP: -20.9+/-3.8%; p=.11). In addition, no significant differences were observed in plasma levels of fibrinogen degradation products generated by neutrophil serine proteinases before and after exposure to ozone. We conclude that neutrophil-derived serine proteinases are not important mediators for ozone-induced hyperresponsiveness.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

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Lewis, John G; Elder, Peter A

2017-07-01

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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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.

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.

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.

The effect of the Z mutation on the ability of alpha 1-antitrypsin to prevent neutrophil mediated tissue damage.

Science.gov (United States)

Llewellyn-Jones, C G; Lomas, D A; Carrell, R W; Stockley, R A

1994-11-29

Recent studies have shown that alpha 1-antitrypsin (alpha 1-AT) from Z antitrypsin deficiency subjects has a slightly lower association rate constant with neutrophil elastase (NE) than alpha 1-AT from normal subjects, although it is unknown whether this is of clinical importance. We have purified alpha 1-AT from a normal (M alpha 1-AT) and from a deficient (Z alpha 1-AT) subject and have confirmed that the association rate constants for NE are different (5.28; S.E. 0.06.10(7) M-1 s-1 and 1.2; S.E. 0.2.10(7) M-1 s-1, respectively). We have assessed the ability of both of these proteins to inhibit neutrophil mediated fibronectin (FN) degradation in vitro. Both proteins inhibited FN degradation in a dose dependant manner although Z alpha 1-AT was less effective than M alpha 1-AT at equivalent concentrations of active inhibitor (P < 0.05). Inhibition by M alpha 1-AT was 28.5% S.E. 3.9 at 0.01 microM; 35.5% S.E. 7.3 at 0.1 microM and 37% S.E. 8.4 at 0.5 microM, whereas inhibition by Z alpha 1-AT was 9.25% S.E. 3.9; 19.25% S.E. 7.7 and 21.2% S.E. 9.7, respectively. When the time course of inhibition of FN degradation was studied the difference (although less at 1.0 microM) became greater over the 3 h period of the assay. These results suggest that Z alpha 1-AT is less able than the M phenotype to inhibit connective tissue degradation by neutrophils at equivalent concentrations. This is probably due to the lower association rate constant although the reduced stability of the Z molecule may play a role. The differences, together with the reduced plasma concentration, may accentuate the susceptibility of deficient subjects to the development of emphysema.

Optimization of Microbial Elastase Production

International Nuclear Information System (INIS)

Abd EI-Aziz, A.B.; Hassan, A.A.

2010-01-01

The extra cellular proteases (caseinase, gelatinase and elastase) and hemolytic activities of the tested microorganisms on agar plates were detected, using different substrates (gelatin, casein, hemoglobin and elastin).The proteolytic activities were detected only from Pseudomonas aeruginosa, Prevotella bivius, Bacillus subtilis and Micrococcus luteus. The production of elastase by Bacillus subtilis (has low hemolysins activity) at various temperatures (30 degree C - 37 degree C) and at exposure to different doses of gamma irradiation (0.25-1.0 kGy) was investigated in shake flask. The results indicated that the incubation temperature 37 degree C was the optimum for cell growth at earlier stage; while maximum elastase activity was obtained when the cells were cultivated at 30 degree C and irradiation dose level of 0.75 kGy. The effects of temperature, substrate content, elastase concentration, ph and different metals ions on elastolysis were investigated as well the elastase amino acids composition was detected by using amino acids analyzer

Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro

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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

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition.

Science.gov (United States)

Casilag, Fiordiligie; Lorenz, Anne; Krueger, Jonas; Klawonn, Frank; Weiss, Siegfried; Häussler, Susanne

2016-01-01

The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation

NARCIS (Netherlands)

Rossaint, Jan; Herter, Jan M.; van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

2014-01-01

There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute

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.

Increased systemic elastase and C-reactive protein in aggressive periodontitis (CLOI-D-00160R2).

Science.gov (United States)

Wohlfeil, Martin; Scharf, Susanne; Siegelin, Yasemin; Schacher, Beate; Oremek, Gerhard M; Sauer-Eppel, Hildegund; Schubert, Ralf; Eickholz, Peter

2012-08-01

The inflammatory mediators, serum elastase and C-reactive protein (CRP), are associated with an increased risk for coronary heart disease. Thus, the aim of this study is to compare systemic inflammatory mediators in periodontally healthy controls (C), patients with untreated aggressive (AgP) and chronic (ChP) periodontitis. C [periodontal pocket probing depth (PPD)  30% of sites; age >35 years), and AgP (clinically healthy; PDD ≥ 3.6 mm at >30% of sites, bone loss ≥50% at ≥2 teeth; age ≤35 years) were examined clinically, and the body mass index was assessed. Blood was sampled for assessment of serum levels of elastase, CRP, lipopolysaccharide binding protein (LBP), interleukin (IL) 6, 8, and leukocyte counts. Thirty C, 31 ChP, and 29 AgP were analyzed. Elastase, CRP, LBP, and IL-6 levels were elevated in AgP compared to C (p C. AgP patients exhibit a stronger systemic inflammatory burden than C patients.

Concomitant elevations of MMP-9, NGAL, proMMP-9/NGAL and neutrophil elastase in serum of smokers with chronic obstructive pulmonary disease.

Science.gov (United States)

Bchir, Sarra; Nasr, Hela Ben; Bouchet, Sandrine; Benzarti, Mohamed; Garrouch, Abdelhamid; Tabka, Zouhair; Susin, Santos; Chahed, Karim; Bauvois, Brigitte

2017-07-01

A growing body of evidence points towards smoking-related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase-9 (pro- and active MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and the proMMP-9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable-phase COPD patients (82 smokers, 18 never-smokers) and 28 healthy adults (21 smokers, 7 never-smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP-9, NGAL and proMMP-9/NGAL in COPD smokers. While the triad discriminated between smokers and non-smokers in the COPD group, MMP-9 and proMMP-9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack-years did not alter the findings. Serum MMP-9, NGAL and proMMP-9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP-3 (but not of IL-6 and MMP-12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack-years. Among COPD smokers, levels of MMP-9, NGAL and proMMP-9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP-9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP-9, NGAL, proMMP-9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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

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

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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.

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

Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

Science.gov (United States)

Xu, Tingshuang; Liu, Wenai; Yang, Chen; Ba, Xueqing; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

2015-02-01

Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein β-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein β-adducin from lipid rafts and actin, as well as phosphorylation of β-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of β-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced β-adducin phosphorylation and relocation. Taken together, these results show that β-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin. © Society for Leukocyte Biology.

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 ...

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.

Elevated Systemic Levels of Eosinophil, Neutrophil, and Mast Cell Granular Proteins in Strongyloides Stercoralis Infection that Diminish following Treatment.

Science.gov (United States)

Rajamanickam, Anuradha; Munisankar, Saravanan; Bhootra, Yukthi; Dolla, Chandra Kumar; Nutman, Thomas B; Babu, Subash

2018-01-01

Infection with the helminth parasite Strongyloides stercoralis ( Ss ) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

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

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.

Proteomic Characterization of Middle Ear Fluid Confirms Neutrophil Extracellular Traps as a Predominant Innate Immune Response in Chronic Otitis Media.

Directory of Open Access Journals (Sweden)

Stephanie Val

Full Text Available Chronic Otitis Media (COM is characterized by middle ear effusion (MEE and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited.This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses.MEEs were collected from children (n = 49 with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis.109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin.Proteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions.

Swell activated chloride channel function in human neutrophils

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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.

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

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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.

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

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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.

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

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

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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.

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

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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

Human milk proresolving mediators stimulate resolution of acute inflammation.

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Arnardottir, Hildur; Orr, Sarah K; Dalli, Jesmond; Serhan, Charles N

2016-05-01

Human milk contains nutrients and bioactive products relevant to infant development and immunological protection. Here, we investigated the proresolving properties of milk using human milk lipid mediator isolates (HLMIs) and determined their impact on resolution programs in vivo and with human macrophages. HLMIs reduced the maximum neutrophil numbers (14.6±1.2 × 10(6)-11.0±1.0 × 10(6) cells per exudate) and shortened the resolution interval (Ri; 50% neutrophil reduction) by 54% compared with peritonitis. Using rigorous liquid-chromatography tandem-mass spectrometry (LC-MS-MS)-based lipid mediator (LM) metabololipidomics, we demonstrated that human milk possesses a proresolving LM-specialized proresolving mediator (LM-SPM) signature profile, containing SPMs (e.g. resolvins (Rv), protectins (PDs), maresins (MaRs), and lipoxins (LXs)) at bioactive levels (pico-nanomolar concentrations) that enhanced human macrophage efferocytosis and bacterial containment. SPMs identified in human milk included D-series Rvs (e.g., RvD1, RvD2, RvD3, AT-RvD3, and RvD4), PD1, MaR1, E-series Rvs (e.g. RvE1, RvE2, and RvE3), and LXs (LXA4 and LXB4). Of the SPMs identified in human milk, RvD2 and MaR1 (50 ng per mouse) individually shortened Ri by ∼75%. Milk from mastitis gave higher leukotriene B4 and prostanoids and lower SPM levels. Taken together, these findings provide evidence that human milk has proresolving actions via comprehensive LM-SPM profiling, describing a potentially novel mechanism in maternal-infant biochemical imprinting.

Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

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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.

A Neutrophil Proteomic Signature in Surgical Trauma Wounds

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Sander Bekeschus

2018-03-01

Full Text Available Non-healing wounds continue to be a clinical challenge for patients and medical staff. These wounds have a heterogeneous etiology, including diabetes and surgical trauma wounds. It is therefore important to decipher molecular signatures that reflect the macroscopic process of wound healing. To this end, we collected wound sponge dressings routinely used in vacuum assisted therapy after surgical trauma to generate wound-derived protein profiles via global mass spectrometry. We confidently identified 311 proteins in exudates. Among them were expected targets belonging to the immunoglobulin superfamily, complement, and skin-derived proteins, such as keratins. Next to several S100 proteins, chaperones, heat shock proteins, and immune modulators, the exudates presented a number of redox proteins as well as a discrete neutrophil proteomic signature, including for example cathepsin G, elastase, myeloperoxidase, CD66c, and lipocalin 2. We mapped over 200 post-translational modifications (PTMs; cysteine/methionine oxidation, tyrosine nitration, cysteine trioxidation to the proteomic profile, for example, in peroxiredoxin 1. Investigating manually collected exudates, we confirmed presence of neutrophils and their products, such as microparticles and fragments containing myeloperoxidase and DNA. These data confirmed known and identified less known wound proteins and their PTMs, which may serve as resource for future studies on human wound healing.

INACTIVITY OF RECOMBINANT ELA2B PROVIDES A NEW EXAMPLE OF EVOLUTIONARY ELASTASE SILENCING IN HUMANS

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Szepessy, Edit; Sahin-Tóth, Miklós

2005-01-01

BACKGROUND. The archetypal mammalian elastase (ELA1) is not expressed in the human pancreas, because evolutionary mutations suppressed transcription of the ELA1 gene. AIMS. In this study we tested the theory that the unique duplication of the ELA2 gene in humans might compensate for the loss of ELA1. METHODS. Recombinant ELA2A and ELA2B were expressed in Escherichia coli, and their activity was tested on Glt-Ala-Ala-Pro-Leu-p-nitroanilide, DQ elastin and bovine milk protein. RESULTS. Surprisi...

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

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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

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

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Paris, Daniel H; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N; Day, Nicholas P J; Zeerleder, Sacha

2015-01-01

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and

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

Neutrophil extracellular trap formation in supragingival biofilms.

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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

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

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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.

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

Serum elastase activity, serum elastase inhibitors, and occurrence of carotid atherosclerotic plaques: the Etude sur le Vieillissement Artériel (EVA) study.

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Zureik, Mahmoud; Robert, Ladislas; Courbon, Dominique; Touboul, Pierre-Jean; Bizbiz, Latifa; Ducimetière, Pierre

2002-06-04

In the last decades, interest has increased in the potential deleterious atherogenic effects of some cellular elastase activities. The results of experimental and clinical investigations were inconsistent. In this report, we assessed the associations of serum elastase activity and serum elastase inhibitors with carotid plaque occurrence during the 4-year follow-up in a population of 859 subjects free of coronary heart disease and stroke (age, 59 to 71 years). Serum elastase activity and serum elastase inhibitors were measured at baseline examination. Carotid B-mode ultrasound examination was performed at baseline and 2 years and 4 years later. The occurrence of carotid plaques in subjects with the lowest serum elastase activity values (quartile 1), in those with the intermediate values (quartiles 2 to 3), and in those with the highest values (quartile 4) was, respectively, 24.6%, 18.9%, and 12.2% (P<0.001 for trend). The multivariate odds ratios of carotid plaque occurrence associated with the three groups (adjusted for major known cardiovascular risk factors) were, respectively, 1.00, 0.67 (CI, 0.44 to 1.02; P<0.06), and 0.40 (CI, 0.23 to 0.70, P<0.001). For serum elastase inhibitors, the occurrence of carotid plaques in quartile 1 (lowest values), quartiles 2 to 3, and quartile 4 (highest values) was, respectively, 11.7%, 18.8%, and 25.2% (P for trend<0.001). The corresponding multivariate adjusted odds ratios were 1.00, 1.98 (CI, 1.19 to 3.31, P<0.01), and 3.18 (CI, 1.80 to 5.60, P<0.001). Low values of serum elastase activity and high values of serum elastase inhibitors were strongly and independently associated with increased 4-year carotid plaque occurrence. Further studies are necessary to elucidate the nature of the associations between elastase parameters and atherosclerosis.

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 ...

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.

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

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.

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.

The action of neutrophil serine proteases on elastin and its precursor

DEFF Research Database (Denmark)

Heinz, Andrea; Jung, Michael C; Jahreis, Günther

2012-01-01

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass...... spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three...... proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr...

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....

Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

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Rosana L. Pagano

2002-01-01

Full Text Available Background: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice.

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

Effects of elastase on fatty liver

International Nuclear Information System (INIS)

Ogura, Kazuo; Shimizu, Yoshikazu; Hihara, Masafumi; Ando, Hideki; Nishiyama, Masateru; Tano, Hironobu

1984-01-01

Elastase (Elaszym 6T) was administered, in addition to the dietary instruction, to three patients with fatty liver. CT scanning revealed marked improvement in fatty liver. Transaminase levels returned to normal, total cholesterol levels tended to decrease, and HDL-cholesterol levels tended to increase. These results suggest that elastase is effective in the treatment of fatty liver. (Namekawa, K.)

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.

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.

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...

Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

International Nuclear Information System (INIS)

Garver, R.I. Jr.; Chytil, A.; Karlsson, S.

1987-01-01

α 2 -Antitrypsin (α 1 AT) deficiency is a hereditary disorder characterized by reduced serum levels of α 1 AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α 1 AT levels in this disorder with physiologically normal human α 1 AT, the authors have integrated a full-length normal human α 1 AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α 1 AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α 1 AT sequences, secreted an α 1 AT molecule recognized by an anti-human α 1 AT antibody, with the same molecular mass as normal human α 1 AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α 1 AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α 1 AT purified from human plasma and markedly longer than that of nonglycosylated human α 1 AT cDNA-directed yeast-produced α 1 AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α 1 AT cDNA into non-α 1 AT-producing cells, resulting in the synthesis and secretion of physiologically normal α 1 AT

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...

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

Intravenous immunoglobulin prevents murine antibody-mediated acute lung injury at the level of neutrophil reactive oxygen species (ROS production.

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John W Semple

Full Text Available Transfusion-related acute lung injury (TRALI is a leading cause of transfusion-associated mortality that can occur with any type of transfusion and is thought to be primarily due to donor antibodies activating pulmonary neutrophils in recipients. Recently, a large prospective case controlled clinical study of cardiac surgery patients demonstrated that despite implementation of male donors, a high incidence of TRALI still occurred and suggested a need for additional interventions in susceptible patient populations. To examine if intravenous immunoglobulin (IVIg may be effective, a murine model of antibody-mediated acute lung injury that approximates human TRALI was examined. When BALB/c mice were injected with the anti-major histocompatibility complex class I antibody 34-1-2s, mild shock (reduced rectal temperature and respiratory distress (dyspnea were observed and pre-treatment of the mice with 2 g/kg IVIg completely prevented these symptoms. To determine IVIg's usefulness to affect severe lung damage, SCID mice, previously shown to be hypersensitive to 34-1-2s were used. SCID mice treated with 34-1-2s underwent severe shock, lung damage (increased wet/dry ratios and 40% mortality within 2 hours. Treatment with 2 g/kg IVIg 18 hours before 34-1-2s administration completely protected the mice from all adverse events. Treatment with IVIg after symptoms began also reduced lung damage and mortality. While the prophylactic IVIg administration did not affect 34-1-2s-induced pulmonary neutrophil accumulation, bone marrow-derived neutrophils from the IVIg-treated mice displayed no spontaneous ROS production nor could they be stimulated in vitro with fMLP or 34-1-2s. These results suggest that IVIg prevents murine antibody-mediated acute lung injury at the level of neutrophil ROS production and thus, alleviating tissue damage.

Effects of brachytherapy on gene expressions of elastin and elastase

International Nuclear Information System (INIS)

Li Junming; Zhou Jingqun; Hu Bin; Li Shuguo

2004-01-01

Objective: To study the effects of brachytherapy on the gene expressions of elastin and elastase in cultured rat vascular smooth muscle cells (VSMCs). Methods: Rat VSMCs cultured in DMEM containing 10% FBS were irradiated by 60 Co γ-rays at 0, 7, 14, 28 Gy respectively. Then mRNA levels of elastin and elastase were determined by reverse transcription competitive PCR(RT-PCR). Results: Brachytherapy inhibited the expressions of elastase. Elastase mRNA decreased 25.3% and 50.1% in VSMC irradiated with 14, 28 Gy, respectively (P<0.05). The elastin mRNA level increased 80.7% and 102.3% in VSMC irradiated with 14, 25 Gy, respectively (P<0.05). Conclusion: Brachytherapy inhabits the expressions of elastase and increased elastin in VSMC cells

Neutrophil transmigration mediated by the neutrophil-specific antigen CD177 is influenced by the endothelial S536N dimorphism of platelet endothelial cell adhesion molecule-1.

Science.gov (United States)

Bayat, Behnaz; Werth, Silke; Sachs, Ulrich J H; Newman, Debra K; Newman, Peter J; Santoso, Sentot

2010-04-01

The human neutrophil-specific adhesion molecule CD177 (also known as the NB1 alloantigen) becomes upregulated on the cell surface in a number of inflammatory settings. We recently showed that CD177 functions as a novel heterophilic counterreceptor for the endothelial junctional protein PECAM-1 (CD31), an interaction that is mediated by membrane-proximal PECAM-1 IgD 6, which is known to harbor an S(536)N single nucleotide polymorphism of two major isoforms V(98)N(536)G(643) and L(98)S(536)R(643) and a yet-to-be-determined region on CD177. In vitro transendothelial migration experiments revealed that CD177(+) neutrophils migrated significantly faster through HUVECs expressing the LSR, compared with the VNG, allelic variant of PECAM-1 and that this correlated with the decreased ability of anti-PECAM-1 Ab of ITIM tyrosine phosphorylation in HUVECs expressing the LSR allelic variant relative to the VNG allelic variant. Moreover, engagement of PECAM-1 with rCD177-Fc (to mimic heterophilic CD177 binding) suppressed Ab-induced tyrosine phosphorylation to a greater extent in cells expressing the LSR isoform compared with the VNG isoform, with a corresponding increased higher level of beta-catenin phosphorylation. These data suggest that heterophilic PECAM-1/CD177 interactions affect the phosphorylation state of PECAM-1 and endothelial cell junctional integrity in such a way as to facilitate neutrophil transmigration in a previously unrecognized allele-specific manner.

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

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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.

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.

IL-17A and serum amyloid A are elevated in a cigarette smoke cessation model associated with the persistence of pigmented macrophages, neutrophils and activated NK cells.

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Michelle J Hansen

Full Text Available While global success in cessation advocacy has seen smoking rates fall in many developed countries, persistent lung inflammation in ex-smokers is an increasingly important clinical problem whose mechanistic basis remains poorly understood. In this study, candidate effector mechanisms were assessed in mice exposed to cigarette smoke (CS for 4 months following cessation from long term CS exposure. BALF neutrophils, CD4+ and CD8+ T cells and lung innate NK cells remained significantly elevated following smoking cessation. Analysis of neutrophil mobilization markers showed a transition from acute mediators (MIP-2α, KC and G-CSF to sustained drivers of neutrophil and macrophage recruitment and activation (IL-17A and Serum Amyoid A (SAA. Follicle-like lymphoid aggregates formed with CS exposure and persisted with cessation, where they were in close anatomical proximity to pigmented macrophages, whose number actually increased 3-fold following CS cessation. This was associated with the elastolytic protease, MMP-12 (macrophage metallo-elastase which remained significantly elevated post-cessation. Both GM-CSF and CSF-1 were significantly increased in the CS cessation group relative to the control group. In conclusion, we show that smoking cessation mediates a transition to accumulation of pigmented macrophages, which may contribute to the expanded macrophage population observed in COPD. These macrophages together with IL-17A, SAA and innate NK cells are identified here as candidate persistence determinants and, we suggest, may represent specific targets for therapies directed towards the amelioration of chronic airway inflammation.

Granule protein processing and regulated secretion in neutrophils

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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.

Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

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Youngwoo Choi

2017-01-01

Full Text Available The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA. However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs, cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE or myeloperoxidase (MPO attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC, nonsevere asthma (NSA, and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

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.

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

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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.

Altered Innate Immune Responses in Neutrophils from Patients with Well- and Suboptimally Controlled Asthma

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Francesca S. M. Tang

2015-01-01

Full Text Available Background. Respiratory infections are a major cause of asthma exacerbations where neutrophilic inflammation dominates and is associated with steroid refractory asthma. Structural airway cells in asthma differ from nonasthmatics; however it is unknown if neutrophils differ. We investigated neutrophil immune responses in patients who have good (AGood and suboptimal (ASubopt asthma symptom control. Methods. Peripheral blood neutrophils from AGood (ACQ 0.75, n=7, and healthy controls (HC (n=9 were stimulated with bacterial (LPS (1 μg/mL, fMLF (100 nM, and viral (imiquimod (3 μg/mL, R848 (1.5 μg/mL, and poly I:C (10 μg/mL surrogates or live rhinovirus (RV 16 (MOI1. Cell-free supernatant was collected after 1 h for neutrophil elastase (NE and matrix metalloproteinase- (MMP- 9 measurements or after 24 h for CXCL8 release. Results. Constitutive NE was enhanced in AGood neutrophils compared to HC. fMLF stimulated neutrophils from ASubopt but not AGood produced 50% of HC levels. fMLF induced MMP-9 was impaired in ASubopt and AGood compared to HC. fMLF stimulated CXCL8 but not MMP-9 was positively correlated with FEV1 and FEV1/FVC. ASubopt and AGood responded similarly to other stimuli. Conclusions. Circulating neutrophils are different in asthma; however, this is likely to be related to airflow limitation rather than asthma control.

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

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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.

The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI Attenuates Elastase-Induced Emphysema in Mice

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Bruno Tadeu Martins-Olivera

2016-01-01

Full Text Available Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD. However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group or saline (SAL group and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups. At day 28, the following analyses were performed: (I lung mechanics, (II exhaled nitric oxide (ENO, (III bronchoalveolar lavage fluid (BALF, and (IV lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment.

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

Science.gov (United States)

Gautam, S; Kirschnek, S; Gentle, I E; Kopiniok, C; Henneke, P; Häcker, H; Malleret, L; Belaaouaj, A; Häcker, G

2013-08-01

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation

Role of the Mycoplasma pneumoniae/Interleukin-8/Neutrophil Axis in the Pathogenesis of Pneumonia.

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Zhengrong Chen

Full Text Available Neutrophil infiltration is the characteristic pathological feature of M. pneumoniae pneumonia (MPP. This study aimed to explore the associations among neutrophil activity, clinical presentation, and role of the M. pneumoniae/interleukin-8 (IL-8/neutrophil axis in the pathogenesis of MPP. A total of 42 patients with MPP were prospectively enrolled in the study. Neutrophil activity, including matrix metalloproteinase-9 (MMP-9, myeloperoxidase (MPO, and neutrophil elastase (NE, were measured. Clinical information was collected for all patients and control group. In vitro, IL-8 production was measured at different time points after M. pneumoniae infection of bronchial epithelial cells, and neutrophil activity was analyzed after IL-8 stimulation. The percentage of neutrophil in the bronchoalveolar lavage fluid was higher in the group of patients with high levels of M. pneumoniae DNA than in those with low levels of M. pneumoniae DNA (P < 0.05. IL-8, MMP-9, and NE in patients with MPP significantly increased compared with controls and decreased after treatment (P < 0.05. MPO and MMP-9 were associated with duration of fever (r = 0.332, P < 0.05 and length of stay (r = 0.342, P < 0.05, respectively. In vitro, M. pneumoniae induced IL-8 production by bronchial epithelial cells in a time dependent manner. MPO, MMP-9 and NE production by neutrophils significantly increased compared with medium controls after IL-8 stimulation. In summary, the M. pneumoniae/IL-8/neutrophil axis likely plays a vital role in the pathogenesis of MPP.

Neutrophils reduce the parasite burden in Leishmania (Leishmania amazonensis-infected macrophages.

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Erico Vinícius de Souza Carmo

2010-11-01

Full Text Available Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L major, whereas less information is available for L. (L amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L. amazonensis (C3H/HePas. In contrast, the susceptible strain (BALB/c displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L. amazonensis-infected macrophages in vitro.Mouse peritoneal macrophages infected with L. (L. amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1 intracellular parasites were efficiently destroyed in the co-cultures; 2 the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas or susceptible (BALB/c to L. (L. amazonensis; 3 parasite destruction did not require contact between infected macrophages and neutrophils; 4 tumor necrosis factor alpha (TNF-α, neutrophil elastase and platelet activating factor (PAF were involved with the leishmanicidal activity, and 5 destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L. amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.

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

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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.

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

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Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

2017-08-01

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg 2+ and Ca 2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs. © 2016 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

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

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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.

Mutations in the ELA2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease.

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Ancliff, P J; Gale, R E; Liesner, R; Hann, I M; Linch, D C

2001-11-01

Severe congenital neutropenia (SCN) was originally described as an autosomal recessive disorder. Subsequently, autosomal dominant and sporadic forms of the disease have been recognized. All forms are manifest by persistent severe neutropenia and recurrent bacterial infection. In contrast, cyclical hematopoiesis is characterized by periodic neutropenia inter-spaced with (near) normal neutrophil counts. Recently, linkage analysis on 13 affected pedigrees identified chromosome 19p13.3 as the likely position for mutations in cyclical hematopoiesis. Heterozygous mutations in the ELA2 gene encoding neutrophil elastase were detected in all families studied. Further work also demonstrated mutations in ELA2 in sporadic and autosomal dominant SCN. However, all mutations described to date are heterozygous and thus appear to act in a dominant fashion, which is inconsistent with an autosomal recessive disease. Therefore, the current study investigated whether mutations in ELA2 could account for the disease phenotype in classical autosomal recessive SCN and in the sporadic and autosomal dominant types. All 5 exons of ELA2 and their flanking introns were studied in 18 patients (3 autosomal recessive, 5 autosomal dominant [from 3 kindreds], and 10 sporadic) using direct automated sequencing. No mutations were found in the autosomal recessive families. A point mutation was identified in 1 of 3 autosomal dominant families, and a base substitution was identified in 8 of 10 patients with the sporadic form, though 1 was subsequently shown to be a low-frequency polymorphism. These results suggest that mutations in ELA2 are not responsible for classical autosomal recessive Kostmann syndrome but provide further evidence for the role of ELA2 in SCN.

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

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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.

Effects of acrolein on leukotriene biosynthesis in human neutrophils.

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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

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

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

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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.

Tamoxifen up-regulates catalase production, inhibits vessel wall neutrophil infiltration, and attenuates development of experimental abdominal aortic aneurysms.

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Grigoryants, Vladimir; Hannawa, Kevin K; Pearce, Charles G; Sinha, Indranil; Roelofs, Karen J; Ailawadi, Gorav; Deatrick, Kristopher B; Woodrum, Derek T; Cho, Brenda S; Henke, Peter K; Stanley, James C; Eagleton, Matthew J; Upchurch, Gilbert R

2005-01-01

Selective estrogen receptor modulators (SERMs), similar to estrogens, possess vasoprotective effects by reducing release of reactive oxygen species. Little is known about the potential effects of SERMs on the pathogenesis of abdominal aortic aneurysms (AAAs). This study's objective was to investigate the growth of experimental AAAs in the setting of the SERM tamoxifen. In the first set of experiments, adult male rats underwent subcutaneous tamoxifen pellet (delivering 10 mg/kg/day) implantation (n = 14) or sham operation (n = 16). Seven days later, all animals underwent pancreatic elastase perfusion of the abdominal aorta. Aortic diameters were determined at that time, and aortas were harvested 7 and 14 days after elastase perfusion for immunohistochemistry, real-time polymerase chain reaction, Western blot analysis, and zymography. In the second set of experiments, a direct irreversible catalase inhibitor, 3-amino-1,2,4-triazole (AT), was administered intraperitoneally (1 mg/kg) daily to tamoxifen-treated (n = 6) and control rats (n = 6), starting on day 7 after elastase perfusion. Aortic diameters were measured on day 14. In a third set of experiments, rats were perfused with catalase (150 mg/kg) after the elastase (n = 5), followed by daily intravenous injections of catalase (150 mg/kg/day) administered for 10 days. A control group of rats (n = 7) received 0.9% NaCl instead of catalase. Mean AAA diameters were approximately 50% smaller in tamoxifen-treated rats compared with sham rats 14 days after elastase perfusion (P = .002). The tamoxifen-treated group's aortas had a five-fold increase in catalase mRNA expression (P = .02) on day 7 and an eight-fold increase in catalase protein on day 14 (P = .04). Matrix metalloprotroteinase-9 activity was 2.4-fold higher (P = .01) on day 7 in the aortas of the controls compared to the tamoxifen-treated group's aortas. Tamoxifen-treated rats had approximately 40% fewer aortic polymorphonuclear neutrophils compared to

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.

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

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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

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

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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

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

International Nuclear Information System (INIS)

Nourshargh, S.; Rampart, M.; Hellewell, P.G.; Jose, P.J.; Harlan, J.M.; Edwards, A.J.; Williams, T.J.

1989-01-01

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: (1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; (2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and (3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules

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...

BACILLUS THURINGIENSIS ELASTASES WITH INSECTICIDE ACTIVITY

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E. V. Matseliukh

2015-10-01

Full Text Available The purpose of the research was a screening of proteases with elastase activity among Bacillus thuringiensis strains, their isolation, partially purification, study of physicochemical properties and insecticide activity in relation to the larvae of the Colorado beetle. The objects of the investigation were 18 strains of B. thuringiensis, isolated from different sources: sea water, dry biological product "Bitoksibatsillin" and also from natural populations of Colorado beetles of the Crimea, Kherson, Odesa, Mykolaiv and Zaporizhiia regions of Ukraine. Purification of enzymes with elastase activity isolated from above mentioned strains was performed by gel-chromatography and insecticide activity was studied on the 3–4 larvae instar of Colorado beetle. The ability of a number of B. thuringiensis strains to synthesize the proteases with elastase activity has been established. The most active were enzymes obtained from strains IMV B-7465, IMV B-7324 isolated from sea water, and strains 9, 902, Bt-H and 0-239 isolated from Colorado beetles. The study of the physicochemical properties of the partially purified proteases of these strains showed that they belonged to enzymes of the serine type. Peptidases of a number of B. thuringiensis strains (IMV B-7324, IMV B-7465, 902, 0-239, 9 are metal-dependent enzymes. Optimal conditions of action of all tested enzymes are the neutral and alkaline рН values and the temperatures of 30–40 °С. The studies of influence of the complex enzyme preparations and partially purified ones of B. thuringiensis strains on the larvae instar of Colorado beetles indicated that enzymes with elastase activity could be responsible for insecticide action of the tested strains.

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

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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.

Neutrophil programming dynamics and its disease relevance.

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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.

Physicochemical properties of elastase isolated from clinical Pseudomonas Aeruginosa

International Nuclear Information System (INIS)

Elbazza, Z.E.; Moroz, A.F.

1989-01-01

Purified elastase was obtained from clinical Pseudomonas Aeruginosa (P.A.-283). The enzyme showed not only elasto lytic activity, but also a broad proteolytic activity against various proteins. The activity of the enzyme on collagen and gelatin was also observed. The optimum pH for elastase was 7.8 to 8.0 for both the proteolytic and elasto lytic activities. The elastase was stable in a pH range from 6.6 to 9.0. Optimum temperature for proteolytic and elasto lytic activities was 40 and inhibition of elastase occurs at 80 . The D 1 0 value of the P.A-283 was found to be 0.11 kGy. Increasing the dose level value of gamma-irradiation decrease the proteolytic activity in the culture filtrate reaching only 16% at the dose level 0.5 kGy. Chelating agents and some metal ions inhibited both proteolytic and elasto lytic activities. Selective inhibition of elasto lytic activity was observed in high concentrations of sodium and ammonium salts without concurrent decrease in the proteolytic activity of the enzyme.4 fig., 3 tab

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

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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.

Fecal pancreatic elastase-1 levels in older individuals without known gastrointestinal diseases or diabetes mellitus

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Idziak Joanna

2011-01-01

Full Text Available Abstract Background Structural changes occur in the pancreas as a part of the natural aging process. With aging, also the incidence of maldigestive symptoms and malnutrition increases, raising the possibility that these might be caused at least in part by inadequate pancreatic enzyme secretion due to degenerative processes and damage of the gland. Fecal elastase-1 is a good marker of pancreatic exocrine secretion. The aim of this study was to investigate the fecal elastase-1 levels among over 60 years old Finnish and Polish healthy individuals without any special diet, known gastrointestinal disease, surgery or diabetes mellitus. Methods A total of 159 patients participated in this cross-sectional study. 106 older individuals (aged 60-92 years were recruited from outpatient clinics and elderly homes. They were divided to three age groups: 60-69 years old (n = 31; 70-79 years old (n = 38 and over 80 years old (n = 37. 53 young subjects (20-28 years old were investigated as controls. Inclusion criteria were age over 60 years, normal status and competence. Exclusion criteria were any special diet, diabetes mellitus, any known gastrointestinal disease or prior gastrointestinal surgery. Fecal elastase-1 concentration was measured from stool samples with an ELISA that uses two monoclonal antibodies against different epitopes of human elastase-1. Results Fecal elastase-1 concentrations correlated negatively with age (Pearson r = -0,3531, P P Conclusion In our study one fifth of healthy older individuals without any gastrointestinal disorder, surgery or diabetes mellitus suffer from pancreatic exocrine insufficiency and might benefit from enzyme supplementation therapy.

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

Earthworm coelomocyte extracellular traps: structural and functional similarities with neutrophil NETs.

Science.gov (United States)

Homa, Joanna

2018-03-01

Invertebrate immunity is associated with natural mechanisms that include cellular and humoral elements, similar to those that play a role in vertebrate innate immune responses. Formation of extracellular traps (ETs) is a newly discovered mechanism to combat pathogens, operating not only in vertebrate leucocytes but also in invertebrate immune cells. The ET components include extracellular DNA (exDNA), antimicrobial proteins and histones. Formation of mammalian ETs depends on enzymes such as neutrophil elastase, myeloperoxidase, the citrullination of histones and protease activity. It was confirmed that coelomocytes-immunocompetent cells of the earthworm Eisenia andrei-are also able to release ETs in a protease-dependent manner, dependent or independent of the formation of reactive oxygen species and rearrangement of the cell cytoskeleton. Similar to vertebrate leukocytes (e.g., neutrophil), coelomocytes are responsible for many immune functions like phagocytosis, cytotoxicity and secretion of humoral factors. ETs formed by coelomocyte analogues to neutrophil ETs consist of exDNA, histone H3 and attached to these structures proteins, e.g., heat shock proteins HSP27. The latter fact confirms that mechanisms of ET release are conserved in evolution. The study on Annelida adds this animal group to the list of invertebrates capable of ET release, but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa.

Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

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H. S. Euzger

1999-01-01

Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

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

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

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Pfundt, R; van Ruissen, F; van Vlijmen-Willems, I M; Alkemade, H A; Zeeuwen, P L; Jap, P H; Dijkman, H; Fransen, J; Croes, H; van Erp, P E; Schalkwijk, J

1996-01-01

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

DEFF Research Database (Denmark)

Bassompierre, Marc; Nielsen, Henrik Hauch; Børresen, Torger

1993-01-01

1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography. 2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic. 3. The pH optimum of this e......1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography. 2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic. 3. The pH optimum...... of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide. 4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45 degree C, and the enzyme was stable up to this temperature. 5. The trout elastase exhibited a higher specific activity than...... porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein. 6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor....

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...

Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of Legionella pneumophila Lung Infection via TNF and ROS.

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Pascal Ziltener

2016-04-01

Full Text Available Legionella pneumophila is a facultative intracellular bacterium that lives in aquatic environments where it parasitizes amoeba. However, upon inhalation of contaminated aerosols it can infect and replicate in human alveolar macrophages, which can result in Legionnaires' disease, a severe form of pneumonia. Upon experimental airway infection of mice, L. pneumophila is rapidly controlled by innate immune mechanisms. Here we identified, on a cell-type specific level, the key innate effector functions responsible for rapid control of infection. In addition to the well-characterized NLRC4-NAIP5 flagellin recognition pathway, tumor necrosis factor (TNF and reactive oxygen species (ROS are also essential for effective innate immune control of L. pneumophila. While ROS are essential for the bactericidal activity of neutrophils, alveolar macrophages (AM rely on neutrophil and monocyte-derived TNF signaling via TNFR1 to restrict bacterial replication. This TNF-mediated antibacterial mechanism depends on the acidification of lysosomes and their fusion with L. pneumophila containing vacuoles (LCVs, as well as caspases with a minor contribution from cysteine-type cathepsins or calpains, and is independent of NLRC4, caspase-1, caspase-11 and NOX2. This study highlights the differential utilization of innate effector pathways to curtail intracellular bacterial replication in specific host cells upon L. pneumophila airway infection.

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

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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

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....

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

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

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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.

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.

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

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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.

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.

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

Evidence for a self-enforcing inflammation in neutrophil-mediated chronic diseases

NARCIS (Netherlands)

Overbeek, S.A.

2011-01-01

In summary, this thesis provides evidence for the self-sustaining role of neutrophils in the inflammatory state in the pathogenesis of COPD and CD. In active disease, neutrophils release proteolytic enzymes that breakdown collagen. One of the collagen fragments can be neutrophilic chemoattractant

Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

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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

Parenteral medium-chain triglyceride-induced neutrophil activation is not mediated by a Pertussis Toxin sensitive receptor.

Science.gov (United States)

Versleijen, Michelle W J; van Esterik, Joantine C J; Roelofs, Hennie M J; van Emst-de Vries, Sjenet E; Willems, Peter H G M; Wanten, Geert J A

2009-02-01

Lipid-induced immune modulation might contribute to the increased infection rate that is observed in patients using parenteral nutrition. We previously showed that emulsions containing medium-chain triglycerides (LCT/MCTs or pure MCTs), but not pure long-chain triglycerides (LCTs), impair neutrophil functions, modulate cell-signaling and induce neutrophil activation in vitro. It has recently been shown that medium-chain fatty acids are ligands for GPR84, a pertussis toxin (PT)-sensitive G-protein-coupled receptor (GPCR). This finding urged us to investigate whether MCT-induced neutrophil activation is mediated by PT-sensitive GPCRs. Neutrophils isolated from blood of healthy volunteers were pre-incubated with PT (0.5-1 microg/mL, 1.5 h) and analyzed for the effect of this pre-incubation on LCT/MCT (2.5 mmol/L)-dependent modulation of serum-treated zymosan (STZ)-induced intracellular Ca(2+) mobilization and on LCT/MCT (5 mmol/L)-induced expression of cell surface adhesion (CD11b) and degranulation (CD66b) markers and oxygen radical (ROS) production. PT did not inhibit the effects of LCT/MCT on the STZ-induced increase in cytosolic free Ca(2+) concentration. LCT/MCT increased ROS production to 146% of unstimulated cells. However, pre-incubation with PT did not inhibit the LCT/MCT-induced ROS production. Furthermore, the LCT/MCT-induced increase in CD11b and CD66b expression (196% and 235% of unstimulated cells, respectively) was not inhibited by pre-incubation with PT. LCT/MCT-induced neutrophil activation does not involve the action of a PT-sensitive G-protein-coupled receptor.

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria.

Science.gov (United States)

Hirschfeld, Josefine; White, Phillipa C; Milward, Michael R; Cooper, Paul R; Chapple, Iain L C

2017-12-01

Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii , stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated. Copyright © 2017 American Society for Microbiology.

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....

Crucial involvement of tumor-associated neutrophils in the regulation of chronic colitis-associated carcinogenesis in mice.

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Kun Shang

Full Text Available Ulcerative colitis (UC is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC. However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM, followed by repeated dextran sulfate sodium (DSS ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP-9 and neutrophil elastase (NE, accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2-CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.

Crucial Involvement of Tumor-Associated Neutrophils in the Regulation of Chronic Colitis-Associated Carcinogenesis in Mice

Science.gov (United States)

Wang, Chen; Wang, Zhen; Gu, Hong-Yu; Du, Xiang; Zhou, Xiao-Yan; Zheng, Chun-Lei; Chi, Ya-Yun; Mukaida, Naofumi; Li, Ying-Yi

2012-01-01

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2–CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer. PMID:23272179

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

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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.

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

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...

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...

Risk factors for admission and the role of respiratory syncytial virus ...

African Journals Online (AJOL)

disease and poor outcomes when exposed to the influenza virus. Studies of CTL responses ... (IL)8 and IL2) and human neutrophil elastase play a significant ~ role in the ..... Prophylaxis against respiratory syncytial virus in premature infants.

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.

Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture

International Nuclear Information System (INIS)

Kispert, J.; Mogayzel, P.J. Jr.; Pratt, C.A.; Toselli, P.; Wolfe, B.L.; Faris, B.; Franzblau, C.

1986-01-01

The effect of porcine pancreatic elastase on the extracellular matrix (ECM) of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Initially, the elastin appeared as non-coalesced material closely associated with filaments, presumably microfibrils. The insoluble elastin accumulated in the ECM of cells in culture for 6 weeks accounted for 40-45% of the total protein. After exposure to elastase for 30-60 minutes, the elastin content was reduced to 14-20%. The reduction in the total protein content of the cultures after elastase treatment was due primarily to the loss of elastin. Although the amino acid compositions of the elastin isolated from cultures both before and after elastase treatment were similar, there were striking ultrastructural differences in the amorphous elastin. The elastin assumed a mottled appearance after elastase exposure, similar to that seen in in vivo emphysema models. Pulse experiments with 3 H-valine demonstrated an increase in protein synthesis by the cells 20 hours after elastase exposure, suggesting the potential for elastin repair. The use of this culture system will aid in clarifying the role of elastolysis in pulmonary and vascular injuries

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.

Vasculitis and antineutrophil cytoplasmic autoantibodies associated with propylthiouracil therapy

NARCIS (Netherlands)

Dolman, K. M.; Gans, R. O.; Vervaat, T. J.; Zevenbergen, G.; Maingay, D.; Nikkels, R. E.; Donker, A. J.; von dem Borne, A. E.; Goldschmeding, R.

1993-01-01

Vasculitis is a rare complication of propylthiouracil therapy. Antineutrophil cytoplasmic antibodies (ANCA) have been described in association with several vasculitic disorders. We report detection of ANCA against human neutrophil elastase, proteinase 3, and myeloperoxidase in serum from six

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...

High-density lipoproteins potentiate α1-antitrypsin therapy in elastase-induced pulmonary emphysema.

Science.gov (United States)

Moreno, Juan-Antonio; Ortega-Gomez, Almudena; Rubio-Navarro, Alfonso; Louedec, Liliane; Ho-Tin-Noé, Benoit; Caligiuri, Giuseppina; Nicoletti, Antonino; Levoye, Angelique; Plantier, Laurent; Meilhac, Olivier

2014-10-01

Several studies report that high-density lipoproteins (HDLs) can carry α1-antitrypsin (AAT; an elastase inhibitor). We aimed to determine whether injection of exogenous HDL, enriched or not in AAT, may have protective effects against pulmonary emphysema. After tracheal instillation of saline or elastase, mice were randomly treated intravenously with saline, human plasma HDL (75 mg apolipoprotein A1/kg), HDL-AAT (75 mg apolipoprotein A1-3.75 mg AAT/kg), or AAT alone (3.75 mg/kg) at 2, 24, 48, and 72 hours. We have shown that HDL-AAT reached the lung and prevented the development of pulmonary emphysema by 59.3% at 3 weeks (alveoli mean chord length, 22.9 ± 2.8 μm versus 30.7 ± 4.5 μm; P pulmonary emphysema than AAT alone, and may represent a significant development for the management of emphysema associated with AAT deficiency.

Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

DEFF Research Database (Denmark)

van der Plas, Mariena J A; Bhongir, Ravi K V; Kjellström, Sven

2016-01-01

Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21...

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

Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity

Directory of Open Access Journals (Sweden)

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.

Establishment of a new murine elastase-induced aneurysm model combined with transplantation.

Directory of Open Access Journals (Sweden)

Zuzanna Rowinska

Full Text Available The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation.Adult male mice (n = 6-9 per group underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting.Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11 ± 0.10 mm vs. 0.75 ± 0.03 mm; p ≤ 0,001. Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts.We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals.

Bottlenose dolphins (Tursiops truncatus do also cast neutrophil extracellular traps against the apicomplexan parasite Neospora caninum

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R. Villagra-Blanco

2017-12-01

Full Text Available Neutrophil extracellular traps (NETs are web-like structures composed of nuclear DNA decorated with histones and cytoplasmic peptides which antiparasitic properties have not previously been investigated in cetaceans. Polymorphonuclear neutrophils (PMN were isolated from healthy bottlenose dolphins (Tursiops truncatus, and stimulated with Neospora caninum tachyzoites and the NETs-agonist zymosan. In vitro interactions of PMN with the tachyzoites resulted in rapid extrusion of NETs. For the demonstration and quantification of cetacean NETs, extracellular DNA was stained by using either Sytox Orange® or Pico Green®. Scanning electron microscopy (SEM and fluorescence analyses demonstrated PMN-derived release of NETs upon exposure to tachyzoites of N. caninum. Co-localization studies of N. caninum induced cetacean NETs proved the presence of DNA adorned with histones (H1, H2A/H2B, H3, H4, neutrophil elastase (NE, myeloperoxidase (MPO and pentraxin (PTX confirming the molecular properties of mammalian NETosis. Dolphin-derived N. caninum-NETosis were efficiently suppressed by DNase I and diphenyleneiodonium (DPI treatments. Our results indicate that cetacean-derived NETs represent an ancient, conserved and relevant defense effector mechanism of the host innate immune system against N. caninum and probably other related neozoan parasites circulating in the marine environment. Keywords: Tursiops truncatus, cetaceans, Neutrophil extracellular traps, Innate immunity, Neospora caninum.

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

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.

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.

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.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Science.gov (United States)

Tanaka, Kento; Yoshitomi, Tomomi; Hirahara, Kazuki

2017-01-01

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

Sphingosine 1-phosphate mediates hyperalgesia via a neutrophil-dependent mechanism.

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Amanda Finley

Full Text Available Novel classes of pain-relieving molecules are needed to fill the void between non-steroidal anti-inflammatory agents and narcotics. We have recently shown that intraplantar administration of sphingosine 1-phosphate (S1P in rats causes peripheral sensitization and hyperalgesia through the S1P(1 receptor subtype (S1PR(1: the mechanism(s involved are largely unknown and were thus explored in the present study. Intraplantar injection of carrageenan in rats led to a time-dependent development of thermal hyperalgesia that was associated with pronounced edema and infiltration of neutrophils in paw tissues. Inhibition of 1 S1P formation with SK-I, a sphingosine kinase inhibitor, 2 S1P bioavailability with the S1P blocking antibody Sphingomab, LT1002 (but not its negative control, LT1017 or 3 S1P actions through S1PR(1 with the selective S1PR(1 antagonist, W146 (but not its inactive enantiomer, W140 blocked thermal hyperalgesia and infiltration of neutrophils. Taken together, these findings identify S1P as an important contributor to inflammatory pain acting through S1PR(1 to elicit hyperalgesia in a neutrophil-dependant manner. In addition and in further support, we demonstrate that the development of thermal hyperalgesia following intraplantar injection of S1P or SEW2871 (an S1PR(1 agonist was also associated with neutrophilic infiltration in paw tissues as these events were attenuated by fucoidan, an inhibitor of neutrophilic infiltration. Importantly, FTY720, an FDA-approved S1P receptor modulator known to block S1P-S1PR(1 signaling, attenuated carrageenan-induced thermal hyperalgesia and associated neutrophil infiltration. Targeting the S1P/S1PR(1 axis opens a therapeutic strategy for the development of novel non-narcotic anti-hyperalgesic agents.

Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

LENUS (Irish Health Repository)

Fanning, N F

2012-02-03

Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

Science.gov (United States)

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

New Insights into the Pro-Inflammatory Activities of Ang1 on Neutrophils: Induction of MIP-1β Synthesis and Release.

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Elizabeth Dumas

Full Text Available We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2 to induce pro-inflammatory activities, such as platelet-activating factor synthesis, β2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1β (MIP-1β by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1β protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1β protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM increased neutrophil intracellular and extracellular MIP-1β concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002, p38 MAPK (SB203580 and MEK (U0126 inhibitors completely inhibited Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D and protein synthesis inhibitor (cycloheximide, did also abrogate Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1β mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1β gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.

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.

Cotton Study: Albumin Binding and its Effect on Elastase Activity in the Chronic Non-Healing Wound

Energy Technology Data Exchange (ETDEWEB)

Castro, N.; Goheen, S.

2005-01-01

Cotton, as it is used in wound dressings is composed of nearly pure cellulose. During the wound-healing process, cotton is exposed to various blood components including water, salts, cells, and blood proteins. Albumin is the most prominent protein in blood. Elastase is an enzyme secreted by white blood cells and takes an active role in tissue reconstruction. In the chronic non-healing wound, elastase is often over-expressed such that this enzyme digests tissue and growth factors, and interferes with the normal healing process. Our goal is to design a cotton wound dressing that will sequester elastase or assist in reducing elastase activity in the presence of other blood proteins such as albumin. The ability of cotton and various cotton derivatives to sequester elastase and albumin has been studied by examining the adsorption of these two proteins separately. We undertook the present work to confirm the binding of albumin to cotton and to quantify the activity of elastase in the presence of various derivatives of cotton. We previously observed a slight increase in elastase activity when exposed to cotton. We also observed a continuous accumulation of albumin on cotton using high-performance liquid chromatography methods. In the present study, we used an open-column-absorption technique coupled with a colorimetric protein assay to confirm losses of albumin to cotton. We have also confirmed increased elastase activity after exposure to cotton. The results are discussed in relation to the porosity of cotton and the use of cotton for treating chronic non-healing wounds.

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.

Localization and Functionality of the Inflammasome in Neutrophils

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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...

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

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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.

Alendronate inhalation ameliorates elastase-induced pulmonary emphysema in mice by induction of apoptosis of alveolar macrophages.

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Ueno, Manabu; Maeno, Toshitaka; Nishimura, Satoshi; Ogata, Fusa; Masubuchi, Hiroaki; Hara, Kenichiro; Yamaguchi, Kouichi; Aoki, Fumiaki; Suga, Tatsuo; Nagai, Ryozo; Kurabayashi, Masahiko

2015-03-10

Alveolar macrophages play a crucial role in the pathogenesis of emphysema, for which there is currently no effective treatment. Bisphosphonates are widely used to treat osteoclast-mediated bone diseases. Here we show that delivery of the nitrogen-containing bisphosphonate alendronate via aerosol inhalation ameliorates elastase-induced emphysema in mice. Inhaled, but not orally ingested, alendronate inhibits airspace enlargement after elastase instillation, and induces apoptosis of macrophages in bronchoalveolar fluid via caspase-3- and mevalonate-dependent pathways. Cytometric analysis indicates that the F4/80(+)CD11b(high)CD11c(mild) population characterizing inflammatory macrophages, and the F4/80(+)CD11b(mild)CD11c(high) population defining resident alveolar macrophages take up substantial amounts of the bisphosphonate imaging agent OsteoSense680 after aerosol inhalation. We further show that alendronate inhibits macrophage migratory and phagocytotic activities and blunts the inflammatory response of alveolar macrophages by inhibiting nuclear factor-κB signalling. Given that the alendronate inhalation effectively induces apoptosis in both recruited and resident alveolar macrophages, we suggest this strategy may have therapeutic potential for the treatment of emphysema.

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

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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...

Regulation of neutrophil senescence by microRNAs.

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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.

Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

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Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai; van der Plas, Mariena J A; Petrlova, Jitka; Kjellström, Sven; Sze, Siu Kwan; Schmidtchen, Artur

2017-10-13

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

Sexy again: the renaissance of neutrophils in psoriasis.

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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.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

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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

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

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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

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

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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.

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

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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...

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

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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...

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)

Role of β1 Integrin in Tissue Homing of Neutrophils During Sepsis

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Sarangi, Pranita P.; Hyun, Young-Min; Lerman, Yelena V.; Pietropaoli, Anthony P.; Kim, Minsoo

2012-01-01

Aberrant activation of neutrophils during sepsis results in the widespread release of pro-inflammatory mediators, leading to multi-organ system failure and death. However, aberrant activation of neutrophils during sepsis results in the widespread release of harmful inflammatory mediators causing host tissue injuries that can lead to multi organ system failure and death. One of the pivotal components of neutrophil migration during inflammation is the expression of surface integrins. In this study, we show that administration of a cyclic analog of RGD peptide (Arg-Gly-Asp) significantly reduced the number of tissue-invading neutrophils and the degree of sepsis-induced lethality in mice as compared to control peptide. Secondly, β1 integrin (CD29) was highly up-regulated on the neutrophils isolated from both septic patients and animals. Finally, conditional genetic ablation of β1 integrin from granulocytes also improved survival and bacterial clearance in septic animals Thus, our results indicate that expression of β1 integrin is important for modulating neutrophil trafficking during sepsis, and that therapeutics designed against β1 integrins may be beneficial. PMID:22683734

Neutrophil beta-2 microglobulin: an inflammatory mediator

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Bjerrum, O W; Nissen, Mogens Holst; Borregaard, N

1990-01-01

Beta-2 microglobulin (beta 2m) constitutes the light invariant chain of HLA class I antigen, and is a constituent of mobilizable compartments of neutrophils. Two forms of beta 2m exist: native beta 2m and proteolytically modified beta 2m (Des-Lys58-beta 2m), which shows alpha mobility in crossed ...

Interval and continuous exercise regimens suppress neutrophil-derived microparticle formation and neutrophil-promoted thrombin generation under hypoxic stress.

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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

With Friends Like These: The Complex Role of Neutrophils in the Progression of Severe Pneumonia

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Roger D. Pechous

2017-05-01

Full Text Available Pneumonia is a leading cause of death from infection in the United States and across the globe. During pulmonary infection, clear resolution of host inflammatory responses occurs in the absence of appreciable lung damage. Neutrophils are the first wave of leukocytes to arrive in the lung upon infection. After activation, neutrophils traffic from the vasculature via transendothelial migration through the lung interstitium and into the alveolar space. Successful pulmonary immunity requires neutrophil-mediated killing of invading pathogens by phagocytosis and release of a myriad of antimicrobial molecules, followed by resolution of inflammation, neutrophil apoptosis, and clearing of dead or dying neutrophils by macrophages. In addition to their antimicrobial role, it is becoming clear that neutrophils are also important modulators of innate and adaptive immune responses, primarily through the release of cytokines and recruitment of additional waves of neutrophils into the airways. Though typically essential to combating severe pneumonia, neutrophil influx into the airways is a double-edged sword: Overzealous neutrophil activation can cause severe tissue damage as a result of the release of toxic agents including proteases, cationic polypeptides, cytokines, and reactive oxygen species (ROS aimed at killing invading microbes. In extreme cases, the damage caused by neutrophils and other innate immune mediators become the primary source of morbidity and mortality. Here, we review the complex role of neutrophils during severe pneumonia by highlighting specific molecules and processes that contribute to pulmonary immunity, but can also drive progression of severe disease. Depending on the identity of the infectious agent, enhancing or suppressing neutrophil-mediated responses may be key to effectively treating severe and typically lethal pneumonia.

Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.

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Rebordão, M R; Carneiro, C; Alexandre-Pires, G; Brito, P; Pereira, C; Nunes, T; Galvão, A; Leitão, A; Vilela, C; Ferreira-Dias, G

2014-12-01

Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment

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Kinoshita, Takayoshi [Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531 (Japan); Tamada, Taro [Molecular Structural Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan); Imai, Keisuke [Lead Discovery Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585 (Japan); Kurihara, Kazuo; Ohhara, Takashi [Molecular Structural Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan); Tada, Toshiji [Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531 (Japan); Kuroki, Ryota, E-mail: kuroki.ryota@jaea.go.jp [Molecular Structural Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan); Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531 (Japan)

2007-04-01

To investigate the structural characteristics of a covalent inhibitor bound to porcine pancreatic elastase (PPE), including H atoms and hydration by water, a crystal of porcine pancreatic elastase with its inhibitor was grown to a size of 1.6 mm{sup 3} for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination owing to the similar atomic scattering properties of deuterium and carbon. Porcine pancreatic elastase (PPE) resembles the attractive drug target leukocyte elastase, which has been implicated in a number of inflammatory disorders. In order to investigate the structural characteristics of a covalent inhibitor bound to PPE, including H atoms and the hydration by water, a single crystal of PPE for neutron diffraction study was grown in D{sub 2}O containing 0.2 M sodium sulfate (pD 5.0) using the sitting-drop vapour-diffusion method. The crystal was grown to a size of 1.6 mm{sup 3} by repeated macroseeding. Neutron diffraction data were collected at room temperature using a BIX-3 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 51.2, b = 57.8, c = 75.6 Å.

Osthol attenuates neutrophilic oxidative stress and hemorrhagic shock-induced lung injury via inhibition of phosphodiesterase 4.

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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.

Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

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Anderson Ronald

2009-10-01

Full Text Available Abstract Background The role of protein kinase C (PKC in regulating the activity of phospholipase C (PLC in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM, was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM and staurosporine (400 nM. Methods Alterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3, and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively. Results Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase. Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils. Conclusion Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.

Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice.

Science.gov (United States)

Bayliss, Jill; Delarosa, Sara; Wu, Jianfeng; Peterson, Jonathan R; Eboda, Oluwatobi N; Su, Grace L; Hemmila, Mark; Krebsbach, Paul H; Cederna, Paul S; Wang, Stewart C; Xi, Chuanwu; Levi, Benjamin

2014-01-01

Extracellular adenosine triphosphate (ATP), present in thermally injured tissue, modulates the inflammatory response and causes significant tissue damage. The authors hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site. Mice were subjected to 30% TBSA partial-thickness scald burn by dorsal skin immersion in a water bath at 60 or 20°C (nonburn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain, elastase, and Ki-67 staining. Tumor necrosis factor (TNF)-α and interferon (IFN)-β expression were measured through quantitative real-time polymerase chain reaction. At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (P burn group at 24 hours and 5 days. TNF-α and IFN-β expression at 24 hours in the apyrase group was lower than in the burn group (P burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially because of TNF-α and IFN-β signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

Non-surgical periodontal therapy decreases serum elastase levels in aggressive but not in chronic periodontitis.

Science.gov (United States)

Eickholz, Peter; Siegelin, Yasemin; Scharf, Susanne; Schacher, Beate; Oremek, Gerhard M; Sauer-Eppel, Hildegund; Schubert, Ralf; Wohlfeil, Martin

2013-04-01

Assessment of the effect of non-surgical periodontal therapy (SRP) on serum inflammatory parameters in patients with untreated aggressive (AgP) and chronic (ChP) periodontitis. Overall, 31 ChP and 29 AgP were examined clinically prior to and 12 weeks after SRP (subgingival scaling of all pockets within 2 days) with systemic antibiotics for patients positive for Aggregatibacter actinomycetemcomitans (14 AgP, 9 ChP). Blood was sampled prior to, one day, 6, and 12 weeks after the first SRP visit. Serum elastase, C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP), interleukin (IL) 6, 8, and leukocyte counts were assessed. At baseline, serum elastase, CRP, and LBP were significantly (p < 0.01) higher in AgP than ChP. Serum elastase, CRP, LBP, and IL-6 were significantly (p < 0.001) elevated one day after scaling in both groups. Both groups showed significant clinical improvement (p < 0.001). A significant difference was observed regarding change of serum elastase 12 weeks after SRP between AgP and ChP (p = 0.015). Multiple regression analysis revealed AgP, African origin, and bleeding on probing to be associated with more pronounced elastase reduction. CRP reduction was associated with African origin, systemic antibiotics, and baseline probing pocket depth. SRP results in serum elastase reduction in AgP but not in ChP. © 2013 John Wiley & Sons A/S.

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.

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

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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.

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.

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.

An evaluation on elastase enzyme activity in gingival crevicular fluid in periodontitis

Directory of Open Access Journals (Sweden)

Qujeq D

2003-08-01

Full Text Available Statement of Problem: Changes in protein levels, host calls enzymes and inflammatory mediators in gingival"ncrevicular Fluid (GCF are considered as diagnostic indicators of Periodontitis."nPurpose: he aim of the present study was to measure the elastase enzyme activity in gingival crevicular Fluid"namong patients with periodontitis."nMaterial and Methods: In this study, 52 periodontitis patients (experimental group and 51 healthy subjects"nwithout any gingival inflammatio (control group were participated. Subjects of the periodontitis group"nshowed pockets of 4-5 mm depth without gingival enlargement and recession or pockets of 1-2 mm depth"nwith gingival recession. For enzyme activity measurement, lOOu,! of gingival fluid of each sample was mixed"nwith lOOu! of enzyme substrate on the tube. The mixture was incubated at 34°c for lh with a buffer solution"nof 1ml volume and absorbance was read at 410nm with spectrophotometer. The enzyme activity differences"nbetween two groups were analyzed by student t test."nResults: The elastase enzyme activity in gingival crevicular fluid in subjects with periodontium destruction"nand control subjects was 153±11.3 and 52.7±10.4 enzyme unit in ml per minute, respectively. The difference"nbetween groups was statistically significant (PO.05."nConclusion: Based on the findings of this study, the measurement of elastae enzyme activity could be a useful"nindication of tissue changes that may ultimately manifest clinically as periodontitis.

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....

Increased galectin-3 levels are associated with abdominal aortic aneurysm progression and inhibition of galectin-3 decrease elastase-induced AAA development

DEFF Research Database (Denmark)

Fernandez-García, Carlos-Ernesto; Tarin, Carlos; Roldan-Montero, Raquel

2017-01-01

Abdominal aortic aneurysm (AAA) evolution is unpredictable. Moreover, no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA have not been addressed. Galectin-3 levels were increased in plasma...... of AAA patients (n=225) compared to controls (n=100). Moreover, galectin-3 concentrations were associated with need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared to healthy aortas. Experimental AAA...... in mice was induced by aortic elastase perfusion. Mice were treated i.v. with the galectin-3 inhibitor modified citrus pectin (MCP, 10mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared to control mice...

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

International Nuclear Information System (INIS)

Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2007-01-01

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 o C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function

Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide.

OpenAIRE

Burns, F R; Paterson, C A; Gray, R D; Wells, J T

1990-01-01

Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL...

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)

The effect of n-3 fatty acids and coenzyme Q10 supplementation on neutrophil leukotrienes, mediators of inflammation resolution and myeloperoxidase in chronic kidney disease.

Science.gov (United States)

Barden, Anne E; Shinde, Sujata; Burke, Valerie; Puddey, Ian B; Beilin, Lawrence J; Irish, Ashley B; Watts, Gerald F; Mori, Trevor A

2018-03-22

Neutrophils release leukotriene (LT)B 4 and myeloperoxidase (MPO) that may be important mediators of chronic inflammation in chronic kidney disease (CKD). The n-3 fatty acids (n-3 FA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have the potential to attenuate inflammation through production of LTB 5 and the Specialized Proresolving Lipid Mediators (SPM) that promote the resolution of inflammation. In animal models, coenzyme Q10 (CoQ) also attenuates inflammation by reducing MPO and LTB 4 . This study evaluated the independent and combined effects of n-3 FA and CoQ supplementation on neutrophil leukotrienes, the pro-inflammatory eicosanoid 5-hydroxyeicosatetraenoic acid (5-HETE), SPM, and plasma MPO, in patients with CKD. In a double-blind, placebo-controlled intervention of factorial design, 85 patients with CKD were randomized to either n-3 FA (4 g), CoQ (200 mg), both supplements, or control (4 g olive oil), daily for 8 weeks. Plasma MPO and calcium ionophore-stimulated neutrophil release of LTs, 5-HETE and SPM were measured at baseline and after 8 weeks. Seventy four patients completed the intervention. n-3 FA, but not CoQ, significantly increased neutrophil LTB 5 (P n-3 FA or CoQ. Plasma MPO was significantly reduced with n-3 FA alone (P = 0.013) but not when given in combination with CoQ. n-3 FA supplementation in patients with CKD leads to increased neutrophil release of LTB 5 and several SPM, as well as a reduction in plasma MPO that may have important implications for limiting chronic inflammation. Copyright © 2018 Elsevier Inc. All rights reserved.

In vivo induction of neutrophil extracellular traps by Mycobacterium tuberculosis in a guinea pig model.

Science.gov (United States)

Filio-Rodríguez, Georgina; Estrada-García, Iris; Arce-Paredes, Patricia; Moreno-Altamirano, María M; Islas-Trujillo, Sergio; Ponce-Regalado, M Dolores; Rojas-Espinosa, Oscar

2017-10-01

In 2004, a novel mechanism of cellular death, called 'NETosis', was described in neutrophils. This mechanism, different from necrosis and apoptosis, is characterized by the release of chromatin webs admixed with microbicidal granular proteins and peptides (NETs). NETs trap and kill a variety of microorganisms. Diverse microorganisms, including Mycobacterium tuberculosis, are NET inducers in vitro. The aim of this study was to examine whether M. tuberculosis can also induce NETs in vivo and if the NETs are bactericidal to the microorganism. Guinea pigs were intradermally inoculated with M. tuberculosis H37Rv, and the production of NETs was investigated at several time points thereafter. NETs were detected as early as 30 min post-inoculation and were clearly evident by 4 h post-inoculation. NETs produced in vivo contained DNA, myeloperoxidase, elastase, histones, ROS and acid-fast bacilli. Viable and heat-killed M. tuberculosis, as well as Mycobacterium bovis BCG were efficient NET inducers, as were unilamellar liposomes prepared with lipids from M. tuberculosis. In vitro, guinea pig neutrophils also produced NETs in response to M. tuberculosis. However, neither the in vivo nor the in vitro-produced NETs were able to kill M. tuberculosis. Nevertheless, in vivo, neutrophils might propitiate recruitment and activation of more efficient microbicidal cells.

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.

Liberation of Desmosine and Isodesmosine as Amino Acids from Insoluble Elastin by Elastolytic Proteases

Science.gov (United States)

Umeda, Hideyuki; Aikawa, Masanori; Libby, Peter

2011-01-01

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation. PMID:21726534

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.

Sulfated caffeic acid dehydropolymer attenuates elastase and cigarette smoke extract-induced emphysema in rats: sustained activity and a need of pulmonary delivery.

Science.gov (United States)

Saluja, Bhawana; Li, Hua; Desai, Umesh R; Voelkel, Norbert F; Sakagami, Masahiro

2014-08-01

Although emphysema destroys alveolar structures progressively and causes death eventually, no drug has been discovered to prevent, intervene, and/or resolve this life-threatening disease. We recently reported that sulfated caffeic acid dehydropolymer CDSO3 is a novel potent triple-action inhibitor of elastolysis, oxidation, and inflammation in vitro, and therefore, a potential anti-emphysema agent. However, the in vivo therapeutic potency, duration and mode of actions, and effective route remain to be demonstrated. Emphysema was induced in rats with human sputum elastase (HSE) combined with cigarette smoke extract (CSE). CDSO3 at 5, 30, or 100 μg/kg was dosed to the lung or injected subcutaneously at 2, 6, or 24 h before or 1 or 24 h or 1 week after the HSE/CSE instillation. At 1 h or 48 h or on day 21-22 or day 28, lungs were examined for airway-to-blood injurious barrier damage; their elastolytic, oxidative, and inflammatory activities; lung luminal leukocytes infiltration; functional treadmill exercise endurance; and/or morphological airspace enlargement. CDSO3, when dosed to the lung at 30 or 100 μg/kg, but not via systemic subcutaneous injection, significantly (43-93 %) attenuated HSE/CSE-induced (1) barrier damage measured by luminal hemorrhage and protein leak; (2) elastolytic, oxidative, and inflammatory activities measured with elastase, reduced glutathione, and TNFα levels, respectively; (3) luminal neutrophil infiltration and tissue myeloperoxidase activity; (4) functional impairment of exercise endurance; and (5) airspace enlargement, in both preventive and interventional dosing protocols. Notably, the effects were shown to last for 24 h at the greater 100-μg/kg dose, and the 1-week-delayed administration was also capable of attenuating the development of emphysema. CDSO3 is a novel, potent, long-acting, nonpeptidic macromolecule that inhibits HSE/CSE-induced elastolysis, oxidation, and inflammation in the lung and thereby attenuates the development

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...

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.

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.

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)

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

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.

The preparation of aneurysm model in rabbits by vessel ligation and elastase-induced technique

International Nuclear Information System (INIS)

Lu Chuan; Xie Qianyu; Liu Linxiang

2010-01-01

Objective: To establish an aneurysm model, which is quite similar to the human intracranial aneurysm in morphology, in rabbits by means of vessel ligation together with elastase-induced technique. Methods: Sixteen New Zealand white rabbits were used in this study. Distal carotid ligation and intraluminal elastase incubation was employed in ten rabbits (study group) to create aneurysm on the right common carotid artery. And surgical suture of a segment of the left carotid common artery was carried out in six rabbits (used as control group) to establish the aneurysm model. DSA exam of the created aneurysms by using catheterization via femoral artery was performed at one week and at one month after surgery. The patency, morphology and pathology of the aneurysms were observed. The results were statistically analyzed. Results: The aneurysms in both groups remained patent after they were created. Angiography one week after the surgery showed that all the aneurysms in study group were patent, while in control group only two aneurysms showed opacification with contrast medium and the remaining four aneurysms were all occluded. DSA at one month after the procedure demonstrated that all the aneurysms in study group remained patent, and the previous two patent aneurysms in control group became occluded. The mean width and length of the aneurysmal cavity in study group immediately after the procedure were (3.70 ± 0.16) mm and (6.53 ± 0.65) mm respectively, which enlarged to (5.06 ± 0.31) mm and (9.0 ± 0.52) mm respectively one month after the surgery. The difference in size changes was statistically significant (P < 0.05). Pathologically, almost complete absence of the internal elastic lamina and medial wall elastin of the aneurysms was observed. Conclusion: The aneurysm model prepared with vessel ligation together with elastase-induced technique carries high patent rate and possesses the feature of spontaneous growing, moreover, its morphology is quite similar to the

Interleukin-17A and Neutrophils in a Murine Model of Bird-Related Hypersensitivity Pneumonitis.

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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.

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.

The effect of garlic extract on the expression of genes elastase and exotoxin A in Pseudomonas aeruginosa

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Batoul Kavyani

2016-11-01

Full Text Available Background: Multidrug-resistant bacteria make many problems in clinical therapy, design and manufacture of synthetic drugs. Pseudomonas aeruginosa is one of the most important multidrug-resistance bacteria leads to variety infections in human especially in immunocompromised, patients with severe burns, and nosocomial infections. It Recent years, this organism makes a big challenge in clinical treatment of infections using a wide range of antibiotics. Medicinal herbs for thousands of years to prevent or treat infectious diseases were considered. Today, pharmacists have high interest of using medicinal herbs to prepare a new antimicrobial compounds. The goal of this study was to investigation the effect of aqueous and alcoholic extract of fresh garlic on the expression of genes encoding elastase and exotoxin A virulence factors, in P. aeruginosa PAO1 strain. Methods: Present study was an experimental study and performed from 2015 to 2016 in Hamadan University of Medical Science, Iran. The minimal inhibitory concentration (MIC and minimal bactericidal concentration (MBC of aqueous and alcoholic extract of garlic was determined. Then in order to investigation the gene expression of elastase and exotoxin A genes, quantitative real-time polymerase chain reaction (qPCR method was performed at sub-MBC concentrations. Results: According to the results aqueous extracts of garlic had better impact in comparison with alcoholic alone. At concentration of 64 and 8 mg/ml of aqueous extract the expression of both elastase and exotoxin A genes were decreased. Although, the expression of elastase gene was most affected by garlic at different concentrations than exotoxin A. Conclusion: The results suggested that the compositions of garlic extracts can inhibit the production of virulence factors in P. aeruginosa. So in order to treat infectious diseases in the near future, medicinal plants known as new antimicrobial drugs can be used alone or with antibiotic drugs

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.

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.

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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

Cell therapy with bone marrow mononuclear cells in elastase-induced pulmonary emphysema.

Science.gov (United States)

Longhini-Dos-Santos, Nathalia; Barbosa-de-Oliveira, Valter Abraão; Kozma, Rodrigo Heras; Faria, Carolina Arruda de; Stessuk, Talita; Frei, Fernando; Ribeiro-Paes, João Tadeu

2013-04-01

Emphysema is characterized by destruction of alveolar walls with loss of gas exchange surface and consequent progressive dyspnea. This study aimed to evaluate the efficiency of cell therapy with bone marrow mononuclear cells (BMMC) in an animal model of elastase-induced pulmonary emphysema. Emphysema was induced in C57Bl/J6 female mice by intranasal instillation of elastase. After 21 days, the mice received bone marrow mononuclear cells from EGFP male mice with C57Bl/J6 background. The groups were assessed by comparison and statistically significant differences (p pulmonary emphysema.

Neutrophil NETs in reproduction: from infertility to preeclampsia and the possibility of fetal loss

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Sinuhe eHahn

2012-11-01

Full Text Available The intention of this review is to provide an overview of the potential role of neutrophil extracellular traps (NETs in mammalian reproduction. Neutrophil NETs appear to be involved in various stages of the reproductive cycle, starting with fertility and possibly ending with fetal loss. The first suggestion that NETs may play a role in pregnancy-related disorders was in preeclampsia, where vast numbers were detected in the intervillous space of affected placentae. The induction of NETosis involved an auto-inflammatory component, mediated by the increased release of placental micro-debris in preeclampsia. This report was the first indicating that NETs may be associated with a human pathology not involving infection.Subsequently, NETs have since then been implicated in bovine or equine infertility, in that semen may become entrapped in the female reproductive during their passage to the oocyte. In this instance interesting species-specific differences are apparent, in that equine sperm evade entrapment via expression of a DNAse-like molecule, whereas highly motile bovine sperm, once free from seminal plasma that promotes interaction with neutrophils, appear impervious to NETs entrapment.Although still in the realm of speculation it is plausible that NETs may be involved in recurrent fetal loss mediated by anti-phospholipid antibodies, or perhaps even in fetal abortion triggered by infections with microorganisms such as L. monocytogenes or B. abortus.

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

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

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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.

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...

Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

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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.

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.

Angiotensin-(1-7 Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

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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.

Neutrophil infiltration and release of IL-8 in airway mucosa from subjects with grain dust-induced occupational asthma.

Science.gov (United States)

Park, H S; Jung, K S; Hwang, S C; Nahm, D H; Yim, H E

1998-06-01

The immuno-pathological mechanism for occupational asthma induced by grain dust (GD) remains to be clarified. There have been few reports suggesting the involvement of neutrophils inducing bronchoconstriction after inhalation of GD. To further understand the role of neutrophil in the pathogenesis of GD-induced asthma. We studied the phenotype of leucocytes of the bronchial mucosa in patients with GD-induced asthma. Bronchial biopsy specimens were obtained by fibreoptic bronchoscopy from six subjects with GD-induced asthma. Six allergic asthma patients sensitive to house dust mite were enrolled as controls. Bronchial biopsy specimens were examined by immunohistochemistry with a panel of monoclonal antibodies to tryptase-containing mast cell (AA1), activated eosinophil (EG2), pan T-lymphocyte (CD3) and neutrophil elastase (NE). Induced sputum was collected before and after the GD-bronchoprovocation test. The IL-8 level in the sputum was measured using ELISA. There was a significant increase in the number of AA1+ and NE+ cells in bronchial mucosa of GD-induced asthma, compared with those of allergic asthma (P=0.01, P=0.01, respectively). No significant differences were observed in the number of EG2+ and CD3+ cells (P = 0.13, P=0.15, respectively). IL-8 was abundant in the sputum of all GD-induced asthma patients and significantly increased after the bronchial challenges compared with the baseline value (P = 0.03). These findings support the view that neutrophil recruitment together with mast cells may contribute to the bronchoconstriction induced by GD. A possible involvement of IL-8 was suggested.

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.

Characterization of a Mouse Model of Emphysema Induced by Multiple Instillations of Low-Dose Elastase

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Milena V. Oliveira

2016-10-01

Full Text Available Many experimental models have been proposed to study the pathophysiological features of emphysema, as well as to search for new therapeutic approaches for acute or chronically injured lung parenchyma. We aimed to characterize an emphysema model induced by multiple instillations of elastase by tracking the changes in inflammation, remodeling, and cardiac function after each instillation. Forty-eight C57BL/6 mice were randomly assigned across 2 groups. Emphysema (ELA animals received 1, 2, 3, or 4 intratracheal instillations of pancreatic porcine elastase (PPE, 0.2 IU with a 1-week interval between them. Controls (C received saline following the same protocol. Before and after implementation of the protocol, animals underwent echocardiographic analysis. After the first instillation of PPE, the percentage of mononuclear cells in the lung parenchyma was increased compared to C (p = 0.0001. The second instillation resulted in hyperinflated alveoli, increased mean linear intercept, and reduced elastic fiber content in lung parenchyma compared to C (p=0.0197. Following the third instillation, neutrophils and collagen fiber content in alveolar septa and airways were increased, whereas static lung elastance was reduced compared to C (p=0.0094. After the fourth instillation, the percentage of M1 macrophages in lungs; levels of interleukin-1beta, keratinocyte-derived chemokine, hepatocyte growth factor, and vascular endothelial growth factor; and collagen fiber content in the pulmonary vessel wall were increased compared to C (p=0.0096. At this time point, pulmonary arterial hypertension was apparent, with increased diastolic right ventricular wall thickness. In conclusion, the initial phase of emphysema was characterized by lung inflammation with predominance of mononuclear cells, whereas at the late stage, impairment of pulmonary and cardiovascular functions was observed. This model enables analysis of therapies at different time points during controlled

The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

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Philippe Behe

2017-05-01

Full Text Available The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl- current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A; the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their “swell activated” Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

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

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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.

Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

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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.

Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro.

LENUS (Irish Health Repository)

Power, C

2012-02-03

Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b\\/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN\\'s or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

Gβ1 is required for neutrophil migration in zebrafish.

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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.

Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

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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.

Diverse novel functions of neutrophils in immunity, inflammation, and beyond.

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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.

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.

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

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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

IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

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Kim, Ji Hyung; Oh, Sae Jin; Ahn, Sehee; Chung, Doo Hyun

2014-07-01

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT-cell-deficient mice of C57BL/6 background during cecal ligation and puncture-induced sepsis. The levels of C5a, IFN-γ, and IL-10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d(-/-) mice, while the mortality rate was lower in CD1d(-/-) mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d(-/-) mice. As assessed by intracellular staining, NKT cells expressed IFN-γ, while neutrophils expressed IL-10. Upon coculture, IL-10-deficient NKT cells enhanced IL-10 production by WT, but not IFN-γR-deficient, neutrophils. Meanwhile, CD1d(-/-) mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL-10 administration into CD1d(-/-) mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN-γ-deficient, NKT cells into CD1d(-/-) mice suppressed CD55 expression on neutrophils, but increased IL-10 and C5a levels. Taken together, IFN-γ-producing NKT cells enhance C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pig but not Human Interferon-γ Initiates Human Cell-Mediated Rejection of Pig Tissue in vivo

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Sultan, Parvez; Murray, Allan G.; McNiff, Jennifer M.; Lorber, Marc I.; Askenase, Philip W.; Bothwell, Alfred L. M.; Pober, Jordan S.

1997-08-01

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ ) induced human CD4+ and CD8+ T cells and macrophages to more extensively infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

IL-23 Is Essential for the Development of Elastase-Induced Pulmonary Inflammation and Emphysema.

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Fujii, Utako; Miyahara, Nobuaki; Taniguchi, Akihiko; Waseda, Koichi; Morichika, Daisuke; Kurimoto, Etsuko; Koga, Hikari; Kataoka, Mikio; Gelfand, Erwin W; Cua, Daniel J; Yoshimura, Akihiko; Tanimoto, Mitsune; Kanehiro, Arihiko

2016-11-01

We recently reported that IL-17A plays a critical role in the development of porcine pancreatic elastase (PPE)-induced emphysema. The proliferation of T-helper type 17 (Th17) cells was induced by IL-23. To determine the contribution of IL-23 to the development of pulmonary emphysema, a mouse model of PPE-induced emphysema was used in which responses of IL-23p19-deficient (IL-23 -/- ) and wild-type (WT) mice were compared. Intratracheal instillation of PPE induced emphysematous changes in the lungs and was associated with increased levels of IL-23 in lung homogenates. Compared with WT mice, IL-23 -/- mice developed significantly lower static compliance values and markedly reduced emphysematous changes on histological analyses after PPE instillation. These changes were associated with lower levels of IL-17A and fewer Th17 cells in the lung. The neutrophilia seen in bronchoalveolar lavage fluid of WT mice was attenuated in IL-23 -/- mice, and the reduction was associated with decreased levels of keratinocyte-derived cytokine and macrophage inflammatory protein-2 in bronchoalveolar lavage fluid. Treatment with anti-IL-23p40 monoclonal antibody significantly attenuated PPE-induced emphysematous changes in the lungs of WT mice. These data identify the important contributions of IL-23 to the development of elastase-induced pulmonary inflammation and emphysema, mediated through an IL-23/IL-17 pathway. Targeting IL-23 in emphysema is a potential therapeutic strategy for delaying disease progression.

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

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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.

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...

18F-fluoro-2-deoxyglucose PET informs neutrophil accumulation and activation in lipopolysaccharide-induced acute lung injury.

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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

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

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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.

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice

OpenAIRE

Almeida-Reis, Rafael; Theodoro-Junior, Osmar A.; Oliveira, Bruno T. M.; Oliva, Leandro V.; Toledo-Arruda, Alessandra C.; Bonturi, Camila R.; Brito, Marlon V.; Lopes, Fernanda D. T. Q. S.; Prado, Carla M.; Florencio, Ariana C.; Martins, Mílton A.; Owen, Caroline A.; Leick, Edna A.; Oliva, Maria L. V.; Tibério, Iolanda F. L. C.

2017-01-01

Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.??C57BL/6 mice received intratracheal elastase (ELA group) or saline (SAL group). One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group). Controls received saline and BbCI (SALBC group). After 28 days, we evaluated respirator...

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

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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.

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...

Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

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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.

Neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ALI/ARDS.

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Li, Haitao; Zhou, Xiaoting; Tan, Hongyi; Hu, Yongbin; Zhang, Lemeng; Liu, Shuai; Dai, Minhui; Li, Yi; Li, Qian; Mao, Zhi; Pan, Pinhua; Su, Xiaoli; Hu, Chengpin

2018-01-05

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a manifestation of systemic inflammation in the lungs, but the factors that trigger inflammation in ALI/ARDS are unclear. We hypothesized that neutrophil extracellular traps (NETs) contribute to the pathogenesis of acid aspiration-induced ALI/ARDS. Analysis of bronchial aspirates from ARDS patients showed that NETs were significantly correlated with the degree of ARDS (r = -0.5846, p = 0.0359). NETs in bronchoalveolar lavage fluid of acid-aspiration mice were significantly higher (141.6 ± 23.08) at 3 h after injury than those in the sham group (1234 ± 101.9; p = 0.003, n = 5 per group). Exogenous NETs aggravated lung injury, while alvelestat and DNase markedly attenuated the intensity of ARDS. We investigated whether NETs are involved in the severity of gastric aspiration-induced ARDS. Then, a hydrochloric acid aspiration-induced ALI murine model was used to assess whether NETs are pathogenic and whether targeting NETs is protective. Exogenous NETs were administered to mice. Alvelestat can inhibit neutrophil elastase (NE), which serves an important role in NET formation, so we investigated whether alvelestat could protect against ALI in cell and mouse models. NETs may contribute to ALI/ARDS by promoting tissue damage and systemic inflammation. Targeting NETs by alvelestat may be a potential therapeutic strategy.

d(− Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

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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.

Inhibitory effects of constituents of Morinda citrifolia seeds on elastase and tyrosinase.

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Masuda, Megumi; Murata, Kazuya; Fukuhama, Akiko; Naruto, Shunsuke; Fujita, Tadashi; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

2009-07-01

A 50% ethanolic extract (MCS-ext) from seeds of Morinda citrifolia ("noni" seeds) showed more potent in vitro inhibition of elastase and tyrosinase, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than extracts of M. citrifolia leaves or flesh. Activity-guided fractionation of MCS-ext using in vitro assays led to the isolation of ursolic acid as an active constituent of elastase inhibitory activity. 3,3'-Bisdemethylpinoresinol, americanin A, and quercetin were isolated as active constituents having both tyrosinase inhibitory and radical scavenging activities. Americanin A and quercetin also showed superoxide dismutase (SOD)-like activity. These active compounds were isolated from noni seeds for the first time.

Yersinia pestis targets neutrophils via complement receptor 3

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Merritt, Peter M.; Nero, Thomas; Bohman, Lesley; Felek, Suleyman; Krukonis, Eric S.; Marketon, Melanie M.

2015-01-01

Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Yersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y. pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins due to reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria toward neutrophils during plague infection. PMID:25359083

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

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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.

Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities.

Science.gov (United States)

Lee, Kwang Sik; Kim, Bo Yeon; Yoon, Hyung Joo; Choi, Yong Soo; Jin, Byung Rae

2016-10-01

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Identifying Network Motifs that Buffer Front-to-Back Signaling in Polarized Neutrophils

Directory of Open Access Journals (Sweden)

Yanqin Wang

2013-05-01

Full Text Available Neutrophil polarity relies on local, mutual inhibition to segregate incompatible signaling circuits to the leading and trailing edges. Mutual inhibition alone should lead to cells having strong fronts and weak backs or vice versa. However, analysis of cell-to-cell variation in human neutrophils revealed that back polarity remains consistent despite changes in front strength. How is this buffering achieved? Pharmacological perturbations and mathematical modeling revealed a functional role for microtubules in buffering back polarity by mediating positive, long-range crosstalk from front to back; loss of microtubules inhibits buffering and results in anticorrelation between front and back signaling. Furthermore, a systematic, computational search of network topologies found that a long-range, positive front-to-back link is necessary for back buffering. Our studies suggest a design principle that can be employed by polarity networks: short-range mutual inhibition establishes distinct signaling regions, after which directed long-range activation insulates one region from variations in the other.

Neutrophil targeted nano-drug delivery system for chronic obstructive lung diseases.

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Vij, Neeraj; Min, Taehong; Bodas, Manish; Gorde, Aakruti; Roy, Indrajit

2016-11-01

The success of drug delivery to target airway cell(s) remains a significant challenge due to the limited ability of nanoparticle (NP) systems to circumvent protective airway-defense mechanisms. The size, density, surface and physical-chemical properties of nanoparticles are the key features that determine their ability to navigate across the airway-barrier. We evaluated here the efficacy of a PEGylated immuno-conjugated PLGA-nanoparticle (PINP) to overcome this challenge and selectively deliver drug to specific inflammatory cells (neutrophils). We first characterized the size, shape, surface-properties and neutrophil targeting using dynamic laser scattering, transmission electron microscopy and flow cytometry. Next, we assessed the efficacy of neutrophil-targeted PINPs in transporting through the airway followed by specific binding and release of drug to neutrophils. Finally, our results demonstrate the efficacy of PINP mediated non-steroidal anti-inflammatory drug-(ibuprofen) delivery to neutrophils in murine models of obstructive lung diseases, based on its ability to control neutrophilic-inflammation and resulting lung disease. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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.

Localization and stretch-dependence of lung elastase activity in development and compensatory growth.

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Young, Sarah Marie; Liu, Sheng; Joshi, Rashika; Batie, Matthew R; Kofron, Matthew; Guo, Jinbang; Woods, Jason C; Varisco, Brian Michael

2015-04-01

Synthesis and remodeling of the lung matrix is necessary for primary and compensatory lung growth. Because cyclic negative force is applied to developing lung tissue during the respiratory cycle, we hypothesized that stretch is a critical regulator of lung matrix remodeling. By using quantitative image analysis of whole-lung and whole-lobe elastin in situ zymography images, we demonstrated that elastase activity increased twofold during the alveolar stage of postnatal lung morphogenesis in the mouse. Remodeling was restricted to alveolar walls and ducts and was nearly absent in dense elastin band structures. In the mouse pneumonectomy model of compensatory lung growth, elastase activity increased threefold, peaking at 14 days postpneumonectomy and was higher in the accessory lobe compared with other lobes. Remodeling during normal development and during compensatory lung growth was different with increased major airway and pulmonary arterial remodeling during development but not regeneration, and with homogenous remodeling throughout the parenchyma during development, but increased remodeling only in subpleural regions during compensatory lung growth. Left lung wax plombage prevented increased lung elastin during compensatory lung growth. To test whether the adult lung retains an innate capacity to remodel elastin, we developed a confocal microscope-compatible stretching device. In ex vivo adult mouse lung sections, lung elastase activity increased exponentially with strain and in peripheral regions of lung more than in central regions. Our study demonstrates that lung elastase activity is stretch-dependent and supports a model in which externally applied forces influence the composition, structure, and function of the matrix during periods of alveolar septation. Copyright © 2015 the American Physiological Society.

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

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.

Neutrophil-Mediated Delivery of Therapeutic Nanoparticles across Blood Vessel Barrier for Treatment of Inflammation and Infection.

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Chu, Dafeng; Gao, Jin; Wang, Zhenjia

2015-12-22

Endothelial cells form a monolayer in lumen of blood vessels presenting a great barrier for delivery of therapeutic nanoparticles (NPs) into extravascular tissues where most diseases occur, such as inflammation disorders and infection. Here, we report a strategy for delivering therapeutic NPs across this blood vessel barrier by nanoparticle in situ hitchhiking activated neutrophils. Using intravital microscopy of TNF-α-induced inflammation of mouse cremaster venules and a mouse model of acute lung inflammation, we demonstrated that intravenously (iv) infused NPs made from denatured bovine serum albumin (BSA) were specifically internalized by activated neutrophils, and subsequently, the neutrophils containing NPs migrated across blood vessels into inflammatory tissues. When neutrophils were depleted using anti-Gr-1 in a mouse, the transport of albumin NPs across blood vessel walls was robustly abolished. Furthermore, it was found that albumin nanoparticle internalization did not affect neutrophil mobility and functions. Administration of drug-loaded albumin NPs markedly mitigated the lung inflammation induced by LPS (lipopolysaccharide) or infection by Pseudomonas aeruginosa. These results demonstrate the use of an albumin nanoparticle platform for in situ targeting of activated neutrophils for delivery of therapeutics across the blood vessel barriers into diseased sites. This study demonstrates our ability to hijack neutrophils to deliver nanoparticles to targeted diseased sites.

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

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.

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

TGF-β1 and granulocyte elastase in the evaluation of activity of inflammatory bowel disease. A pilot study

Directory of Open Access Journals (Sweden)

Irena Ciećko-Michalska

2014-01-01

Full Text Available Introduction: The aim was to assess the usefulness of TGF-β1 and elastase in the evaluation of activity of ulcerative colitis (UC and Crohn’s disease (CD.Material and Methods: 32 patients diagnosed with UC, 31 with CD and 30 healthy volunteers were enrolled in this study. Diagnosis of the disease was confirmed by videocolonoscopy and histopathological evaluation of intestinal biopsies. Disease activity was assessed by use of the Mayo Scoring System for Assessment of Ulcerative Colitis Activity in UC patients and by CDAI in CD patients. hsCRP was determined by the immunonephelometric method, TGF-β1 and elastase plasma concentration by ELISA. The results of the study were analyzed using Statistica and R statistical language.Results: In UC a positive correlation between disease activity and platelet level, hsCRP and TGF-β1 concentration was noted. Elastase concentration in UC patients was significantly higher than in CD, but there was no correlation with the activity of the disease. In CD patients we observed a positive correlation between disease activity and leukocytes, platelet levels and elastase concentration, and a very low correlation with hsCRP and TGF-β1.Discussion: Determination of TGF-β1 can be used for evaluation of inflammatory activity in UC and it is connected with elevated concentrations of CRP and platelets. To a lower extent TGF-β1 can also be used for evaluation of inflammatory activity in CD. Examination of elastase concentration may be useful in the assessment of CD activity. Plasma elastase concentration may be helpful in UC and CD differentiation. The preliminary results of this investigation seem promising; nevertheless, more studies are necessary.

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

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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

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

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target

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Knight, Jason S.; Meng, He; Coit, Patrick; Yalavarthi, Srilakshmi; Sule, Gautam; Gandhi, Alex A.; Grenn, Robert C.; Mazza, Levi F.; Ali, Ramadan A.; Renauer, Paul; Wren, Jonathan D.; Bockenstedt, Paula L.; Wang, Hui; Eitzman, Daniel T.; Sawalha, Amr H.

2017-01-01

Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils — neutrophil extracellular traps (NETs), in particular — in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1–KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1–deficient mice by infusion of WT neutrophils, while an anti–PSGL-1 monoclonal antibody inhibited APS IgG–mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS. PMID:28931754

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

Insights into the degradation of human elastin by matrilysin-1

DEFF Research Database (Denmark)

Heinz, Andrea; Taddese, Samuel; Sippl, Wolfgang

2011-01-01

Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describin...... into the degradation of human elastin by MMP-7 and may aid in the development of approaches to treat elastin-degrading diseases.......Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describing...... the enzymatic turnover of matrix components helps to understand the molecular basis of disease processes. Hence, in this work, the cleavage behavior of MMP-7 with respect to its natural substrate human elastin was investigated using mass spectrometric (MS) techniques and molecular modeling. Elastin peptides...

CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation

NARCIS (Netherlands)

Marcos, Veronica; Zhou, Zhe; Yildirim, Ali Onder; Bohla, Alexander; Hector, Andreas; Vitkov, Ljubomir; Wiedenbauer, Eva-Maria; Krautgartner, Wolf Dietrich; Stoiber, Walter; Belohradsky, Bernd H.; Rieber, Nikolaus; Kormann, Michael; Koller, Barbara; Roscher, Adelbert; Roos, Dirk; Griese, Matthias; Eickelberg, Oliver; Döring, Gerd; Mall, Marcus A.; Hartl, Dominik

2010-01-01

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases.

A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

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Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E; Araujo-Hernandez, Sophia A; Harrison, Joe J; MacCoss, Michael J; Parsek, Matthew R

2018-04-10

Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. IMPORTANCE Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix

Multiple lupus-associated ITGAM variants alter Mac-1 functions on neutrophils.

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Zhou, Yebin; Wu, Jianming; Kucik, Dennis F; White, Nathan B; Redden, David T; Szalai, Alexander J; Bullard, Daniel C; Edberg, Jeffrey C

2013-11-01

Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the nonsynonymous SNPs rs1143679, rs1143678, and rs1143683) are associated with systemic lupus erythematosus (SLE). ITGAM encodes the protein CD11b, a subunit of the β2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biologic functions of neutrophil Mac-1. Neutrophils from ITGAM-genotyped and -sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement-coated erythrocytes, serum-treated zymosan, heat-treated zymosan, and IgG-coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells, was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry. Mac-1-mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the β-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation. The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE

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

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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.

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

International Nuclear Information System (INIS)

Wan Haiyan; Korzh, Svitlana; Li Zhen; Mudumana, Sudha Puttur; Korzh, Vladimir; Jiang Yunjin; Lin Shuo; Gong Zhiyuan

2006-01-01

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development

The third serine proteinase with chymotrypsin specificity isolated from Atlantic cod (Gadus morhua) is a type-II elastase

DEFF Research Database (Denmark)

Asgeirsson, B; Leth-Larsen, Rikke; Thórólfsson, M

1998-01-01

-Ala-Ala-Pro-Phe-p-nitroanilide, but inactive against the typical elastase substrates succinyl-Ala-Ala-Ala-p-nitroanilide and orcein-elastin. Comparison of the kinetic properties of the cod elastase C with bovine chymotrypsin and cod chymotrypsin variants A and B, using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, showed a lower catalytic...

Severe congenital neutropenia

DEFF Research Database (Denmark)

Borregaard, Niels

2014-01-01

In this issue of Blood, Tidwell et al1 demonstrate that mutations in the start codon (protein synthesis is initiated at the codon ATG) of neutrophil elastase (ELANE) result in the production of N-terminally truncated elastase, which mislocates to the nucleus and results in severe congenital neutr...... neutropenia (SCN)....

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.

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

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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.

Association of Low Fecal Elastase-1 and Non-Ulcer Dyspepsia

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Mustafa Tahtaci

2018-06-01

Full Text Available Non-ulcer dyspepsia (NUD is a term used to define a set of symptoms that are believed to originate from the gastroduodenal region, and no underlying organic, systemic, or metabolic reason can be found. The majority of patients suffer from chronic symptoms although half of the patients report improvement in symptoms with time. The potential role exocrine pancreatic insufficiency in NUD patients has not been clarified yet. We aimed to identify exocrine pancreas function with pancreatic fecal elastase-1 in patients diagnosed with non-ulcer dyspepsia and no typical exocrine pancreatic insufficiency (EPI symptoms. Thirty-five patients referred to gastroenterology clinics with NUD and 35 people with no dyspeptic symptoms as a control group were included in this prospective study. Non-ulcer dyspepsia patients were classified as group 1 and control subjects classified as group 2. Upper gastrointestinal endoscopies were performed in both groups. Assessment of exocrine pancreatic function was performed by measuring fecal elastase-1 concentration with a commercial ELISA kit using polyclonal antibodies (BioServ Diagnostics in NUD patients compared to control subjects. Mean fecal elastase-1 levels were significantly lower in group 1 patients compared with group 2 (367.47 ± 43.27; 502.48 ± 50.94 respectively; p = 0.04. The percentage of the patients with EPI was significantly higher in group 1 (p = 0.02. Patients with NUD should be re-evaluated if they do not show satisfactory improvement with treatment. Exocrine pancreatic insufficiency was significantly higher in patients with NUD in our study. Evaluation for the presence of EPI can be a cost effective approach in management of refractory patients during the process of ruling out organic reasons.

Application of chemical arrays in screening elastase inhibitors.

Science.gov (United States)

Gao, Feng; Du, Guan-Hua

2006-06-01

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).

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)

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)

NKT cells mediate the recruitment of neutrophils by stimulating epithelial chemokine secretion during colitis.

Science.gov (United States)

Huang, Enyu; Liu, Ronghua; Lu, Zhou; Liu, Jiajing; Liu, Xiaoming; Zhang, Dan; Chu, Yiwei

2016-05-27

Ulcerative colitis (UC) is a kind of inflammatory bowel diseases characterized by chronic inflammation and ulcer in colon, and UC patients have increased risk of getting colorectal cancer. NKT cells are cells that express both NK cell markers and semi-invariant CD1d-restricted TCRs, can regulate immune responses via secreting a variety of cytokines upon activation. In our research, we found that the NKT cell-deficient CD1d(-/-) mice had relieved colitis in the DSS-induced colitis model. Further investigations revealed that the colon of CD1d(-/-) mice expressed less neutrophil-attracting chemokine CXCL 1, 2 and 3, and had decreased neutrophil infiltration. Infiltrated neutrophils also produced less reactive oxygen species (ROS) and TNF-α, indicating they may cause less epithelial damage. In addition, colitis-associated colorectal cancer was also relieved in CD1d(-/-) mice. During colitis, NKT cells strongly expressed TNF-α, which could stimulate CXCL 1, 2, 3 expressions by the epithelium. In conclusion, NKT cells can regulate colitis via the NKT cell-epithelium-neutrophil axis. Targeting this mechanism may help to improve the therapy of UC and prevent colitis-associated colorectal cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Poly herbal formulation with anti-elastase and anti-oxidant properties for skin anti-aging.

Science.gov (United States)

Kalyana Sundaram, Induja; Sarangi, Deepika Deeptirekha; Sundararajan, Vignesh; George, Shinomol; Sheik Mohideen, Sahabudeen

2018-01-29

Skin forms an important part of human innate immune system. Wrinkles, thinning and roughening of skin are some of the symptoms that affect the skin as it ages. Reactive oxygen species induced oxidative stress plays a major role in skin aging by modulating the elastase enzyme level in the skin. Extrinsic factors that affect skin aging such as UV radiation can also cause malignant melanoma. Here we selected four medicinal plant materials, namely, leaves of Nyctanthes arbor-tristis, unripe and ripe Aegle marmelos fruit pulp and the terminal meristem of Musa paradisiaca flower and investigated their anti-aging properties and cytotoxicity in vitro individually as well as in a poly herbal formulation containing the four plant extracts in different ratios. The phytochemical contents of the plant extracts were investigated for radical scavenging activity and total reducing power. Based upon its anti-oxidant properties, a poly herbal formulation containing leaves of Nyctanthes arbor-tristis, unripe and ripe fruit pulp of Aegle marmelos, and the terminal meristem of Musa paradisiaca flower in the ratio 6:2:1:1 (Poly Herbal Formulation 1) and 1:1:1:1 (Poly Herbal Formulation 2), respectively were formulated. It has been observed that the Poly Herbal Formulation 1 was more potent than Poly Herbal Formulation 2 due to better anti-oxidant and anti-elastase activities in NIH3T3 fibroblast cells. In addition Poly Herbal formulation 1 also had better anti-cancer activity in human malignant melanoma cells. Based on these results these beneficial plant extracts were identified for its potential application as an anti-aging agent in skin creams as well as an anti-proliferation compound against cancer cells.

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.

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.

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.

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

Alterations in pulmonary structure by elastase administration in a model of emphysema in mice is associated with functional disturbances

Directory of Open Access Journals (Sweden)

D. Vidal

2012-05-01

Full Text Available Several experimental studies of pulmonary emphysema using animal models have been described in the literature. However, only a few of these studies have focused on the assessment of ergometric function as a non-invasive technique to validate the methodology used for induction of experimental emphysema. Additionally, functional assessments of emphysema are rarely correlated with morphological pulmonary abnormalities caused by induced emphysema. The present study aimed to evaluate the effects of elastase administered by tracheal puncture on pulmonary parenchyma and their corresponding functional impairment. This was evaluated by measuring exercise capacity in C57Bl/6 mice in order to establish a reproducible and safe methodology of inducing experimental emphysema. Thirty six mice underwent ergometric tests before and 28 days after elastase administration. Pancreatic porcine elastase solution was administered by tracheal puncture, which resulted in a significantly decreased exercise capacity, shown by a shorter distance run (−30.5% and a lower mean velocity (−15%, as well as in failure to increase the elimination of carbon dioxide. The mean linear intercept increased significantly by 50% in tracheal elastase administration. In conclusion, application of elastase by tracheal function in C57Bl/6 induces emphysema, as validated by morphometric analyses, and resulted in a significantly lower exercise capacity, while resulting in a low mortality rate. Resumo: Vários estudos experimentais de enfisema pulmonar em modelos animais têm sido descritos na literatura científica. No entanto, apenas alguns destes estudos têm sido concentrados na avaliação da função ergométrica como técnica não-invasiva para validar a metodologia utilizada para a indução do enfisema experimental. Além disso, as avaliações funcionais de enfisema raramente se encontram correlacionadas com anomalias morfológicas pulmonares

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

International Nuclear Information System (INIS)

Traynor-Kaplan, A.E.; Thompson, B.L.; Harris, A.L.; Taylor, P.; Omann, G.M.; Sklar, L.A.

1989-01-01

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation

Neutrophil trails guide influenza-specific CD8+ T cells in the airways

Science.gov (United States)

Lim, Kihong; Hyun, Young-Min; Lambert-Emo, Kris; Capece, Tara; Bae, Seyeon; Miller, Richard; Topham, David J.; Kim, Minsoo

2016-01-01

During viral infections, chemokines guide activated effector T cells to infection sites. However, the cells responsible for producing these chemokines and how such chemokines recruit T cells is unknown. Here, we show that the early recruitment of neutrophils into influenza-infected trachea is essential for CD8+ T cell-mediated immune protection in mice. We observed that migrating neutrophils leave behind long-lasting trails that are enriched in the chemokine CXCL12. Experiments with granulocyte-specific CXCL12 conditional knock-out mice and a CXCR4 antagonist revealed that CXCL12 derived from neutrophil trails is critical for virus-specific CD8+ T cell recruitment and effector functions. Collectively, these results suggest neutrophils deposit long-lasting, chemokine-containing trails, which may provide both chemotactic and haptotactic cues for efficient CD8+ T cell migration and localization in influenza-infected tissues. PMID:26339033

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

Science.gov (United States)

Yan, Meiping; Zhang, Xinhua; Chen, Ao; Gu, Wei; Liu, Jie; Ren, Xiaojiao; Zhang, Jianping; Wu, Xiaoxiong; Place, Aaron T; Minshall, Richard D; Liu, Guoquan

2017-11-01

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2- via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction. © FASEB.

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

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)

Increased galectin-3 levels are associated with abdominal aortic aneurysm progression and inhibition of galectin-3 decreases elastase-induced AAA development.

Science.gov (United States)

Fernandez-García, Carlos-Ernesto; Tarin, Carlos; Roldan-Montero, Raquel; Martinez-Lopez, Diego; Torres-Fonseca, Monica; Lindhot, Jes S; Vega de Ceniga, Melina; Egido, Jesus; Lopez-Andres, Natalia; Blanco-Colio, Luis-Miguel; Martín-Ventura, Jose-Luis

2017-11-15

Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients ( n =225) compared with the control group ( n =100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Inhibitory effect of burdock leaves on elastase and tyrosinase activity

Science.gov (United States)

Horng, Chi-Ting; Wu, Hsing-Chen; Chiang, Ni-Na; Lee, Chiu-Fang; Huang, Yu-Syuan; Wang, Hui-Yun; Yang, Jai-Sing; Chen, Fu-An

2017-01-01

Burdock (Arctium lappa L.) leaves generate a considerable amount of waste following burdock root harvest in Taiwan. To increase the use of burdock leaves, the present study investigated the optimal methods for producing burdock leaf extract (BLE) with high antioxidant polyphenolic content, including drying methods and solvent extraction concentration. In addition, the elastase and tyrosinase inhibitory activity of BLE was examined. Burdock leaves were dried by four methods: Shadow drying, oven drying, sun drying and freeze-drying. The extract solution was then subjected to total polyphenol content analysis and the method that produced BLE with the highest amount of total antioxidant components was taken forward for further analysis. The 1,1-diphenyl-2-pycrylhydrazyl scavenging, antielastase and antityrosinase activity of the BLE were measured to enable the evaluation of the antioxidant and skin aging-associated enzyme inhibitory activities of BLE. The results indicated that the total polyphenolic content following extraction with ethanol (EtOH) was highest using the freeze-drying method, followed by the oven drying, shadow drying and sun drying methods. BLE yielded a higher polyphenol content and stronger antioxidant activity as the ratio of the aqueous content of the extraction solvent used increased. BLE possesses marked tyrosinase and elastase inhibitory activities, with its antielastase activity notably stronger compared with its antityrosinase activity. These results indicate that the concentration of the extraction solvent was associated with the antioxidant and skin aging-associated enzyme inhibitory activity of BLE. The reactive oxygen species scavenging theory of skin aging may explain the tyrosinase and elastase inhibitory activity of BLE. In conclusion, the optimal method for obtaining BLE with a high antioxidant polyphenolic content was freeze-drying followed by 30–50% EtOH extraction. In addition, the antielastase and antityrosinase activities of the

The LPS-induced neutrophil recruitment into rat air pouches is mediated by TNFα: likely macrophage origin

Directory of Open Access Journals (Sweden)

C-D. Arreto

1997-01-01

Full Text Available The role of resident cells during the lipopolysaccharide (LPS-induced neutrophil recruitment into rat air pouches was investigated. In this model, LPS (Escherichia coli, O55: B5 strain; 2–2000 ng induced a dose– and time-dependent neutrophil recruitment accompanied by the generation of a tumour necrosis factor-α (TNFα-like activity. Dexamethasone (0.05–5 mug and cycloheximide (6 ng, injected 2 h before LPS into the pouches, inhibited the neutrophil recruitment and the generation of the TNFα-like activity, while the H1-receptor antagonist mepyramine (1 and 4 mg/kg, i.p., 0.5 h before LPS and the PAF-receptor antagonist WEB 2170 (0.05 and 1 mg/kg, i.p., 0.5 h before LPS had no effect. Purified alveolar macrophages (AM were used to replenish the pouches of cycloheximide-treated recipient rats. AM provided by PBS-treated animals led to the recovery of the LPS-induced neutrophil recruitment and of the TNFα-like formation contrasting with those from cycloheximide-treated animals (1 mg/kg, i.p.. When delivered in situ, liposome-encapsulated clodronate, a macrophage depletor, significantly impaired both the LPSinduced neutrophil recruitment and the TNFα-like activity. An anti-murine TNFα polyclonal antibody (0.5 h before LPS was also effective. These results emphasize the pivotal role of macrophages for LPS-induced neutrophil recruitment via the formation of TNFα.

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

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

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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

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.

Pulmonary deposition and disappearance of aerosolised secretory leucocyte protease inhibitor.

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Stolk, J.; Camps, J.; Feitsma, H. I.; Hermans, J.; Dijkman, J. H.; Pauwels, E. K.

1995-01-01

BACKGROUND--The neutrophil elastase inhibitor, secretory leucocyte protease inhibitor (SLPI), is a potential therapeutic tool in inflammatory lung diseases such as cystic fibrosis and pulmonary emphysema. The distribution and disappearance in the lung of aerosolised recombinant SLPI (rSLPI) was investigated in healthy humans and in patients with cystic fibrosis or alpha 1-antitrypsin-associated emphysema. METHODS--To distinguish aerosolised rSLPI from endogenous SLPI the recombinant inhibitor was radiolabelled with 99m-technetium (99mTc) pertechnetate. Distribution and disappearance of aerosolised 99mTc-rSLPI in the lungs were studied by gamma radiation imaging. RESULTS--The deposition of 99mTc-rSLPI in normal volunteers was homogeneous in all lung lobes, while in patients with cystic fibrosis or emphysema only well ventilated areas showed deposition of the aerosol. The disappearance rate of 99mTc-rSLPI was biexponential. The half life of the rapid phase was 0.2-2.8 hours, while that of the slow phase was more than 24 hours. CONCLUSIONS--Future aerosol therapy with rSLPI will be most beneficial for well ventilated lung tissue that needs protection against neutrophil derived elastase. It may be more difficult to neutralise the burden of elastase in poorly ventilated, highly inflamed areas as are seen in cystic fibrosis. Images PMID:7638807

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 ...

Hypercholesterolemia Increases the Production of Leukotriene B4 in Neutrophils by Enhancing the Nuclear Localization of 5-Lipoxygenase

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Xiao-Feng Lai

2014-11-01

Full Text Available Aims: Neutrophils can synthesize leukotriene B4 (LTB4 by activating the 5-lipoxygenase (5-LOsignaling pathway. LTB4 is a pro-inflammatory mediator associated with the etiology and progression of atherosclerosis. It can increase function and number of neutrophils in an autocrine manner. Since hypercholesterolemia is associated with an increase in the number and function of neutrophils, we hypothesized that this effect could be mediated through increased production of LTB4 in neutrophils. Methods/Results: Hypercholesterolemia was modeled in Wistar rats by feeding them with a high cholesterol diet. The induction of hypercholesterolemia caused an increase in the plasma levels of LTB4, following lipopolysaccharide stimulation. This effect was recapitulated in vitro, both in the presence and absence of stimulation with the activator of 5-LO, A23187. Neutrophils in hypercholesterolemia rats expressed similar total levels of 5-LO as control rats, but displayed increased nuclear localization of 5-LO, as well as elevated levels of phosphorylated 5-LO and ERK1/2. In vitro, MβCD/cholesterol complexes enriched cholesterol in neutrophils, resulted in similar changes in 5-LO/LTB4. In addition, these alterations could be inhibited with the ERK inhibitor PD98059. Conclusion: Hypercholesterolemia increases LTB4 production in neutrophils by increasing the nuclear localization of 5-LO, which is the result of its phosphorylation by activated ERK1/2.

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

Foveolar cells phagocytose apoptotic neutrophils in chronic active Helicobacter pylori gastritis.

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Caruso, R A; Fedele, F; Di Bella, C; Mazzon, E; Rigoli, L

2012-11-01

The recognition and removal of apoptotic inflammatory cells by tissue macrophages and non-professional phagocytes, in a process called efferocytosis, is required for resolution of inflammation and is actively anti-inflammatory. We have previously demonstrated phagocytosis of apoptotic neutrophils by tumor cells in human gastric carcinoma, but to date, there have been no studies investigating this process in chronic active Helicobacter pylori gastritis. Biopsy specimens from 28 subjects with or without H. pylori infection and active inflammation were examined and graded according to the updated Sydney system. Light microscopy, electron microscopy, and Terminal Deoxynucleotidyltransferase-Mediated UTP End Labeling staining were used to identify apoptosis. H. pylori infection was detected by histology and by molecular assay in 16 out of 28 cases. DNA from paraffin-embedded gastric biopsies was amplified using primers specific for cagA, for the cag "empty site" as well as for the s and m alleles of vacA. The more virulent cagA-positive strains were found in five out of nine patients with chronic active gastritis. The vacA s1/m1 and s2/m1 genotypes were more common in nine patients with chronic active gastritis, while the vacA s2/m2 genotype was more frequent in seven patients with chronic inactive gastritis. Apoptotic neutrophils were also detected within the cytoplasmic vacuoles of the foveolar cells of nine cases with chronic active gastritis. Transmission electron micrographs revealed further apoptotic neutrophils within spacious phagosomes of foveolar cells in a similar manner to those described in late-phase efferocytosis both in vivo and in vitro. These new observations expand the morphological spectrum of gastritis in patients infected with more virulent H. pylori strains, compatible with an anti-inflammatory role for the gastric epithelial cells in their removal of apoptotic neutrophils during active chronic gastritis.

Preoperative neutrophil response as a predictive marker of clinical outcome following open heart surgery and the impact of leukocyte filtration.

LENUS (Irish Health Repository)

Soo, Alan W

2010-11-01

Open heart surgery is associated with a massive systemic inflammatory response. Neutrophils, are the main mediator of this response. We hypothesised that the degree of neutrophil activation and inflammatory response to open heart surgery varies individually and correlates with clinical outcome. The aim of this study was to determine if individual clinical outcome can be predicted preoperatively through assessment of in-vitro stimulated neutrophil responses. Following that, the effects of neutrophil depletion through leukocyte filters are examined.

Inflammatory Ly6Chigh Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.

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Domínguez-Andrés, Jorge; Feo-Lucas, Lidia; Minguito de la Escalera, María; González, Leticia; López-Bravo, María; Ardavín, Carlos

2017-06-20

Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6C high monocytes in NK cell-mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti-Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon-dependent IL-15 production by spleen inflammatory Ly6C high monocytes to drive efficient activation and GM-CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL-15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL-15-based antifungal immunotherapies. Copyright © 2017 Elsevier Inc. All rights reserved.

G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling

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Bajrami, Besnik; Zhu, Haiyan; Zhang, Yu C.

2016-01-01

Cytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation. PMID:27551153

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos

NARCIS (Netherlands)

Paris, Daniel H.; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N.; Day, Nicholas P. J.; Zeerleder, Sacha

2015-01-01

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil

Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.

Science.gov (United States)

Zhang, Yanlin; Guan, Li; Yu, Jie; Zhao, Zanmei; Mao, Lijun; Li, Shuqiang; Zhao, Jinyuan

2016-11-21

During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating

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.

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...

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

Damaging role of neutrophilic infiltration in a mouse model of progressive tuberculosis.

Science.gov (United States)

Marzo, Elena; Vilaplana, Cristina; Tapia, Gustavo; Diaz, Jorge; Garcia, Vanessa; Cardona, Pere-Joan

2014-01-01

Tuberculosis was studied using an experimental model based on the C3HeB/FeJ mouse strain, which mimics the liquefaction of caseous necrosis occurring during active disease in immunocompetent adults. Mice were intravenously infected with 2 × 10(4) Colony Forming Units of Mycobacterium tuberculosis and their histopathology, immune response, bacillary load, and survival were evaluated. The effects of the administration of drugs with anti-inflammatory activity were examined, and the C3H/HeN mouse strain was also included for comparative purposes. Massive intra-alveolar neutrophilic infiltration led to rapid granuloma growth and coalescence of lesions into superlesions. A central necrotic area appeared showing progressive cellular destruction, the alveoli cell walls being initially conserved (caseous necrosis) but finally destroyed (liquefactive necrosis). Increasing levels of pro-inflammatory mediators were detected in lungs. C3HeB/FeJ treated with anti-inflammatory drugs and C3H/HeN animals presented lower levels of pro-inflammatory mediators such as TNF-α, IL-17, IL-6 and CXCL5, a lower bacillary load, better histopathology, and increased survival compared with untreated C3HeB/FeJ. The observation of massive neutrophilic infiltration suggests that inflammation may be a key factor in progression towards active tuberculosis. On the basis of our findings, we consider that the C3HeB/FeJ mouse model would be useful for evaluating new therapeutic strategies against human tuberculosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans

DEFF Research Database (Denmark)

Pedersen, Corinna C.; Borup, Rehannah; Fischer-Nielsen, Anne

2016-01-01

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF...... stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy...... individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors...

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

A Neutrophil Phenotype Model for Extracorporeal Treatment of Sepsis.

Directory of Open Access Journals (Sweden)

Alexander D Malkin

2015-10-01

Full Text Available Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage in sepsis. Interleukin-8 (IL-8, a key modulator of neutrophil function, signals through neutrophil specific surface receptors CXCR-1 and CXCR-2. In this study a mechanistic computational model was used to evaluate and deploy an extracorporeal sepsis treatment which modulates CXCR-1/2 levels. First, a simplified mechanistic computational model of IL-8 mediated activation of CXCR-1/2 receptors was developed, containing 16 ODEs and 43 parameters. Receptor level dynamics and systemic parameters were coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils which reduce pathogen load, and/or primed neutrophils which cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a two-hour infusion of E coli and followed for a maximum of 28 days. Ensembles of parameters were generated using a Bayesian parallel tempering approach to produce model fits that could recreate experimental outcomes. Stepwise logistic regression identified seven model parameters as key determinants of mortality. Sensitivity analysis showed that parameters controlling the level of killer cell neutrophils affected the overall systemic damage of individuals. To evaluate rescue strategies and provide probabilistic predictions of their impact on mortality, time of onset, duration, and capture efficacy of an extracorporeal device that modulated neutrophil phenotype were explored. Our findings suggest that interventions aiming to modulate phenotypic composition are time sensitive. When introduced between 3-6 hours of infection for a 72 hour duration, the survivor population increased from 31% to 40-80%. Treatment efficacy quickly diminishes if not introduced within 15 hours of infection. Significant harm is possible with treatment durations ranging from 5

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.