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Sample records for reduces neutrophil activation

  1. Human neutrophil antimicrobial activity.

    Science.gov (United States)

    Thomas, E L; Lehrer, R I; Rest, R F

    1988-01-01

    Polymorphonuclear neutrophilic leukocytes (PMNs) take up opsonized microorganisms into phagosomes that fuse with secretory granules in the PMN cytoplasm to form phagolysosomes. Killing and digestion of microorganisms take place within phagolysosomes. Antimicrobial activities in phagolysosomes are divided into two classes. Oxygen (O2)-dependent mechanisms are expressed when PMNs undergo the "respiratory burst." An NADPH oxidase in the phagolysosome membrane is activated and reduces O2 to superoxide (O2-). O2 reduction is the first step in a series of reactions that produce toxic oxidants. For example, .O2- dismutases to hydrogen peroxide (H2O2), and the azurophil granule enzyme myeloperoxidase catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The reaction of HOCl with ammonia and amines modulates the toxicity of this oxidant. O2-independent antimicrobial mechanisms include the activities of lysosomal proteases, other hydrolytic enzymes, and proteins and peptides that bind to microorganisms and disrupt essential processes or structural components. For example, the bactericidal/permeability-increasing protein, cathepsin G, and the defensins are released into phagolysosomes from the azurophil granules. Proposed mechanisms of action of neutrophil antimicrobial agents, their range of microbial targets, and their possible interactions within phagolysosomes are discussed.

  2. Combined anti CXC receptors 1 and 2 therapy is a promising anti-inflammatory treatment for respiratory diseases by reducing neutrophil migration and activation.

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    Planagumà, A; Domènech, T; Pont, M; Calama, E; García-González, V; López, R; Aulí, M; López, M; Fonquerna, S; Ramos, I; de Alba, J; Nueda, A; Prats, N; Segarra, V; Miralpeix, M; Lehner, M D

    2015-10-01

    Neutrophil infiltration and activation in the lung are important pathophysiological features in COPD, severe asthma and bronchiectasis mostly mediated by CXCL8 and CXCL1 via CXCR1 and CXCR2. No thorough study to date has been performed to compare the anti-inflammatory effect profile of dual CXCR1/2 vs. selective CXCR2 antagonists in relevant human neutrophil assays and pulmonary inflammation models. Dual CXCR1/2 (SCH527123, diaminocyclobutandione-1) and selective CXCR2 (SB265610, thiopyrimidine-1) antagonist activity and receptor residence time were determined by [(35)S]GTPγS binding in human (h)- and guinea pig (gp)-CXCR1 and CXCR2 overexpressing membranes. h-neutrophil chemotaxis, degranulation and ROS production were established using CXCL8 or CXCL1 to evaluate dual CXCR1/2- or selective CXCR2-dependent activities. LPS-induced lung inflammation in gp was selected to assess in vivo potency. Dual CXCR1/2 antagonists blocked both CXCL8 and CXCL1-induced h-neutrophil functions and [(35)S]GTPγS binding. In contrary, selective CXCR2 antagonists displayed significantly reduced potency in CXCL8 -mediated h-neutrophil responses despite being active in CXCR2 assays. Upon LPS challenge in gp, administration of SCH527123 inhibited the increase of neutrophils in BALF, modestly reduced blood neutrophils and induced minor neutrophil accumulation in bone marrow. Differentiation of CXCR1/2 vs. CXCR2 antagonists could not be extended to in vivo due to differences in CXCR1 receptor homology between h and gp. Dual CXCR1/2 therapy may represent a promising anti-inflammatory treatment for respiratory diseases reducing more effectively neutrophil migration and activation in the lung than a CXCR2 selective treatment. However, the in vivo confirmation of this claim is still missing due to species differences in CXCR1.

  3. Proteolytic Activity Present in House-Dust-Mite Extracts Degrades ENA-78/CXCL5 and Reduces Neutrophil Migration

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    Laura Keglowich

    2014-01-01

    Full Text Available Background. Bronchial smooth muscle cells (BSMC are a major source of proinflammatory and proangiogenic cytokines and chemokines, including VEGF and CXC-chemokines. CXC-chemokines act primarily on neutrophils, mediating their recruitment to and activation at the site of inflammation. In humans, house-dust mite (HDM allergens can cause asthmatic exacerbations and trigger an inflammatory response through protease-dependent mechanisms. Objective. We investigated the effect HDM extract on the release of pro-angiogenic and proinflammatory cytokines from BSMC. Methods. Human primary BSMC were stimulated with HDM extract in the absence or presence of fetal calf serum (FCS. Twenty angiogenic cytokines were detected by a specific antibody array and modified protein levels were confirmed by ELISA. Neutrophil migration was measured using a 96-well Boyden chamber. Results. ENA-78/CXCL5 protein levels in conditioned medium of BSMC stimulated with HDM extract were significantly reduced (n=10, P<0.05 but restored in the presence of 5% FCS. HDM extracts did not affect ENA-78/CXCL5 mRNA levels. Recombinant ENA-78/CXCL5 was degraded after incubation with HDM extracts (n=7, P<0.05 but restored after the addition of the serine protease AEBSF. Neutrophil migration towards recombinant ENA-78/CXCL5 was also reduced in the presence of HDM extract. Conclusion. HDM proteases degrade ENA-78/CXCL5. Thus exposure to HDM allergens may alter ENA-78/CXCL5 levels in the lungs and may affect angiogenesis and the inflammatory response in the airways of asthma patients.

  4. Proteolytic Activity Present in House-Dust-Mite Extracts Degrades ENA-78/CXCL5 and Reduces Neutrophil Migration.

    Science.gov (United States)

    Keglowich, Laura; Tamm, Michael; Zhong, Jun; Miglino, Nicola; Borger, Pieter

    2014-01-01

    Background. Bronchial smooth muscle cells (BSMC) are a major source of proinflammatory and proangiogenic cytokines and chemokines, including VEGF and CXC-chemokines. CXC-chemokines act primarily on neutrophils, mediating their recruitment to and activation at the site of inflammation. In humans, house-dust mite (HDM) allergens can cause asthmatic exacerbations and trigger an inflammatory response through protease-dependent mechanisms. Objective. We investigated the effect HDM extract on the release of pro-angiogenic and proinflammatory cytokines from BSMC. Methods. Human primary BSMC were stimulated with HDM extract in the absence or presence of fetal calf serum (FCS). Twenty angiogenic cytokines were detected by a specific antibody array and modified protein levels were confirmed by ELISA. Neutrophil migration was measured using a 96-well Boyden chamber. Results. ENA-78/CXCL5 protein levels in conditioned medium of BSMC stimulated with HDM extract were significantly reduced (n = 10, P ENA-78/CXCL5 mRNA levels. Recombinant ENA-78/CXCL5 was degraded after incubation with HDM extracts (n = 7, P ENA-78/CXCL5 was also reduced in the presence of HDM extract. Conclusion. HDM proteases degrade ENA-78/CXCL5. Thus exposure to HDM allergens may alter ENA-78/CXCL5 levels in the lungs and may affect angiogenesis and the inflammatory response in the airways of asthma patients.

  5. Ticagrelor reduces neutrophil recruitment and lung damage in abdominal sepsis.

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    Rahman, Milladur; Gustafsson, David; Wang, Yongzhi; Thorlacius, Henrik; Braun, Oscar Ö

    2014-01-01

    Abstract Platelets play an important role in abdominal sepsis and P2Y12 receptor antagonists have been reported to exert anti-inflammatory effects. Herein, we assessed the impact of platelet inhibition with the P2Y12 receptor antagonist ticagrelor on pulmonary neutrophil recruitment and tissue damage in a model of abdominal sepsis. Wild-type C57BL/6 mice were subjected to cecal ligation and puncture (CLP). Animals were treated with ticagrelor (100 mg/kg) or vehicle prior to CLP induction. Edema formation and bronchoalveolar neutrophils as well as lung damage were quantified. Flow cytometry was used to determine expression of platelet-neutrophil aggregates, neutrophil activation and CD40L expression on platelets. CLP-induced pulmonary infiltration of neutrophils at 24 hours was reduced by 50% in ticagrelor-treated animals. Moreover, ticagrelor abolished CLP-provoked lung edema and decreased lung damage score by 41%. Notably, ticagrelor completely inhibited formation of platelet-neutrophil aggregates and markedly reduced thrombocytopenia in CLP animals. In addition, ticagrelor reduced platelet shedding of CD40L in septic mice. Our data indicate that ticagrelor can reduce CLP-induced pulmonary neutrophil recruitment and lung damage suggesting a potential role for platelet antagonists, such as ticagrelor, in the management of patients with abdominal sepsis.

  6. Activation of Neutrophils by Nanoparticles

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    David M. Goncalves

    2011-01-01

    Full Text Available The use of nanoparticles (NPs has increased in the past few years in various fields, including defence, aerospace, electronics, biology, medicine, and so forth. and in applications such as diagnostic technology, bioimaging, and drug/gene delivery. Thus, human exposure to NPs and nanomaterials is unavoidable and will certainly expand in the future resulting in a growing interest in nanotoxicology, the study of toxicity of nanomaterials. A number of studies have reported the effects of NPs in respect to pulmonary inflammation by investigating in vitro activation of pulmonary cells with NPs and in vivo in a variety of models in which neutrophils appear to be the predominant leukocyte cell type in lungs and in bronchoalveolar lavages following inhalation or intratracheal instillation of NPs. Despite the fact that several studies have reported an increased number of neutrophils, the literature dealing with the direct activation of neutrophils by a given NP is poorly documented. This paper will summarize the current literature in this latter area of research and will end with a perspective view in which our laboratory will be involved in the following years.

  7. CFTR targeting during activation of human neutrophils.

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    Ng, Hang Pong; Valentine, Vincent G; Wang, Guoshun

    2016-12-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, plays critical roles in phagocytic host defense. However, how activated neutrophils regulate CFTR channel distribution subcellularly is not well defined. To investigate, we tested multiple Abs against different CFTR domains, to examine CFTR expression in human peripheral blood neutrophils by flow cytometry. The data confirmed that resting neutrophils had pronounced CFTR expression. Activation of neutrophils with soluble or particulate agonists did not significantly increase CFTR expression level, but induced CFTR redistribution to cell surface. Such CFTR mobilization correlated with cell-surface recruitment of formyl-peptide receptor during secretory vesicle exocytosis. Intriguingly, neutrophils from patients with ΔF508-CF, despite expression of the mutant CFTR, showed little cell-surface mobilization upon stimulation. Although normal neutrophils effectively targeted CFTR to their phagosomes, ΔF508-CF neutrophils had impairment in that process, resulting in deficient hypochlorous acid production. Taken together, activated neutrophils regulate CFTR distribution by targeting this chloride channel to the subcellular sites of activation, and ΔF508-CF neutrophils fail to achieve such targeting, thus undermining their host defense function.

  8. Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase.

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    Ribeiro-Gomes, Flavia L; Moniz-de-Souza, Maria Carolina A; Alexandre-Moreira, Magna S; Dias, Wagner B; Lopes, Marcela F; Nunes, Marise P; Lungarella, Giuseppe; DosReis, George A

    2007-09-15

    We investigated the role of neutrophil elastase (NE) in interactions between murine inflammatory neutrophils and macrophages infected with the parasite Leishmania major. A blocker peptide specific for NE prevented the neutrophils from inducing microbicidal activity in macrophages. Inflammatory neutrophils from mutant pallid mice were defective in the spontaneous release of NE, failed to induce microbicidal activity in wild-type macrophages, and failed to reduce parasite loads upon transfer in vivo. Conversely, purified NE activated macrophages and induced microbicidal activity dependent on secretion of TNF-alpha. Induction of macrophage microbicidal activity by either neutrophils or purified NE required TLR4 expression by macrophages. Injection of purified NE shortly after infection in vivo reduced the burden of L. major in draining lymph nodes of TLR4-sufficient, but not TLR4-deficient mice. These results indicate that NE plays a previously unrecognized protective role in host responses to L. major infection.

  9. Polyphenol derivatives – potential regulators of neutrophil activity

    OpenAIRE

    2012-01-01

    The study provides new information on the effect of natural polyphenols (derivatives of stilbene – resveratrol, pterostilbene, pinosylvin and piceatannol and derivatives of ferulic acid – curcumin, N-feruloylserotonin) on the activity of human neutrophils in influencing oxidative burst. All the polyphenols tested were found to reduce markedly the production of reactive oxygen species released by human neutrophils on extra-and intracellular levels as well as in cell free system. Moreover, pino...

  10. Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J2, reduces neutrophil migration via a nitric oxide pathway.

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    Napimoga, Marcelo H; Vieira, Silvio M; Dal-Secco, Daniela; Freitas, Andressa; Souto, Fabrício O; Mestriner, Fabiola L; Alves-Filho, José C; Grespan, Renata; Kawai, Toshihisa; Ferreira, Sérgio H; Cunha, Fernando Q

    2008-01-01

    Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ2-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ2 administration decreases leukocyte rolling and adhesion to the inflamed mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS-/- mice were not susceptible to 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ2-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-1 expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ2, whereas 15d-PGJ2 inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ2 suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

  11. NET amyloidogenic backbone in human activated neutrophils.

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    Pulze, L; Bassani, B; Gini, E; D'Antona, P; Grimaldi, A; Luini, A; Marino, F; Noonan, D M; Tettamanti, G; Valvassori, R; de Eguileor, M

    2016-03-01

    Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role.

  12. Cryptococcal capsular glucuronoxylomannan reduces ischaemia-related neutrophil influx

    NARCIS (Netherlands)

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

    2004-01-01

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

  13. Activation of bovine neutrophils by Brucella spp.

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    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.

  14. Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

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    Irundika H K Dias

    Full Text Available The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. - by the nicotinamide adenine dinucleotide (NADPH oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2, a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH/oxidised glutathione (GSSG ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC, and modifier (GCLM subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

  15. Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

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    Dias, Irundika H K; Chapple, Ian L C; Milward, Mike; Grant, Melissa M; Hill, Eric; Brown, James; Griffiths, Helen R

    2013-01-01

    The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

  16. Nitrite attenuated peroxynitrite and hypochlorite generation in activated neutrophils.

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    Ren, Xiaoming; Ding, Yun; Lu, Naihao

    2016-03-15

    Oxidative stress is usually considered as an important factor to the pathogenesis of various diseases. Peroxynitrite (ONOO(-)) and hypochlorite (OCl(-)) are formed in immune cells as a part of the innate host defense system, but excessive reactive oxygen species generation can cause progressive inflammation and tissue damage. It has been proven that through mediating nitric oxide (NO) homeostasis, inorganic nitrite (NO2(-)) shows organ-protective effects on oxidative stress and inflammation. However, the effects of NO2(-) on the function of immune cells were still not clear. The potential role of NO2(-) in modulating ONOO(-) and OCl(-) generation in neutrophil cells was investigated in this study. As an immune cell activator, lipopolysaccharide (LPS) increased both ONOO(-) and OCl(-) production in neutrophils, which was significantly attenuated by NO2(-). NO2(-) reduced superoxide (O2(·-)) generation via a NO-dependent mechanism and increased NO formation in activated neutrophils, suggesting a crucial role of O2(·-) in NO2(-)-mediated reduction of ONOO(-). Moreover, the reduced effect of NO2(-) on OCl(-) production was attributed to that NO2(-) reduced H2O2 production in activated neutrophils without influencing the release of myeloperoxidase (MPO), thus limiting OCl(-) production by MPO/H2O2 system. Therefore, NO2(-) attenuates ONOO(-) and OCl(-) formation in activated neutrophils, opening a new direction to modulate the inflammatory response.

  17. Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation.

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    Jochen Grommes

    Full Text Available INTRODUCTION: Treatment of acute lung injury (ALI remains an unsolved problem in intensive care medicine. As simvastatin exerts protective effects in inflammatory diseases we explored its effects on development of ALI and due to the importance of neutrophils in ALI also on neutrophil effector functions. METHODS: C57Bl/6 mice were exposed to aerosolized LPS (500 µg/ml for 30 min. The count of alveolar, interstitial, and intravasal neutrophils were assessed 4 h later by flow cytometry. Lung permeability changes were assessed by FITC-dextran clearance and albumin content in the BAL fluid. In vitro, we analyzed the effect of simvastatin on neutrophil adhesion, degranulation, apoptosis, and formation of reactive oxygen species. To monitor effects of simvastatin on bacterial clearance we performed phagocytosis and bacterial killing studies in vitro as well as sepsis experiments in mice. RESULTS: Simvastatin treatment before and after onset of ALI reduces neutrophil influx into the lung as well as lung permeability indicating the protective role of simvastatin in ALI. Moreover, simvastatin reduces the formation of ROS species and adhesion of neutrophils without affecting apoptosis, bacterial phagocytosis and bacterial clearance. CONCLUSION: Simvastatin reduces recruitment and activation of neutrophils hereby protecting from LPS-induced ALI. Our results imply a potential role for statins in the management of ALI.

  18. Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents.

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    Tan, A S; Berridge, M V

    2000-04-21

    Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 microg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 microM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay

  19. 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.

  20. Neutrophil activator of matrix metalloproteinase-2 (NAM).

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    Rollo, Ellen E; Hymowitz, Michelle; Schmidt, Cathleen E; Montana, Steve; Foda, Hussein; Zucker, Stanley

    2006-01-01

    We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

  1. Reduced PU.1 expression underlies aberrant neutrophil maturation and function in β-thalassemia mice and patients.

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    Siwaponanan, Panjaree; Siegers, Jurre Ynze; Ghazali, Razi; Ng, Thian; McColl, Bradley; Ng, Garrett Zhen-Wei; Sutton, Philip; Wang, Nancy; Ooi, Isabelle; Thiengtavor, Chayada; Fucharoen, Suthat; Chaichompoo, Pornthip; Svasti, Saovaros; Wijburg, Odilia; Vadolas, Jim

    2017-06-08

    β-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbb(th3/+) β-thalassemia mouse and hemoglobin E (HbE)/β-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbb(th3/+) mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in β-thalassemia patients. In Hbb(th3/+) mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbb(th3/+) neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6G(hi)Ly6C(low)) appeared as band form cells, and a striking expansion of immature (Ly6G(low)Ly6C(low)) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbb(th3/+) neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbb(th3/+) mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/β-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in β-thalassemia patients, which contributes to deficiencies in neutrophil effector functions. © 2017 by The American Society of Hematology.

  2. Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

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    Kenne Ellinor

    2012-01-01

    Full Text Available Abstract Background Brain edema as a result of secondary injury following traumatic brain injury (TBI is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. Methods In this study we used controlled cortical impact (CCI as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI. Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. Results Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. Conclusion Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice.

  3. Exposure to Leishmania braziliensis triggers neutrophil activation and apoptosis.

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    Sarah A C Falcão

    2015-03-01

    Full Text Available BACKGROUND: Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. METHODS AND FINDINGS: Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. CONCLUSIONS: We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.

  4. Reduction of neutrophil activity decreases early microvascular injury after subarachnoid haemorrhage

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    Bi Weina

    2011-08-01

    Full Text Available Abstract Background Subarachnoid haemorrhage (SAH elicits rapid pathological changes in the structure and function of parenchymal vessels (≤ 100 μm. The role of neutrophils in these changes has not been determined. This study investigates the role of neutrophils in early microvascular changes after SAH Method Rats were either untreated, treated with vinblastine or anti-polymorphonuclear (PMN serum, which depletes neutrophils, or treated with pyrrolidine dithiocarbamate (PDTC, which limits neutrophil activity. SAH was induced by endovascular perforation. Neutrophil infiltration and the integrity of vascular endothelium and basement membrane were assessed immunohistochemically. Vascular collagenase activity was assessed by in situ zymography. Results Vinblastine and anti-PMN serum reduced post-SAH accumulation of neutrophils in cerebral vessels and in brain parenchyma. PDTC increased the neutrophil accumulation in cerebral vessels and decreased accumulation in brain parenchyma. In addition, each of the three agents decreased vascular collagenase activity and post-SAH loss of vascular endothelial and basement membrane immunostaining. Conclusions Our results implicate neutrophils in early microvascular injury after SAH and indicate that treatments which reduce neutrophil activity can be beneficial in limiting microvascular injury and increasing survival after SAH.

  5. Morphine reduces local cytokine expression and neutrophil infiltration after incision

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    Li Xiangqi

    2007-10-01

    Full Text Available Abstract Background Inflammation and nociceptive sensitization are hallmarks of tissue surrounding surgical incisions. Recent studies demonstrate that several cytokines may participate in the enhancement of nociception near these wounds. Since opioids like morphine interact with neutrophils and other immunocytes, it is possible that morphine exerts some of its antinociceptive action after surgical incision by altering the vigor of the inflammatory response. On the other hand, keratinocytes also express opioid receptors and have the capacity to produce cytokines after injury. Our studies were directed towards determining if opioids alter cytokine production near incisions and to identify cell populations responsible for producing these cytokines. Results A murine incisional model was used to measure the effects of acute morphine administration (0.1–10 mg/kg on nociceptive thresholds, neutrophil infiltration and cytokine production in hind paw skin 30 minutes and 2 hours after incision. Incised hind paws displayed profound allodynia which was reduced by morphine (0.1–10 mg/kg in the 2 hours following incision. Skin samples harvested from these mice showed enhanced levels of 5 cytokines: IL-1β, IL-6, tumor necrosis factor alpha (TNFα, granulocyte colony stimulating factor (G-CSF and keratinocyte-derived cytokine (KC. Morphine reduced these incision-stimulated levels. Separate analyses measuring myeloperoxidase (MPO and using immunohistochemistry demonstrated that morphine dose-dependently reduced the infiltration of neutrophils into the peri-incisional tissue. The dose of morphine required for reduction of cytokine accumulation, however, was below that required for inhibition of peri-incisional neutrophil infiltration. Additional immunohistochemical studies revealed wound edge keratinocytes as being an important source of cytokines in the acute phase after incision. Conclusion Acute morphine administration of doses as low as 0.1 mg/kg reduces

  6. Cell Intrinsic Galectin-3 Attenuates Neutrophil ROS-Dependent Killing of Candida by Modulating CR3 Downstream Syk Activation

    Science.gov (United States)

    Wu, Sheng-Yang; Huang, Juin-Hua; Chen, Wen-Yu; Chan, Yi-Chen; Lin, Chun-Hung; Chen, Yee-Chun; Liu, Fu-Tong; Wu-Hsieh, Betty A.

    2017-01-01

    Invasive candidiasis is a leading cause of nosocomial bloodstream infection. Neutrophils are the important effector cells in host resistance to candidiasis. To investigate the modulation of neutrophil fungicidal function will advance our knowledge on the control of candidiasis. While recombinant galectin-3 enhances neutrophil phagocytosis of Candida, we found that intracellular galectin-3 downregulates neutrophil fungicidal functions. Co-immunoprecipitation and immunofluorescence staining reveal that cytosolic gal3 physically interacts with Syk in neutrophils after Candida stimulation. Gal3−/− neutrophils have higher level of Syk activation as well as greater abilities to generate reactive oxygen species (ROS) and kill Candida than gal3+/+ cells. While galectin-3 deficiency modulates neutrophil and macrophage activation and the recruitment of monocytes and dendritic cells, the deficiency does not affect the numbers of infiltrating neutrophils or macrophages. Galectin-3 deficiency ameliorates systemic candidiasis by reducing fungal burden, renal pathology, and mortality. Adoptive transfer experiments demonstrate that cell intrinsic galectin-3 negatively regulates neutrophil effector functions against candidiasis. Reducing galectin-3 expression or activity by siRNA or gal3 inhibitor TD139 enhances human neutrophil ROS production. Mice treated with TD139 have enhanced ability to clear the fungus. Our work unravels the mechanism by which galectin-3 regulates Syk-dependent neutrophil fungicidal functions and raises the possibility that blocking gal3 in neutrophils may be a promising therapeutic strategy for treating systemic candidiasis. PMID:28217127

  7. Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

    Science.gov (United States)

    Tamarozzi, F; Wright, H L; Johnston, K L; Edwards, S W; Turner, J D; Taylor, M J

    2014-10-01

    The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism.

  8. Staphylococcus aureus SaeR/S-regulated factors reduce human neutrophil reactive oxygen species production.

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    Guerra, Fermin E; Addison, Conrad B; de Jong, Nienke W M; Azzolino, Joseph; Pallister, Kyler B; van Strijp, Jos A G; Voyich, Jovanka M

    2016-11-01

    Neutrophils are the first line of defense after a pathogen has breached the epithelial barriers, and unimpaired neutrophil functions are essential to clear infections. Staphylococcus aureus is a prevalent human pathogen that is able to withstand neutrophil killing, yet the mechanisms used by S. aureus to inhibit neutrophil clearance remain incompletely defined. The production of reactive oxygen species (ROS) is a vital neutrophil antimicrobial mechanism. Herein, we test the hypothesis that S. aureus uses the SaeR/S two-component gene regulatory system to produce virulence factors that reduce neutrophil ROS production. With the use of ROS probes, the temporal and overall production of neutrophil ROS was assessed during exposure to the clinically relevant S. aureus USA300 (strain LAC) and its isogenic mutant LACΔsaeR/S Our results demonstrated that SaeR/S-regulated factors do not inhibit neutrophil superoxide (O2(-)) production. However, subsequent neutrophil ROS production was significantly reduced during exposure to LAC compared with LACΔsaeR/S In addition, neutrophil H2O2 production was reduced significantly by SaeR/S-regulated factors by a mechanism independent of catalase. Consequently, the reduction in neutrophil H2O2 resulted in decreased production of the highly antimicrobial agent hypochlorous acid/hypochlorite anion (HOCl/(-)OCl). These findings suggest a new evasion strategy used by S. aureus to diminish a vital neutrophil antimicrobial mechanism. © Society for Leukocyte Biology.

  9. Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1.

    Science.gov (United States)

    Liu, Fu-Chao; Yu, Huang-Ping; Syu, Yu-Ting; Fang, Jia-You; Lin, Chwan-Fwu; Chang, Shih-Hsin; Lee, Yen-Tung; Hwang, Tsong-Long

    2017-07-27

    Formyl peptide receptor 1 (FPR1) mediates bacterial and mitochondrial N-formyl peptides-induced neutrophil activation. Therefore, FPR1 is an important therapeutic target for drugs to treat septic or sterile inflammatory diseases. Honokiol, a major bioactive compound of Magnoliaceae plants, possesses several anti-inflammatory activities. Here, we show that honokiol exhibits an inhibitory effect on FPR1 binding in human neutrophils. Honokiol inhibited superoxide anion generation, reactive oxygen species formation, and elastase release in bacterial or mitochondrial N-formyl peptides (FPR1 agonists)-activated human neutrophils. Adhesion of FPR1-induced human neutrophils to cerebral endothelial cells was also reduced by honokiol. The receptor-binding results revealed that honokiol repressed FPR1-specific ligand N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein binding to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells. However, honokiol did not inhibit FPR2-specific ligand binding to FPR2 in human neutrophils. Furthermore, honokiol inhibited FPR1 agonist-induced calcium mobilization as well as phosphorylation of p38 MAPK, ERK, and JNK in human neutrophils. In conclusion, our data demonstrate that honokiol may have therapeutic potential for treating FPR1-mediated inflammatory diseases.

  10. Activation of the Small GTPase Rap1 in Human Neutrophils

    NARCIS (Netherlands)

    M'Rabet, Laura; Coffer, P.J.; Zwartkruis, G.J.T.; Franke, Barbara; Segal, Anthony W.; Koenderman, L.; Bos, J.L.

    2002-01-01

    The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1- binding domain of RalGDS (RalGDS-RBD) as an activationspecific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a varie

  11. Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration.

    Science.gov (United States)

    Kornerup, Kristin N; Salmon, Gary P; Pitchford, Simon C; Liu, Wai L; Page, Clive P

    2010-09-01

    Previous studies in our laboratory have shown that platelets are essential for the migration of eosinophils into the lungs of allergic mice, and that this is dependent on the functional expression of platelet P-selectin. We sought to investigate whether the same is true for nonallergic, acute inflammatory stimuli administered to distinct anatomic compartments. Neutrophil trafficking was induced in two models, namely zymosan-induced peritonitis and LPS-induced lung inflammation, and the platelet dependence of these responses investigated utilizing mice rendered thrombocytopenic. The relative contribution of selectins was also investigated. The results presented herein clearly show that platelet depletion (>90%) significantly inhibits neutrophil recruitment in both models. In addition, we show that P-selectin glycoprotein ligand-1, but not P-selectin, is essential for neutrophil recruitment in mice in vivo, thus suggesting the existence of different regulatory mechanisms for the recruitment of leukocyte subsets in response to allergic and nonallergic stimuli. Further studies in human blood demonstrate that low-dose prothrombotic and pro-inflammatory stimuli (CCL17 or CCL22) synergize to induce platelet and neutrophil activation, as well as the formation of platelet-neutrophil conjugates. We conclude that adhesion between platelets and neutrophils in vivo is an important event in acute inflammatory responses. Targeting this interaction may be a successful strategy for inflammatory conditions where current therapy fails to provide adequate treatment.

  12. Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis.

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    Keqing Wang

    Full Text Available Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

  13. Structural divergence of GPI-80 in activated human neutrophils.

    Science.gov (United States)

    Nitto, Takeaki; Takeda, Yuji; Yoshitake, Hiroshi; Sendo, Fujiro; Araki, Yoshihiko

    2007-07-27

    GPI-80 is a glycosylphosphatidylinositol (GPI)-anchored protein that is mainly expressed in human neutrophils. Previous studies using 3H9, a monoclonal antibody (mAb) against GPI-80, suggested that GPI-80 regulates leukocyte adherence and migration through Mac-1. GPI-80, which is anchored at the plasma membrane in resting neutrophils, moves into the pseudopodia and is released from activated human neutrophils. Here, we demonstrate that neutrophil activation affects GPI-80 dynamics using a new anti-GPI-80 mAb, designated 4D4, which is directed against the form of GPI-80 found on resting human neutrophils. Similar to 3H9, 4D4 influences Mac-1-dependent neutrophil adhesion. Treatment of purified GPI-80 with periodic acid and trypsin indicated that 3H9 and 4D4 recognize peptide and carbohydrate moieties, respectively. Stimulation with fMLP decreased the binding of 4D4 to GPI-80 on the neutrophil surface but increased the overall expression of GPI-80, as visualized by the 3H9 signal. Confocal laser microscopy revealed the 4D4 signal mainly on cell bodies and at a low level on pseudopodia during migration toward increasing concentrations of fMLP, whereas the 3H9 signal was observed in both areas. In addition, soluble GPI-80 released from activated neutrophils did not bind 4D4. These results suggest that there are two populations of GPI-80 that differ in the ability to bind 4D4. The 4D4-recognized form may regulate Mac-1-dependent neutrophil adhesion, and may subsequently be converted to a 4D4-unrecognized form during neutrophil activation.

  14. Activation of AMPK enhances neutrophil chemotaxis and bacterial killing.

    Science.gov (United States)

    Park, Dae Won; Jiang, Shaoning; Tadie, Jean-Marc; Stigler, William S; Gao, Yong; Deshane, Jessy; Abraham, Edward; Zmijewski, Jaroslaw W

    2013-11-08

    An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5' adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.

  15. Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.

    Science.gov (United States)

    Neufert, C; Pai, R K; Noss, E H; Berger, M; Boom, W H; Harding, C V

    2001-08-01

    Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.

  16. Activation of the neutrophil NADPH oxidase by Aspergillus fumigatus.

    Science.gov (United States)

    Boyle, Keith B; Stephens, Len R; Hawkins, Phillip T

    2012-12-01

    Upon infection of the respiratory system with the fungus Aspergillus fumigatus various leukoctytes, in particular neutrophils, are recruited to the lung to mount an immune response. Neutrophils respond by both phagocytosing conidia and mediating extracellular killing of germinated, invasive hyphae. Of paramount importance to an appropriate immune response is the neutrophil NADPH oxidase enzyme, which mediates the production of various reactive oxygen species (ROS). This is evidenced by the acute sensitivity of both oxidase-deficient humans and mice to invasive aspergillosis. Herein we briefly review the mechanisms and functions of oxidase activation and discuss our recent work identifying at least some of the important players in hyphal-induced oxidase activation and neutrophil function. Among these we define the phosphoinositide 3-kinase enzyme and the regulatory protein Vav to be of critical importance and allude to a kinase-independent role for Syk.

  17. Tanshinone IIA Protects against Dextran Sulfate Sodium- (DSS-) Induced Colitis in Mice by Modulation of Neutrophil Infiltration and Activation.

    Science.gov (United States)

    Liu, Xiaowei; He, Haiyue; Huang, Tingting; Lei, Zhen; Liu, Fuquan; An, Guangyu; Wen, Tao

    2016-01-01

    Neutrophils play a critical role in the initiation and maintenance of intestinal inflammation. However, conventional neutrophil-targeted therapies can impair normal host defense. Tanshinone IIA has been recently revealed to act directly on neutrophils. Hence, we aimed at investigating whether Tanshinone IIA can protect against experimental colitis through modulation of neutrophils. We induced colitis in C57BL/6 mice by giving 3% dextran sulfate sodium (DSS) orally, and meanwhile, we treated mice daily with Tanshinone IIA intraperitoneally. The severity of colitis was evaluated by calculating disease activity index (DAI) and histological parameters. Neutrophil infiltration and activation in the colons of mice were measured. Moreover, whether Tanshinone IIA has direct effects on neutrophil migration and activation was determined in vitro. Our data showed that Tanshinone IIA significantly ameliorated the severity of DSS-induced colitis in mice, evidenced by the reduced DAI and improved colonic inflammation. In addition, Tanshinone IIA decreased neutrophil infiltration of intestinal mucosa and activation and reduced colonic inflammatory cytokines in DSS-treated mice. Furthermore, Tanshinone IIA was demonstrated to significantly suppress neutrophil migration and activation. These results provide compelling evidence that Tanshinone IIA has a therapeutic potential for alleviating inflammatory colitis in mice, which is possibly mediated by the immunomodulation of neutrophils.

  18. The Role of Neutrophil Activation in Pathogenesis of Preeclampsia

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To investigate the effect of neutrophil activation on pathogenesis of pre-eclampsia, neutrophil activation was examined by using flow cytometry to assess the CD11b expression and the levels of plasma endothelin-1 (ET-1) and serum NO-2 were also measured by using non-equilibrium radioimmunoassay and by Griess assay in 29 pregnant women with pre-eclampsia and 31 normal pregnant women at third trimester. The expression of neutrophil CD11b was significantly elevated in women with pre-eclampsia as compared with that of normal pregnant women at third trimester. The mean fluorescence index of CD11b was 438.38±179.91 and 326.97±170.14 respectively (P<0.05). The plasma ET-1 level and serum NO 2 concentration in pre-eclampsic women (63.69±48.33 pg/ml and 20.03±4.77 μmol/L, respectively) were both significantly increased as compared with those in the normal pregnancy women (29.98±20.25 pg/ml and 15.47±5.47 μmol/L, respectively, P<0.01). The neutrophil CD11b expression was significantly elevated in pre-eclampsia. The increased neutrophil activation may cause the damage of vascular endothelium and result in NO release compensatory increase in endothelial cells, suggesting that the neutrophil activation may play a key role in pathogenesis of pre-eclampsia.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    Pneumocystis carinii pneumonia (PCP) is accompanied by an acute inflammatory infiltration of the lung parenchyma. The cellular infiltrate is characterized by inflammatory cells including neutrophils, lymphocytes and macrophages. Furthermore, neutrophilia in bronchoalveolar lavage (BAL) fluid has...... been shown to confer a poor prognosis in PCP. We therefore investigated the potential of BAL fluid from 17 patients with PCP to induce neutrophil chemotaxis. BAL fluid from patients induced considerable neutrophil chemotactic activity compared to normal controls. Elevated levels of IL-8 were detected...... in patient samples as compared to controls. A specific anti-IL-8 antibody significantly reduced chemotactic activity of patient samples by more than 50%. In conclusion, IL-8 appears to be a significant participant of neutrophil chemotaxis in AIDS-associated PCP, and may participate in the recruitment...

  20. Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.

    Science.gov (United States)

    Burnett, D; Adams, D H; Martin, T J; Liu, Q; Grant, R A; Stockley, R A; Lord, J M

    1994-09-15

    The macrolide FK506 inhibited, by up to 50%, neutrophil migration and the production of the superoxide radical in response to the formyl peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The production of the superoxide radical in response to phorbol 12-myristate 13-acetate (PMA) was unaffected by FK506. The inhibition of neutrophil functions was accompanied by a partial reversal of FMLP-induced synthesis of cellular proteins, despite a rise in intracellular Ca2+. Neutrophils treated with FK506 demonstrated a small (average 23%) though significant decrease in formyl-peptide receptor numbers but receptor binding affinity was unaffected. The effects of FK506 on neutrophil activation appear to be analogous to those in T-lymphocytes. The incomplete inhibition, by FK506, of neutrophil responses suggests further that activation by FMLP is mediated via distinct multiple signalling pathways, including protein kinase activation and protein synthesis. The inability of FK506 to reduce FMLP-induced rises in cellular Ca2+ or PMA-induced activation of neutrophils suggests that its action is distal to Ca2+ mobilization and distinct from pathways relying on PKC activation. Thus the immunosuppressive effects of FK506 in vivo might be mediated through the inhibition of inflammatory cells other than lymphocytes and the drug therefore has therapeutic potential in a variety of inflammatory conditions. The drug also has potential in vitro for the characterization of signalling pathways from the plasma membrane to the nucleus.

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

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    Kathryn L Butler

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

  2. Chemotactic Activity on Human Neutrophils to Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    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

  3. [Purification and antimicrobial activity of human neutrophil defensins].

    Science.gov (United States)

    Qu, X; Wang, A

    1991-11-01

    Neutrophils are one of the weapons of host defenses against microbial infection. Their ability to kill the invading microorganisms depends on two principle mechanisms. One depends on production of reactive oxygen intermediates (ROI) by stimulated neutrophils, and the other depends on the delivery of antimicrobial contents of the neutrophils' cytoplasmic granules, oxygen-independent. The defensins have the highest concentration in the neutrophils, and the broadest antimicrobial spectrum, being capable of killing gram-positive and gram-negative bacteria, fungi and some envelope viruses. We purified human defensins from the neutrophils' granules by gel permeation chromatography and SDS-preparative acrylamide gel electrophoresis. The molecular weight of human defensins is between 3,000-4,000 daltons. After testing, C. neoformans was susceptible to these defensins. Under condition of 37 degrees C, pH 7.4 and low ionic strength, antifungal activity by human defensins was related to its concentration and incubating time. All of these illustrate that nonoxidative killing mechanism of neutrophils, especially the function of defensins is very important in host defenses.

  4. Solonamide B Inhibits Quorum Sensing and Reduces Staphylococcus aureus Mediated Killing of Human Neutrophils

    DEFF Research Database (Denmark)

    Nielsen, Anita; Månsson, Maria; Bojer, Martin S.

    2014-01-01

    histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like a-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of a......-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and Agr...

  5. Solubilization of the O2(-)-forming activity responsible for the respiratory burst in human neutrophils.

    Science.gov (United States)

    Gabig, T G; Kipnes, R S; Babior, B M

    1978-10-10

    On exposure to suitable activating agents, neutrophils sharply alter their oxygen metabolism, showing large increases in oxygen uptake, O2 and H2O2 production, and glucose consumption via the hexose monophosphate shunt. These metabolic alterations, which together are designated the "respiratory burst," are due to the activation of a system which catalyzes the reaction: 2O2 + NADPH leads to 2O2(-) + NADP. This O2(-)-forming system is found in a particulate fraction isolated from neutrophils which had been activated with opsonized zymosan. When these particles were treated with detergent under suitable conditions, the O2(-)-forming activity was released in a form which passed through a membrane filter capable of retaining species of Mr greater than 3000,000. Soluble O2(-)-forming activity was obtained from normal activated neutrophils, but not from normal resting neutrophils or from activated neutrophils obtained from patients with chronic granulomatous disease, an inherited condition in which the respiratory burst is defective. O2(-)production by the soluble system required a reduced pyridine nucleotide as electron donor, and showed a quadratic dependence on the concentration of the solubilized preparation.

  6. Inhibition of neutrophil activity improves cardiac function after cardiopulmonary bypass

    Directory of Open Access Journals (Sweden)

    Grünwald Frank

    2007-10-01

    Full Text Available Abstract Background The arterial in line application of the leukocyte inhibition module (LIM in the cardiopulmonary bypass (CPB limits overshooting leukocyte activity during cardiac surgery. We studied in a porcine model whether LIM may have beneficial effects on cardiac function after CPB. Methods German landrace pigs underwent CPB (60 min myocardial ischemia; 30 min reperfusion without (group I; n = 6 or with LIM (group II; n = 6. The cardiac indices (CI and cardiac function were analyzed pre and post CPB with a Swan-Ganz catheter and the cardiac function analyzer. Neutrophil labeling with technetium, scintigraphy, and histological analyses were done to track activated neutrophils within the organs. Results LIM prevented CPB-associated increase of neutrophil counts in peripheral blood. In group I, the CI significantly declined post CPB (post: 3.26 ± 0.31; pre: 4.05 ± 0.45 l/min/m2; p 2; p = 0.23. Post CPB, the intergroup difference showed significantly higher CI values in the LIM group (p Conclusion Our data provides strong evidence that LIM improves perioperative hemodynamics and cardiac function after CPB by limiting neutrophil activity and inducing accelerated sequestration of neutrophils in the spleen.

  7. [Murine peritoneal neutrophil activation upon tungsten nanoparticles exposure in vivo].

    Science.gov (United States)

    Martinova, E A; Baranov, V I

    2014-01-01

    Two examples of tungsten carbide nanoparticles (d = 15 nm, 50 nm) and tungsten carbide nanoparticles with 8% cobalt (d = 50 nm) have been found to induce the neutrophil activation 3 h and 36 h after intraperitoneal administration in the doses 0.005; 0.025; 0.05; 0.25; 0.5; 1; 2.5 and 5 microgram per 1 gram body weight to FVB mice. Neutrophil activation was calculated based on the CD11b and S100 antigen expression. Effect of nanoparticles is bimodal for all tested examples.

  8. Spontaneous neutrophil activation in HTLV-1 infected patients

    Directory of Open Access Journals (Sweden)

    Jaqueline B. Guerreiro

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

  9. Spontaneous neutrophil activation in HTLV-1 infected patients

    Directory of Open Access Journals (Sweden)

    Jaqueline B. Guerreiro

    2005-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

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

  11. Vanadium promotes hydroxyl radical formation by activated human neutrophils.

    Science.gov (United States)

    Fickl, Heidi; Theron, Annette J; Grimmer, Heidi; Oommen, Joyce; Ramafi, Grace J; Steel, Helen C; Visser, Susanna S; Anderson, Ronald

    2006-01-01

    This study was undertaken to investigate the effects of vanadium in the +2, +3, +4, and +5 valence states on superoxide generation, myeloperoxidase (MPO) activity, and hydroxyl radical formation by activated human neutrophils in vitro, using lucigenin-enhanced chemiluminescence (LECL), autoiodination, and electron spin resonance with 5,5-dimethyl-l-pyrroline N-oxide as the spin trap, respectively. At concentrations of up to 25 microM, vanadium, in the four different valence states used, did not affect the LECL responses of neutrophils activated with either the chemoattractant, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), or the phorbol ester, phorbol 12-myristate 12-acetate (25 ng/ml). However, exposure to vanadium in the +2, +3, and +4, but not the +5, valence states was accompanied by significant augmentation of hydroxyl radical formation by activated neutrophils and attenuation of MPO-mediated iodination. With respect to hydroxyl radical formation, similar effects were observed using cell-free systems containing either hydrogen peroxide (100 microM) or xanthine/xanthine oxidase together with vanadium (+2, +3, +4), while the activity of purified MPO was inhibited by the metal in these valence states. These results demonstrate that vanadium in the +2, +3, and +4 valence states interacts prooxidatively with human neutrophils, competing effectively with MPO for hydrogen peroxide to promote formation of the highly toxic hydroxyl radical.

  12. PKC-δ activation in neutrophils promotes fungal clearance.

    Science.gov (United States)

    Li, Xun; Cullere, Xavier; Nishi, Hiroshi; Saggu, Gurpanna; Durand, Enrique; Mansour, Michael K; Tam, Jenny M; Song, Xiu-Yu; Lin, Xin; Vyas, Jatin M; Mayadas, Tanya

    2016-09-01

    The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-β-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ-deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.

  13. 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.

  14. Activity of neutrophil elastase reflects the progression of acute pancreatitis

    DEFF Research Database (Denmark)

    Novovic, Srdan; Andersen, Anders M; Nord, Magnus

    2013-01-01

    Abstract Objective. Neutrophil elastase (NE) concentration is associated with progression of acute pancreatitis (AP), but measuring total NE concentration includes biologically inactive NE. This study aims to investigate the relationship between NE activity and the aetiology and severity of AP...... was associated with predicted severity of AP and AP-associated respiratory failure. Specific NE inhibitors may have therapeutic potential in acute pancreatitis....

  15. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation.

    Directory of Open Access Journals (Sweden)

    Bram J van Raam

    Full Text Available BACKGROUND: Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Deltapsi(m, which is usually generated by the respiratory chain complexes. We investigated the source of Deltapsi(m in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Deltapsi(m for the generation of ATP. METHODS AND PRINCIPAL FINDINGS: Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Deltapsi(m was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Deltapsi(m. Complex V did not maintain Deltapsi(m by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils. CONCLUSIONS: We show that neutrophils can maintain Deltapsi(m via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.

  16. Production of interleukin-1 like activity by neutrophils derived from rat lung.

    Science.gov (United States)

    Kusaka, Y; Donaldson, K

    1990-01-01

    Interleukin-1 like activity was produced by neutrophils obtained by bronchoalveolar lavage from experimentally inflamed rat lung. Activity was released spontaneously from neutrophils at high levels but it was enhanced by stimulation with endotoxin in vitro. PMID:2141440

  17. Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis.

    Science.gov (United States)

    Gaudreault, Eric; Stankova, Jana; Rola-Pleszczynski, Marek

    2005-01-01

    Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.

  18. Functional activity of neutrophils in diabetes mellitus and coronary heart disease: role of myeloperoxidase in the development of oxidative stress.

    Science.gov (United States)

    Gorudko, I V; Kostevich, V A; Sokolov, A V; Shamova, E V; Buko, I V; Konstantinova, E E; Vasiliev, V B; Cherenkevich, S N; Panasenko, O M

    2012-11-01

    We performed a comparative analysis of functional activity of neutrophils in patients with type 2 diabetes mellitus with and without symptoms of CHD. Enhanced H2O2 production by neutrophils in response to N-formyl-Met-Leu-Phe (fMLP) was found in patients with type 2 diabetes mellitus. In patients with type 2 diabetes mellitus associated with CHD, fMLP-induced release of myeloperoxidase from azurophilic granules of neutrophils was reduced and plasma myeloperoxidase level was elevated. Increased peroxidase activity of myeloperoxidase, reduced plasma catalase activity, and increased levels of TBA-reactive lipid peroxidation products and oxidized glutathione were detected in patients of both groups. Since myeloperoxidase is an important neutrophilic mediator of oxidative stress, its increased activity in the blood can be an additional marker of oxidative stress and cardiovascular risk in patients with diabetes mellitus.

  19. Repressor and activator protein accelerates hepatic ischemia reperfusion injury by promoting neutrophil inflammatory response.

    Science.gov (United States)

    Li, Chang Xian; Lo, Chung Mau; Lian, Qizhou; Ng, Kevin Tak-Pan; Liu, Xiao Bing; Ma, Yuen Yuen; Qi, Xiang; Yeung, Oscar Wai Ho; Tergaonkar, Vinay; Yang, Xin Xiang; Liu, Hui; Liu, Jiang; Shao, Yan; Man, Kwan

    2016-05-10

    Repressor and activator protein (Rap1) directly regulates nuclear factor-κB (NF-κB) dependent signaling, which contributes to hepatic IRI. We here intended to investigate the effect of Rap1 in hepatic ischemia reperfusion injury (IRI) and to explore the underlying mechanisms. The association of Rap1 expression with hepatic inflammatory response were investigated in both human and rat liver transplantation. The effect of Rap1 in hepatic IRI was studied in Rap1 knockout mice IRI model in vivo and primary cells in vitro. Our results showed that over expression of Rap1 was associated with severe liver graft inflammatory response, especially in living donor liver transplantation. The results were also validated in rat liver transplantation model. In mice hepatic IRI model, the knockout of Rap1 reduced hepatic damage and hepatic inflammatory response. In primary cells, the knockout of Rap1 suppressed neutrophils migration activity and adhesion in response to liver sinusoidal endothelial cells through down-regulating neutrophils F-Actin expression and CXCL2/CXCR2 pathway. In addition, the knockout of Rap1 also decreased production of pro-inflammatory cytokines/chemokines in primary neutrophils and neutrophils-induced hepatocyte damage. In conclusion, Rap1 may induce hepatic IRI through promoting neutrophils inflammatory response. Rap1 may be the potential therapeutic target of attenuating hepatic IRI.

  20. Reduced expression of C5a receptors on neutrophils from cord blood

    DEFF Research Database (Denmark)

    Nybo, Mads; Sørensen, O; Leslie, R;

    1998-01-01

    MLP was tested by measuring migration and exocytosis of myeloperoxidase and lactoferrin. RESULTS: C5a mean fluorescence on neutrophils from neonates was significantly lower (22.4 (SD 3.5)) than in adult controls (31.5 (3.1)). Neutrophils from neonates migrated poorly towards both C5a and fMLP compared with those...... from adult controls. Exocytosis of myeloperoxidase, but not lactoferrin from neonatal neutrophils stimulated with C5a, was significantly lower than in adult controls. fMLP stimulation, on the other hand, resulted in significantly higher exocytosis in neonates. CONCLUSION: The lower expression of C5a...... receptors on neutrophils from neonates could be related to reduced C5a mediated exocytosis of myeloperoxidase....

  1. Factor H and factor H-related protein 1 bind to human neutrophils via complement receptor 3, mediate attachment to Candida albicans, and enhance neutrophil antimicrobial activity.

    Science.gov (United States)

    Losse, Josephine; Zipfel, Peter F; Józsi, Mihály

    2010-01-15

    The host complement system plays an important role in protection against infections. Several human-pathogenic microbes were shown to acquire host complement regulators, such as factor H (CFH), that downregulate complement activation at the microbial surface and protect the pathogens from the opsonic and lytic effects of complement. Because CFH can also bind to host cells, we addressed the role of CFH and CFH-related proteins as adhesion ligands in host-pathogen interactions. We show that the CFH family proteins CFH, CFH-like protein 1 (CFHL1), CFH-related protein (CFHR) 1, and CFHR4 long isoform bind to human neutrophil granulocytes and to the opportunistic human-pathogenic yeast Candida albicans. Two major binding sites, one within the N-terminus and one in the C-terminus of CFH, were found to mediate binding to neutrophils. Complement receptor 3 (CD11b/CD18; alpha(M)beta2 integrin) was identified as the major cellular receptor on neutrophils for CFH, CFHL1, and CFHR1, but not for CFHR4 long isoform. CFH and CFHR1 supported cell migration. Furthermore, CFH, CFHL1, and CFHR1 increased attachment of neutrophils to C. albicans. Adhesion of neutrophils to plasma-opsonized yeasts was reduced when CFH binding was inhibited by specific Abs or when using CFH-depleted plasma. Yeast-bound CFH and CFHR1 enhanced the generation of reactive oxygen species and the release of the antimicrobial protein lactoferrin by human neutrophils, and resulted in a more efficient killing of the pathogen. Thus, CFH and CFHR1, when bound on the surface of C. albicans, enhance antimicrobial activity of human neutrophils.

  2. Adiponectin inhibits neutrophil apoptosis via activation of AMP kinase, PKB and ERK 1/2 MAP kinase.

    Science.gov (United States)

    Rossi, Alessandra; Lord, Janet M

    2013-12-01

    Neutrophils are abundant, short-lived leukocytes that play a key role in the immune defense against microbial infections. These cells die by apoptosis following activation and uptake of microbes and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Adiponectin exerts anti-inflammatory effects on neutrophil antimicrobial functions, but whether this abundant adipokine influences neutrophil apoptosis is unknown. Here we report that adiponectin in the physiological range (1-10 μg/ml) reduced apoptosis in resting neutrophils, decreasing caspase-3 cleavage and maintaining Mcl-1 expression by stabilizing this anti-apoptotic protein. We show that adiponectin induced phosphorylation of AMP-activated kinase (AMPK), protein kinase B (PKB), extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen activated protein kinase (MAPK). Pharmacological inhibition of AMPK, PKB and ERK 1/2 ablated the pro-survival effects of adiponectin and treatment of neutrophils with an AMPK specific activator (AICAR) and AMPK inhibitor (compound C) respectively decreased and increased apoptosis. Finally, activation of AMPK by AICAR or adiponectin also decreased ceramide accumulation in the neutrophil cell membrane, a process involved in the early stages of spontaneous apoptosis, giving another possible mechanism downstream of AMPK activation for the inhibition of neutrophil apoptosis.

  3. P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals.

    Science.gov (United States)

    Sil, Payel; Hayes, Craig P; Reaves, Barbara J; Breen, Patrick; Quinn, Shannon; Sokolove, Jeremy; Rada, Balázs

    2017-01-01

    Human neutrophils (polymorphonuclear leukocytes [PMNs]) generate inflammatory responses within the joints of gout patients upon encountering monosodium urate (MSU) crystals. Neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid of gout patients. The detailed mechanism of MSU crystal-induced NET formation remains unknown. Our goal was to shed light on possible roles of purinergic signaling and neutrophil migration in mediating NET formation induced by MSU crystals. Interaction of human neutrophils with MSU crystals was evaluated by high-throughput live imaging using confocal microscopy. We quantitated NET levels in gout synovial fluid supernatants and detected enzymatically active neutrophil primary granule enzymes, myeloperoxidase, and human neutrophil elastase. Suramin and PPADS, general P2Y receptor blockers, and MRS2578, an inhibitor of the purinergic P2Y6 receptor, blocked NET formation triggered by MSU crystals. AR-C25118925XX (P2Y2 antagonist) did not inhibit MSU crystal-stimulated NET release. Live imaging of PMNs showed that MRS2578 represses neutrophil migration and blocked characteristic formation of MSU crystal-NET aggregates called aggregated NETs. Interestingly, the store-operated calcium entry channel inhibitor (SK&F96365) also reduced MSU crystal-induced NET release. Our results indicate that the P2Y6/store-operated calcium entry/IL-8 axis is involved in MSU crystal-induced aggregated NET formation, but MRS2578 could have additional effects affecting PMN migration. The work presented in the present study could lead to a better understanding of gouty joint inflammation and help improve the treatment and care of gout patients.

  4. Low molecular weight heparin alters porcine neutrophil responses to platelet-activating factor.

    Science.gov (United States)

    Kruse-Elliott, K T; Chaban, K; Grossman, J E; Tomasko, S; Kamke, C; Darien, B

    1998-09-01

    Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has anti-inflammatory effects, we hypothesized that low molecular weight heparin (LMWH) would inhibit PAF-induced activation and chemotaxis in porcine neutrophils. Citrated blood was obtained from pentobarbital-anesthetized pigs, and neutrophils were isolated over a 55%/65% Percoll gradient. The effect of LMWH on basal phorbol myristate acetate (PMA)-induced superoxide (SO) release, as well as its effect on PAF priming for PMA-induced SO release, were investigated. Additionally, the effect of LMWH on PAF-induced chemotaxis of neutrophils across transwell membranes was evaluated. Baseline SO release in response to PMA was .351+/-.046 nmol/10(6) cells/min, and this was decreased to .289+/-.034 nmol/10(6) cells/min by pretreatment with 50 U/mL LMWH. PMA-induced SO production was increased by .240+/-.042 nmol/10(6) cells/min when cells were primed with 10 microM PAF. This priming effect of PAF was reduced significantly by pretreatment of neutrophils with LMWH at 10 and 50 U/mL. Chemotaxis of neutrophils in response to 100 microM PAF was significantly decreased to 70.02+/-6.4% (n = 8) of the control response by pretreatment of cells with 50 U/mL LMWH. We conclude that LMWH has anti-inflammatory effects on porcine neutrophils, which includes attenuation of cell activation and chemotaxis in response to the lipid-derived inflammatory mediator, PAF.

  5. Homocysteine enhances superoxide anion release and NADPH oxidase assembly by human neutrophils. Effects on MAPK activation and neutrophil migration.

    Science.gov (United States)

    Alvarez-Maqueda, Moisés; El Bekay, Rajaa; Monteseirín, Javier; Alba, Gonzalo; Chacón, Pedro; Vega, Antonio; Santa María, Consuelo; Tejedo, Juan R; Martín-Nieto, José; Bedoya, Francisco J; Pintado, Elisabeth; Sobrino, Francisco

    2004-02-01

    Hyperhomocysteinaemia has recently been recognized as a risk factor of cardiovascular disease. However, the action mechanisms of homocysteine (Hcy) are not well understood. Given that Hcy may be involved in the recruitment of monocytes and neutrophils to the vascular wall, we have investigated the role of Hcy in essential functions of human neutrophils. We show that Hcy increased superoxide anion (O2*-) release by neutrophils to the extracellular medium, and that this effect was inhibited by superoxide dismutase and diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase activity. The enzyme from rat peritoneal macrophages displayed a similar response. These effects were accompanied by a time-dependent increased translocation of p47phox and p67phox subunits of NADPH oxidase to the plasma membrane. We also show that Hcy increased intracellular H2O2 production by neutrophils, that Hcy enhanced the activation and phosphorylation of mitogen-activated protein kinases (MAPKs), specifically p38-MAPK and ERK1/2, and that the migration of neutrophils was increased by Hcy. Present results are the first evidence that Hcy enhances the oxidative stress of neutrophils, and underscore the potential role of phagocytic cells in vascular wall injury through O2*- release in hyperhomocysteinaemia conditions.

  6. Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils.

    Science.gov (United States)

    Creed, T Michael; Tandon, Shweta; Ward, Richard A; McLeish, Kenneth R

    2017-06-21

    Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47(phox) phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47(phox) phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey-Kramer multiple-comparison test. Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47(phox) phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

  7. Treatment with selectin blocking antibodies after lengthening contractions of mouse muscle blunts neutrophil accumulation but does not reduce damage.

    Science.gov (United States)

    Sloboda, Darcée D; Brooks, Susan V

    2016-01-01

    P- and E-selectins are expressed on the surface of endothelial cells and may contribute to neutrophil recruitment following injurious lengthening contractions of skeletal muscle. Blunting neutrophil, but not macrophage, accumulation after lengthening contractions may provide a therapeutic benefit as neutrophils exacerbate damage to muscle fibers, while macrophages promote repair. In this study, we tested the hypothesis that P- and E-selectins contribute to neutrophil, but not macrophage, accumulation in muscles after contraction-induced injury, and that reducing neutrophil accumulation by blocking the selectins would be sufficient to reduce damage to muscle fibers. To test our hypothesis, we treated mice with antibodies to block P- and E-selectin function and assessed leukocyte accumulation and damage in muscles 2 days after lengthening contractions. Treatment with P/E-selectin blocking antibodies reduced neutrophil content by about half in muscles subjected to lengthening contractions. In spite of the reduction in neutrophil accumulation, we did not detect a decrease in damage 2 days after lengthening contractions. We conclude that P- and/or E-selectin contribute to the neutrophil accumulation associated with contraction-induced muscle damage and that only a portion of the neutrophils that typically accumulate following injurious lengthening contractions is sufficient to induce muscle fiber damage and force deficits. Thus, therapeutic interventions based on blocking the selectins or other adhesion proteins will have to reduce neutrophil numbers by more than 50% in order to provide a benefit.

  8. The natural stilbenoid pinosylvin and activated neutrophils: effects on oxidative burst, protein kinase C, apoptosis and efficiency in adjuvant arthritis

    Institute of Scientific and Technical Information of China (English)

    Viera JAN(C)INOV(A); Tomá(s) PERE(C)KO; Rado NOS(A)(L); Juraj HARMATHA; Jan (S)MIDRKAL; Katarína DR(A)BIKOV(A)

    2012-01-01

    Aim:To investigate the effects of the naturally occurring stilbenoid pinosylvin on neutrophil activity in vitro and in experimental arthritis,and to examine whether protein kinase C (PKC) activation served as an assumed target of pinosylvin action.Methods:Fresh human blood neutrophils were isolated.The oxidative burst of neutrophils was evaluated on the basis of enhanced chemiluminescence.Neutrophil viability was evaluated with flow cytometry,and PKC phosphorylation was assessed by Western blotting analysis.Adjuvant arthritis was induced in Lewis rats with heat-killed Mycobacterium butyricum,and the animals were administered with pinosylvin (30 mg/kg,po) daily for 21 d after arthritis induction.Results:In isolated human neutrophils,pinosylvin (10 and 100 μmol/L) significantly decreased the formation of oxidants,both extraand intracellularly,and effectively inhibited PKC activation stimulated by phorbol myristate acetate (0.05 μmol/L).The inhibition was not due to neutrophil damage or increased apoptosis.In arthritic rats,the number of neutrophils in blood was dramatically increased,and whole blood chemiluminescence (spontaneous and PMA-stimulated) was markedly enhanced.Pinosylvin administration decreased the number of neutrophils (from 69 671±5588/μL to 51 293±3947/μL,P=0.0198) and significantly reduced the amount of reactive oxygen species in blood.Conclusion:Pinosylvin is an effective inhibitor of neutrophil activity,and is potentially useful as a complementary medicine in states associated with persistent inflammation.

  9. IL-1α-induced microvascular endothelial cells promote neutrophil killing by increasing MMP-9 concentration and lysozyme activity.

    Science.gov (United States)

    Liu, Xiaoye; Dong, Hong; Wang, Mingming; Gao, Ying; Zhang, Tao; Hu, Ge; Duan, Huiqing; Mu, Xiang

    2016-02-01

    The recruitment of neutrophils by endothelial cells during infection has been extensively studied, but little is known about the regulation of neutrophils activity by endothelial cells. To examine the role of microvascular endothelial cells in neutrophil killing, we established a transmigration model using rat intestinal microvascular endothelial cells (RIMVECs) and measured the extracellular and intracellular killing of Escherichia coli, Lactobacillus acidophilus, and Staphylococcus aureus by transendothelial neutrophils. We observed that blood neutrophils engulfed bacteria but did not kill them, and lipopolysaccharide- or hemolysin-injured RIMVECs inhibited the extracellular and intracellular bactericidal activity of transendothelial neutrophils. In comparison, interleukin-1α-induced RIMVECs promoted the extracellular and intracellular killing activity of transendothelial neutrophils and significantly increased MMP-9 concentration and lysozyme activity in transendothelial neutrophils (p neutrophils and bacterial toxin damage of endothelial cells led to reduction in bactericidal activity of transendothelial neutrophils. These findings offered new insight into the role of endothelial cells in the bactericidal activity of neutrophils.

  10. [Investigation of microbicidal activity of neutrophil defensins against leptospires].

    Science.gov (United States)

    Wu, Q; Xu, L; Wang, X; Li, S; Wang, B

    1992-06-01

    Defensins play an important role in oxygen-independent microbicidal mechanisms of neutrophils. They are effective against many bacteria, fungi and enveloped viruses. However, the effect of defensins upon leptospires has not been studied. In the present report, human defensins (i.e. HNP, a mixture of HNP1, HNP2 and HNP3 were prepared from human polymorphonuclear neutrophils by chromatography on Sephadex G-100 and then on Biogel P-10. Rabbit defensin NP1 was purified from rabbit peritoneal granulocytes by preparative acid urea polyacrylamide gel electrophoresis. By using the most-probable-number procedure, HNP and NP1 were tested in vitro for the killing of leptospira interrogans serogroup icteroheamorrhagiae serovar lai strain 017. NP1 was highly effective. When leptospires of strain 017 were incubated with 1 microgram/ml of NP1 at 30 degrees C for 4 hours, > 99% of these organisms were killed. HNP was less potent than NP1, and at 50 micrograms/ml, it killed > 90% of leptospires. As is also the case for the killing of bacteria, NP1 was active against leptospires in nutrient-free buffer, whereas HNP required the addition of glucose. The data suggest that defensins could play a major role in the killing of leptospires by neutrophils.

  11. Neutrophil infiltration favors colitis-associated tumorigenesis by activating the interleukin-1 (IL-1)/IL-6 axis.

    Science.gov (United States)

    Wang, Y; Wang, K; Han, G-C; Wang, R-X; Xiao, H; Hou, C-M; Guo, R-F; Dou, Y; Shen, B-F; Li, Y; Chen, G-J

    2014-09-01

    Neutrophil infiltration is a key event in chronic intestinal inflammation and associated colorectal cancer, but how these cells support cancer development is poorly understood. In this study, using a mouse model of colitis-associated cancer (CAC), we have demonstrated that infiltrated neutrophils produce large amounts of interleukin-1 (IL)-1β that is critical for the development of CAC. Depletion of neutrophil or blockade of IL-1β activity significantly reduced mucosal damage and tumor formation. This protumorigenic function of IL-1β was mainly attributed to increased IL-6 secretion by intestine-resident mononuclear phagocytes (MPs). Furthermore, commensal flora-derived lipopolysaccharide (LPS) was identified to trigger IL-1β expression in neutrophils. Importantly, accumulation of IL-1β-expressing neutrophils was seen in lesions of patients suffering from ulceratic CAC and these infiltrated neutrophils induced IL-6 production by intestinal MPs in an IL-1β-dependent manner. Overall, these findings reveal that in CAC milieu, infiltrating neutrophils secrete IL-1β that promotes tumorigenesis by inducing IL-6 production by intestinal MPs.

  12. Activated human neutrophil response to perfluorocarbon nanobubbles: oxygen-dependent and -independent cytotoxic responses.

    Science.gov (United States)

    Hwang, Tsong-Long; Fang, Chia-Lang; Al-Suwayeh, Saleh A; Yang, Li-Jia; Fang, Jia-You

    2011-06-10

    Nanobubbles, a type of nanoparticles with acoustically active properties, are being utilized as diagnostic and therapeutic nanoparticles to better understand, detect, and treat human diseases. The objective of this work was to prepare different nanobubble formulations and investigate their physicochemical characteristics and toxic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated human neutrophils. The nanobubbles were prepared using perfluoropentane and coconut oil as the respective core and shell, with soybean phosphatidylcholine (SPC) and/or cationic surfactants as the interfacial layers. The cytotoxic effect of the nanobubbles on neutrophils was determined by extracellular O₂(.)⁻ release, intracellular reactive oxygen species (ROS), lactate dehydrogenase (LDH), and elastase release. Particle sizes of the nanobubbles with different percentages of perfluorocarbon, oil, and surfactants in ranged 186-432 nm. The nanobubbles were demonstrated to inhibit the generation of superoxide and intracellular ROS. The cytotoxicity of nanobubbles may be mainly associated with membrane damage, as indicated by the high LDH leakage. Systems with Forestall (FE), a cationic surfactant, or higher SPC contents exhibited the greatest LDH release by 3-fold compared to the control. The further addition of an oil component reduced the cytotoxicity induced by the nanobubbles. Exposure to most of the nanobubble formulations upregulated elastase release by activated neutrophils. Contrary to this result, stearylamine (SA)-containing systems slightly but significantly suppressed elastase release. FE and SA in a free form caused stronger responses by neutrophils than when they were incorporated into nanobubbles. In summary, exposure to nanobubbles resulted in a formulation-dependent toxicity toward human neutrophils that was associated with both oxygen-dependent and -independent pathways. Clinicians should therefore exercise caution when using nanobubbles in patients

  13. Impairment of adenosine A3 receptor activity disrupts neutrophil migratory capacity and impacts innate immune function in vivo.

    Science.gov (United States)

    Butler, Matt; Sanmugalingam, Devika; Burton, Victoria J; Wilson, Tammy; Pearson, Ruth; Watson, Robert P; Smith, Philip; Parkinson, Scott J

    2012-12-01

    Adenosine possesses potent anti-inflammatory properties which are partly mediated by G(i) -coupled adenosine A3 receptors (A3Rs). A3R agonists have shown clinical benefit in a number of inflammatory conditions although some studies in A3R-deficient mice suggest a pro-inflammatory role. We hypothesised that, in addition to cell signalling effects, A3R compounds might inhibit neutrophil chemotaxis by disrupting the purinergic feedback loop controlling leukocyte migration. Human neutrophil activation triggered rapid upregulation of surface A3R expression which was disrupted by pre-treatment with either agonist (Cl-IB-MECA) or antagonist (MRS1220). Both compounds reduced migration velocity and neutrophil transmigration capacity without impacting the response to chemokines per se. Similar effects were observed in murine neutrophils, while cells from A3R-deficient mice displayed a constitutively impaired migratory phenotype indicating compound-induced desensitisation and genetic ablation had the same functional outcome. In a dextran sodium sulphate-induced colitis model, A3R-deficient mice exhibited reduced colon pathology and decreased tissue myeloperoxidase levels at day 8 - consistent with reduced neutrophil recruitment. However, A3R-deficient mice were unable to resolve the dextran sodium sulphate-induced inflammation and had elevated numbers of tissue-associated bacteria by day 21. Our data indicate that A3Rs play a role in neutrophil migration and disrupting this function has the potential to adversely affect innate immune responses.

  14. Mycobacterium abscessus induces a limited pattern of neutrophil activation that promotes pathogen survival.

    Directory of Open Access Journals (Sweden)

    Kenneth C Malcolm

    Full Text Available Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy.

  15. Neutrophil depletion-but not prevention of Kupffer cell activation-decreases the severity of cerulein-induced acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Catherine M Pastor; Alain Vonlaufen; Fabianna Georgi; Antoine Hadengue; Philippe Morel; Jean-Louis Frossard

    2006-01-01

    AIM: To determine whether neutrophil depletion and Kupffer cell inhibition might combine their protective effects to decrease the severity of acute pancreatitis.METHODS: Mice had cerulein administration to induce acute pancreatitis and were pretreated with either anti-mouse neutrophil serum or gadolinium chloride (GdCl3) to prevent Kupffer cell activation, or both treatments. Injury was assessed in pancreas and lungs.Myeloperoxidases (MPO) assessed neutrophil infiltration.Interleukin-6 (IL-6) and IL-10 were measured in serum,pancreas, lungs and liver.RESULTS: In mice with acute pancreatitis, neutrophil depletion reduced the severity of pancreatitis and pancreatitis-associated lung injury. Kupffer cell inactivation by GdCl3 had less protective effect, although IL-6 and IL-10 concentrations were significantly decreased. The protective treatment brought by neutrophil depletion was not enhanced by Kupffer cell inactivation and both treatments did not combine their protective effects.CONCLUSION: Our results confirm the role of activated neutrophils in aggravating organ injury in acute pancreatitis while the role of Kupffer cell activation is less obvious.

  16. Estrogen effect on post-exercise skeletal muscle neutrophil infiltration and calpain activity

    National Research Council Canada - National Science Library

    Tiidus P.M; Holden D; Bombardier E; Zajchowski S; Enns D; Belcastro A

    2001-01-01

    We hypothesized that estrogen administration would attenuate skeletal muscle neutrophil infiltration, indices of muscle membrane disruption, and muscle calpain activity shortly after the termination of exercise...

  17. Helicobacter pylori neutrophil activating protein as target for new drugs against H.pylori inflammation

    Institute of Scientific and Technical Information of China (English)

    Theodora Choli-Papadopoulou; Filippos Kottakis; Georgios Papadopoulos; Stefanos Pendas

    2011-01-01

    Helicobacter pylori (H. pylori ) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. Within this work we present the implication of C-terminal region of H. pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evidence that the C-terminal region of H. pylori activating protein is indispensable for neutrophil adhesion to endothelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins derived from chios mastic gum, the natural resin of the plant Pistacia lentiscus var. Chia inhibit neutrophil activation in vitro .

  18. Solonamide B inhibits quorum sensing and reduces Staphylococcus aureus mediated killing of human neutrophils.

    Science.gov (United States)

    Nielsen, Anita; Månsson, Maria; Bojer, Martin S; Gram, Lone; Larsen, Thomas O; Novick, Richard P; Frees, Dorte; Frøkiær, Hanne; Ingmer, Hanne

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

  19. Neutrophils from Patients with Primary Ciliary Dyskinesia Display Reduced Chemotaxis to CXCR2 Ligands

    Directory of Open Access Journals (Sweden)

    Maaike Cockx

    2017-09-01

    Full Text Available Primary ciliary dyskinesia (PCD, cystic fibrosis (CF, and chronic obstructive airway disease are characterized by neutrophilic inflammation in the lungs. In CF and chronic obstructive airway disease, improper functioning of neutrophils has been demonstrated. We hypothesized that the pulmonary damage in PCD might be aggravated by abnormal functioning neutrophils either as a primary consequence of the PCD mutation or secondary to chronic inflammation. We analyzed chemotactic responses and chemoattractant receptor expression profiles of peripheral blood neutrophils from 36 patients with PCD, 21 healthy children and 19 healthy adults. We stimulated peripheral blood monocytes from patients and healthy controls and measured CXCL8 and IL-1β production with ELISA. PCD neutrophils displayed reduced migration toward CXCR2 ligands (CXCL5 and CXCL8 in the shape change, microchamber and microslide chemotaxis assays, whereas leukotriene B4 and complement component 5a chemotactic responses were not significantly different. The reduced response to CXCL8 was observed in all subgroups of patients with PCD (displaying either normal ultrastructure, dynein abnormalities or central pair deficiencies and correlated with lung function. CXCR2 was downregulated in about 65% of the PCD patients, suggestive for additional mechanisms causing CXCR2 impairment. After treatment with the TLR ligands lipopolysaccharide and peptidoglycan, PCD monocytes produced more CXCL8 and IL-1β compared to controls. Moreover, PCD monocytes also responded stronger to IL-1β stimulation in terms of CXCL8 production. In conclusion, we revealed a potential link between CXCR2 and its ligand CXCL8 and the pathogenesis of PCD.

  20. Cefodizime (HR 221) potentiation of human neutrophil oxygen-independent bactericidal activity.

    Science.gov (United States)

    Labro, M T; Amit, N; Babin-Chevaye, C; Hakim, J

    1987-03-01

    The enhanced bactericidal activity of human neutrophils induced by cefotaxime and cefodizime, two methoxy-imino-amino- 2-thiazolyl cephalosporins, is linked to the cell stimulation of oxygen-dependent and oxygen-independent killing systems, respectively. Cefotaxime enhances both the killing and the oxidative response of neutrophils to opsonized particulate stimuli (bacteria for both activities and opsonized zymosan for the oxidative burst). These effects were not observed with non-opsonized particles (bacteria or zymosan) or soluble stimuli. On the contrary, cefodizime enhances killing of opsonized and non-opsonized bacteria by neutrophils regardless of treatment with phenylbutazone which blocks neutrophil oxidative metabolism. Cefodizime does not universally alter the oxidative burst induced by various stimuli, but has been shown to enhance the bactericidal activity of crude extracts of neutrophil granules. The data suggest that cefodizime and non O2-dependent killing systems of neutrophils cooperate in killing bacteria.

  1. The effect of midazolam on neutrophil mitogen-activated protein kinase.

    LENUS (Irish Health Repository)

    Ghori, Kamran

    2010-06-01

    Neutrophil p38 mitogen-activated protein kinase (MAPK) is a key enzyme in the intracellular signalling pathway that is responsible for many neutrophil functions, which are important in neutrophil-endothelial interaction. The imidazole compounds are inhibitors of this enzyme system. The objectives of this in-vitro investigation were to examine the effect of midazolam on neutrophil p38 MAPK activation (phosphorylation) following in-vitro ischaemia-reperfusion injury, and the expression of adhesion molecule CD11b\\/CD18.

  2. FUNCTIONAL AND METABOLIC ACTIVITY OF NEUTROPHILIC GRANULOCYTES IN CASE OF ACUTE BACTERIAL RHINOSINUSITIS

    Directory of Open Access Journals (Sweden)

    O. A. Kolenchukova

    2013-01-01

    Full Text Available Abstract. The functional and metabolic activities of neutrophilic granulocytes in patients with acute bacterial rhinosinusitis (ABRS have been studied. Characteristics of the indices of chemiluminescence and bioluminescence for neutrophils, extracted from venous blood and maxillary sinus were compared. It was demonstrated the decrease of intensity of APK production in neutrophils, extracted from inflammation point, with simultaneous decrease of intensity of plastic processes and increasing of energy processes in compare with the same indices in blood cells.

  3. Thermal injury induces impaired function in polymorphonuclear neutrophil granulocytes and reduced control of burn wound infection

    DEFF Research Database (Denmark)

    Calum, H.; Moser, C.; Jensen, P. O.;

    2009-01-01

    with infected burn wound. Furthermore, the oxidative burst and the phagocytic capacity of the PMNs were reduced in the group of mice with burn wound. Using this novel mouse model of thermal injury a decline of peripheral leucocytes was observed, whereas the increased local inflammatory response at the site...... of mice with burn wound. The reduction was ascribed to the decline in concentration of polymorphonuclear neutrophil leucocytes and monocytes. When infecting the skin with Pseudomonas aeruginosa, a dissemination of bacteria was observed only in the burn wound group. Histological characterization...... of the skin showed a more polymorphonuclear neutrophil granulocytes (PMNs)-dominated inflammation in the group of mice with infected burn wound compared with the with burn wound group. In contrast, a higher degree of inflammation was observed in the burn wound group compared with the group of mice...

  4. Opa+ Neisseria gonorrhoeae exhibits reduced survival in human neutrophils via Src family kinase-mediated bacterial trafficking into mature phagolysosomes.

    Science.gov (United States)

    Johnson, M Brittany; Ball, Louise M; Daily, Kylene P; Martin, Jennifer N; Columbus, Linda; Criss, Alison K

    2015-05-01

    During gonorrhoeal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial contents. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signalling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signalling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils' full antimicrobial arsenal.

  5. Mincle activation enhances neutrophil migration and resistance to polymicrobial septic peritonitis

    Science.gov (United States)

    Lee, Wook-Bin; Yan, Ji-Jing; Kang, Ji-Seon; Zhang, Quanri; Choi, Won Young; Kim, Lark Kyun; Kim, Young-Joon

    2017-01-01

    Sepsis is a systemic inflammatory response to bacterial infection. The therapeutic options for treating sepsis are limited. Impaired neutrophil recruitment into the infection site is directly associated with severe sepsis, but the precise mechanism is unclear. Here, we show that Mincle plays a key role in neutrophil migration and resistance during polymicrobial sepsis. Mincle-deficient mice exhibited lower survival rates in experimental sepsis from cecal ligation and puncture and Escherichia coli–induced peritonitis. Mincle deficiency led to higher serum inflammatory cytokine levels and reduced bacterial clearance and neutrophil recruitment. Transcriptome analyses revealed that trehalose dimycolate, a Mincle ligand, reduced the expression of G protein–coupled receptor kinase 2 (GRK2) in neutrophils. Indeed, GRK2 expression was upregulated, but surface expression of the chemokine receptor CXCR2 was downregulated in blood neutrophils from Mincle-deficient mice with septic injury. Moreover, CXCL2-mediated adhesion, chemotactic responses, and F-actin polymerization were reduced in Mincle-deficient neutrophils. Finally, we found that fewer Mincle-deficient neutrophils infiltrated from the blood circulation into the peritoneal fluid in bacterial septic peritonitis compared with wild-type cells. Thus, our results indicate that Mincle plays an important role in neutrophil infiltration and suggest that Mincle signaling may provide a therapeutic target for treating sepsis. PMID:28112221

  6. Anti-inflammatory Effects of Quercetin and Vitexin on Activated Human Peripheral Blood Neutrophils - The effects of quercetin and vitexin on human neutrophils -

    Directory of Open Access Journals (Sweden)

    Bahareh Abd Nikfarjam

    2017-06-01

    Full Text Available Objectives: Polymorphonuclear neutrophils (PMNs constitute the first line of defense against invading microbial pathogens. Early events in inflammation involve the recruitment of neutrophils to the site of injury or damage where changes in intracellular calcium can cause the activation of pro-inflammatory mediators from neutrophils including superoxide generation, degranulation and release of myeloperoxidase (MPO, productions of interleukin (IL-8 and tumor necrosis factor α (TNF-α, and adhesion to the vascular endothelium. To address the anti-inflammatory role of flavonoids, in the present study, we investigated the effects of the flavonoids quercetin and vitexin on the stimulus-induced nitric oxide (NO, TNF-α, and MPO productions in human neutrophils. Methods: Human peripheral blood neutrophils were isolated, and their viabilities were determined by using the Trypan Blue exclusion test. The polymorphonuclear leukocyte (PMNL preparations contained more than 98% neutrophils as determin

  7. Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

    Science.gov (United States)

    Yum, H K; Arcaroli, J; Kupfner, J; Shenkar, R; Penninger, J M; Sasaki, T; Yang, K Y; Park, J S; Abraham, E

    2001-12-01

    Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.

  8. The Interleukin-17 Induced Activation and Increased Survival of Equine Neutrophils Is Insensitive to Glucocorticoids.

    Directory of Open Access Journals (Sweden)

    Ruby Yoana Murcia

    Full Text Available Glucocorticoids (GCs are the most effective drugs for the treatment of human asthma. However, a subgroup of asthmatic patients with neutrophilic airway inflammation is insensitive to GCs. Interleukin-17 (IL-17, a cytokine upregulated in the airways of a subset of human asthmatic patients, contributes to the recruitment of neutrophils and induces a glucocorticoid resistance in human airway epithelial cells. We hypothesized that IL-17 similarly activates neutrophils and contributes to their persistence in the asthmatic airways in spite of glucocorticoid therapy.To determine whether IL-17 directly activates neutrophils and whether this response is attenuated by GCs.Neutrophils were isolated from the blood of horses and incubated in the presence of recombinant equine IL-17, LPS and dexamethasone. mRNA and protein expression of IL-17 receptors (IL-17RA/IL-17RC were assessed by qPCR and immunoblot, respectively. Pro-inflammatory cytokine expression, cell viability and apoptosis were determined by qPCR, Trypan Blue exclusion test, and flow cytometry, respectively.Equine neutrophils express both IL-17RA and IL-17RC at the mRNA and protein levels. Neutrophil stimulation with IL-17 increases the mRNA expression of IL-8, which is not attenuated by dexamethasone (p = 0.409. Also, neutrophil viability is significantly increased (p<0.0001 by IL-17 in the presence of LPS when compared to LPS alone. Flow cytometry and light microscopy revealed that LPS-induced apoptosis is decreased by IL-17 (p = 0.02 and p = 0.006 respectively.These results indicate that IL-17 directly activates equine neutrophils at 24 hours, and that the expression of IL-8 thus induced is not attenuated by GCs. Additionally, IL-17 increases neutrophil viability and decreases apoptosis. These findings suggest an important role of IL-17 in pulmonary persistence of neutrophils in the asthmatic airways.

  9. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    OpenAIRE

    Hermann, M.; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-01-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (m...

  10. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

    Science.gov (United States)

    Smiley, P L; Stremler, K E; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1991-06-15

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows

  11. Increased metabolic activity of neutrophils in patients with chronic obstructive pulmonary disease

    Science.gov (United States)

    Vaidyanathan, Ashwin; Damodar, Komaladevi Sampath

    2015-01-01

    Aims: To compare the metabolic activity of peripheral neutrophils in patients diagnosed with chronic obstructive pulmonary disease (COPD) with that of healthy, nonsmoking volunteers. Materials and Methods: Venous blood samples were taken from patients diagnosed with COPD as well as from healthy nonsmokers. Each sample was subjected to the nitro blue tetrazolium (NBT) test in which neutrophils exhibiting elevated metabolic activity were detected by light microscopy. The test was repeated after stimulation with Escherichia coli (E. coli) endotoxin with fresh samples. Neutrophils showing dye uptake were then counted in each case. Results: We found that the mean numbers of activated neutrophils without and with the addition of endotoxin were 19% and 23%, respectively, in the control group and 56% and 62%, respectively, in the test group. Two-sample t-test statistic revealed that there was a significant (P < 0.01) increase in neutrophilic metabolic activity in patients with COPD as compared to that in healthy volunteers. This significance remained even after stimulation using E. coli endotoxin. Conclusion: The results hint at a potentially relevant pathogenic mechanism in COPD related to the metabolic activity of neutrophils. By exhibiting enhanced metabolic activity, neutrophils in the COPD patients are more likely to be involved in damaging lung tissues. PMID:26664165

  12. Activated Neutrophils Are Associated with Pediatric Cerebral Malaria Vasculopathy in Malawian Children

    Science.gov (United States)

    Feintuch, Catherine Manix; Saidi, Alex; Seydel, Karl; Chen, Grace; Goldman-Yassen, Adam; Mita-Mendoza, Neida K.; Kim, Ryung S.; Frenette, Paul S.; Taylor, Terrie

    2016-01-01

    ABSTRACT Most patients with cerebral malaria (CM) sustain cerebral microvascular sequestration of Plasmodium falciparum-infected red blood cells (iRBCs). Although many young children are infected with P. falciparum, CM remains a rare outcome; thus, we hypothesized that specific host conditions facilitate iRBC cerebral sequestration. To identify these host factors, we compared the peripheral whole-blood transcriptomes of Malawian children with iRBC cerebral sequestration, identified as malarial-retinopathy-positive CM (Ret+CM), to the transcriptomes of children with CM and no cerebral iRBC sequestration, defined as malarial-retinopathy-negative CM (Ret-CM). Ret+CM was associated with upregulation of 103 gene set pathways, including cytokine, blood coagulation, and extracellular matrix (ECM) pathways (P < 0.01; false-discovery rate [FDR] of <0.05). Neutrophil transcripts were the most highly upregulated individual transcripts in Ret+CM patients. Activated neutrophils can modulate diverse host processes, including the ECM, inflammation, and platelet biology to potentially facilitate parasite sequestration. Therefore, we compared plasma neutrophil proteins and neutrophil chemotaxis between Ret+CM and Ret-CM patients. Plasma levels of human neutrophil elastase, myeloperoxidase, and proteinase 3, but not lactoferrin or lipocalin, were elevated in Ret+CM patients, and neutrophil chemotaxis was impaired, possibly related to increased plasma heme. Neutrophils were rarely seen in CM brain microvasculature autopsy samples, and no neutrophil extracellular traps were found, suggesting that a putative neutrophil effect on endothelial cell biology results from neutrophil soluble factors rather than direct neutrophil cellular tissue effects. Meanwhile, children with Ret-CM had lower levels of inflammation, higher levels of alpha interferon, and upregulation of Toll-like receptor pathways and other host transcriptional pathways, which may represent responses that do not favor

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-03-01

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

  14. Blood neutrophil bactericidal activity against methicillin-resistant and methicillin-sensitive Staphylococcus aureus during cardiac surgery.

    Science.gov (United States)

    Mekontso-Dessap, Armand; Honoré, Stéphanie; Kirsch, Matthias; Plonquet, Anne; Fernandez, Eric; Touqui, Lhousseine; Farcet, Jean-Pierre; Soussy, Claude-James; Loisance, Daniel; Delclaux, Christophe

    2005-08-01

    Whether methicillin-resistant Staphylococcus aureus (MRSA) constitutes per se an independent risk factor for morbidity and mortality after surgery as compared with methicillin-sensitive Staphylococcus aureus (MSSA) remains a subject of debate. The aim of this study was to assess whether innate defenses against MRSA and MSSA strains are similarly impaired after cardiac surgery. Both intracellular (isolated neutrophil functions) and extracellular (plasma) defenses of 12 patients undergoing scheduled cardiac surgery were evaluated preoperatively (day 0) and postoperatively (day 3) against two MSSA strains with a low level of catalase secretion and two MRSA strains with a high level of catalase secretion, inasmuch as SA killing by neutrophils relies on oxygen-dependent mechanisms. After surgery, an increase in plasma concentration of IL-10, an anti-inflammatory cytokine able to inhibit reactive oxygen species secretion and bactericidal activity of neutrophils, was evidenced. Despite the fact that univariate analysis suggested a specific impairment of neutrophil functions against MRSA strains, two-way repeated-measures ANOVA failed to demonstrate that the effect of S. aureus phenotype was significant. On the other hand, an increase in type-II secretory phospholipase A2 activity, a circulating enzyme involved in SA lysis, was evidenced and was associated with an enhancement of extracellular defenses (bactericidal activity of plasma) against MRSA. Overall, cardiac surgery and S. aureus phenotype had a significant effect on plasma bactericidal activity. Cardiac surgery was characterized by enhanced antibacterial defenses of plasma, whereas neutrophil killing properties were reduced. The overall effect of S. aureus phenotype on neutrophil functions did not seem significant.

  15. Modulation of γδ T cell activation by neutrophil elastase.

    Science.gov (United States)

    Towstyka, Nadia Yasmín; Shiromizu, Carolina Maiumi; Keitelman, Irene; Sabbione, Florencia; Salamone, Gabriela Verónica; Geffner, Jorge Raúl; Trevani, Analía Silvina; Jancic, Carolina Cristina

    2017-09-09

    γδ T cells are non-conventional, innate-like T cells, characterized by a restricted TCR repertoire. They participate in protective immunity response against extracellular and intracellular pathogens, tumor surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance, and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of IFN-γ and TNF-α induced by anti-CD3 antibodies were potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of IL-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine proteases inhibitors prevented the potentiation of γδ T cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the proteases-activated receptor, PAR1, since the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T cell activation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity.

    Science.gov (United States)

    Boly, Raïnatou; Dessy, Stéphanie; Kohnen, Stephan; Kini, Félix; Lompo, Marius; Mouithys-Mickalad, Ange; Guissou, Innocent Pierre; Dubois, Jacques; Deby-Dupont, Ginette; Serteyn, Didier; Franck, Thierry

    2011-08-01

    Agelanthus dodoneifolius DC Danser (Loranthaceae) is used for the treatment of various diseases including asthma. The aqueous and hydroalcoholic extracts have been reported to have anti-inflammatory, spasmolytic and bronchorelaxant activities. The present study investigates the effects of the aqueous decoction and the diethyl ether, ethyl acetate and butanolic fractions of Agelanthus dodoneifolius DC Danser (Loranthaceae) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by phorbol 12-myristate 13-acetate (PMA)-stimulated equine neutrophils and on purified equine MPO activity. ROS production and MPO release by the PMA-stimulated neutrophils were measured by the lucigenin-enhanced chemiluminescence and ELISA assays, respectively. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to specifically measure the equine MPO activity. Identification and quantification of the individual and total phenolic and flavonoid compounds were performed using UPLC-MS/MS equipment and colorimetric methods involving Folin-Ciocalteu and AlCl₃, respectively. All the tested extracts displayed dose-dependent inhibitory effects on the oxidant activities of neutrophils; a stronger effect was observed with the organic fractions than the aqueous decoction. These findings could be correlated with a high content of phenolic and flavonoid compounds. The results confirm the previously shown anti-inflammatory effect of Agelanthus dodoneifolius and its potential use for the treatment of neutrophil-dependent inflammatory diseases.

  17. Staphylococcus aureus SaeR/S-regulated factors reduce human neutrophil reactive oxygen species production

    NARCIS (Netherlands)

    Guerra, Fermin E.; Addison, Conrad B.; de Jong, Nienke W M; Azzolino, Joseph; Pallister, Kyler B.; van Strijp, Jos A G; Voyich, Jovanka M.

    2016-01-01

    Neutrophils are the first line of defense after a pathogen has breached the epithelial barriers, and unimpaired neutrophil functions are essential to clear infections. Staphylococcus aureus is a prevalent human pathogen that is able to withstand neutrophil killing, yet the mechanisms used by S. aure

  18. Genetic Deletion and Pharmacological Inhibition of PI3Kγ Reduces Neutrophilic Airway Inflammation and Lung Damage in Mice with Cystic Fibrosis-Like Lung Disease

    Directory of Open Access Journals (Sweden)

    Maria Galluzzo

    2015-01-01

    Full Text Available Purpose. Neutrophil-dominated airway inflammation is a key feature of progressive lung damage in cystic fibrosis (CF. Thus, reducing airway inflammation is a major goal to prevent lung damage in CF. However, current anti-inflammatory drugs have shown several limits. PI3Kγ plays a pivotal role in leukocyte recruitment and activation; in the present study we determined the effects of genetic deletion and pharmacologic inhibition of PI3Kγ on airway inflammation and structural lung damage in a mouse model of CF lung disease. Methods. βENaC overexpressing mice (βENaC-Tg were backcrossed with PI3Kγ-deficient (PI3KγKO mice. Tissue damage was assessed by histology and morphometry and inflammatory cell number was evaluated in bronchoalveolar lavage fluid (BALF. Furthermore, we assessed the effect of a specific PI3Kγ inhibitor (AS-605240 on inflammatory cell number in BALF. Results. Genetic deletion of PI3Kγ decreased neutrophil numbers in BALF of PI3KγKO/βENaC-Tg mice, and this was associated with reduced emphysematous changes. Treatment with the PI3Kγ inhibitor AS-605240 decreased the number of neutrophils in BALF of βENaC-Tg mice, reproducing the effect observed with genetic deletion of the enzyme. Conclusions. These results demonstrate the biological efficacy of both genetic deletion and pharmacological inhibition of PI3Kγ in reducing chronic neutrophilic inflammation in CF-like lung disease in vivo.

  19. Demodex-associated bacterial proteins induce neutrophil activation.

    LENUS (Irish Health Repository)

    2012-02-01

    Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea thus suggesting a possible role for bacterial proteins in the etiology of this condition. Objectives: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. Methods: Bacterial cells were lysed and proteins were partially purified by AKTA-FPLC. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. Results: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of MMP-9, an enzyme known to degrade collagen and cathelicidin, an antimicrobial peptide. In addition neutrophils exposed to the bacterial proteins demonstrated elevated rates of Il-8 and TNF-alpha production. Conclusions: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

  20. Microparticle production, neutrophil activation, and intravascular bubbles following open-water SCUBA diving.

    Science.gov (United States)

    Thom, Stephen R; Milovanova, Tatyana N; Bogush, Marina; Bhopale, Veena M; Yang, Ming; Bushmann, Kim; Pollock, Neal W; Ljubkovic, Marko; Denoble, Petar; Dujic, Zeljko

    2012-04-01

    The goal of this study was to evaluate annexin V-positive microparticles (MPs) and neutrophil activation in humans following decompression from open-water SCUBA diving with the hypothesis that changes are related to intravascular bubble formation. Sixteen male volunteer divers followed a uniform profile of four daily SCUBA dives to 18 m of sea water for 47 min. Blood was obtained prior to and at 80 min following the first and fourth dives to evaluate the impact of repetitive diving, and intravascular bubbles were quantified by trans-thoracic echocardiography carried out at 20-min intervals for 2 h after each dive. MPs increased by 3.4-fold after each dive, neutrophil activation occurred as assessed by surface expression of myeloperoxidase and the CD18 component of β(2)-integrins, and there was an increased presence of the platelet-derived CD41 protein on the neutrophil surface indicating interactions with platelet membranes. Intravascular bubbles were detected in all divers. Surprisingly, significant inverse correlations were found among postdiving bubble scores and MPs, most consistently at 80 min or more after the dive on the fourth day. There were significant positive correlations between MPs and platelet-neutrophil interactions after the first dive and between platelet-neutrophil interactions and neutrophil activation documented as an elevation in β(2)-integrin expression after the fourth dive. We conclude that MPs- and neutrophil-related events in humans are consistent with findings in an animal decompression model. Whether there are causal relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation remains obscure and requires additional study.

  1. Effects of endogenous and exogenous catecholamines on LPS-induced neutrophil trafficking and activation.

    Science.gov (United States)

    Abraham, E; Kaneko, D J; Shenkar, R

    1999-01-01

    Endotoxemia produces elevations in catecholamine levels in the pulmonary and systemic circulation as well as rapid increases in neutrophil number and proinflammatory cytokine expression in the lungs. In the present experiments, we examined the effects of endogenous and exogenous adrenergic stimulation on endotoxin-induced lung neutrophil accumulation and activation. Levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and macrophage inflammatory protein (MIP)-2 mRNAs were increased in lung neutrophils from endotoxemic mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic or control mice. Treatment with the beta-adrenergic antagonist propranolol before endotoxin administration did not affect trafficking of neutrophils to the lungs or the expression of IL-1beta, TNF-alpha, or MIP-2 by lung neutrophils. Administration of the alpha-adrenergic antagonist phentolamine before endotoxemia did not alter lung neutrophil accumulation as measured by myeloperoxidase (MPO) levels but did result in significant increases in IL-1beta, TNF-alpha, and MIP-2 mRNA expression by lung neutrophils compared with endotoxemia alone. Administration of the alpha1-adrenergic agonist phenylephrine before endotoxin did not affect trafficking of neutrophils to the lungs but was associated with significantly increased expression of TNF-alpha and MIP-2 mRNAs by lung neutrophils compared with that found after endotoxin alone. In contrast, treatment with the alpha2-adrenergic agonist UK-14304 prevented endotoxin-induced increases in lung MPO and lung neutrophil cytokine mRNA levels. The suppressive effects of UK-14304 on endotoxin-induced increases in lung MPO were not affected by administration of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester. These data demonstrate that the initial accumulation and activation of neutrophils in the lungs after endotoxemia can be significantly diminished by alpha

  2. 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.

  3. A taurine-supplemented vegan diet may blunt the contribution of neutrophil activation to acute coronary events.

    Science.gov (United States)

    McCarty, Mark F

    2004-01-01

    Neutrophils are activated in the coronary circulation during acute coronary events (unstable angina and myocardial infarction), often prior to the onset of ischemic damage. Moreover, neutrophils infiltrate coronary plaque in these circumstances, and may contribute to the rupture or erosion of this plaque, triggering thrombosis. Activated neutrophils secrete proteolytic enzymes in latent forms which are activated by the hypochlorous acid (HOCl) generated by myeloperoxidase. These phenomena may help to explain why an elevated white cell count has been found to be an independent coronary risk factor. Low-fat vegan diets can decrease circulating leukocytes--neutrophils and monocytes--possibly owing to down-regulation of systemic IGF-I activity. Thus, a relative neutropenia may contribute to the coronary protection afforded by such diets. However, vegetarian diets are devoid of taurine - the physiological antagonist of HOCl--and tissue levels of this nutrient are relatively low in vegetarians. Taurine has anti-atherosclerotic activity in animal models, possibly reflecting a role for macrophage-derived myeloperoxidase in the atherogenic process. Taurine also has platelet-stabilizing and anti-hypertensive effects that presumably could reduce coronary risk. Thus, it is proposed that a taurine-supplemented low-fat vegan diet represents a rational strategy for diminishing the contribution of activated neutrophils to acute coronary events; moreover, such a regimen would work in a number of other complementary ways to promote cardiovascular health. Moderate alcohol consumption, the well-tolerated drug pentoxifylline, and 5-lipoxygenase inhibitors--zileuton, boswellic acids, fish oil--may also have potential in this regard. Copyright 2004 Elsevier Ltd.

  4. Do neutrophils actively participate in airway inflammation and remodeling in asthma?

    Institute of Scientific and Technical Information of China (English)

    SUN Yong-chang 孙永昌; Hong Wei Chu

    2004-01-01

    @@ Previous studies have demonstrated an increase of neutrophils in lung tissues, bronchoalveolar lavage fluid (BALF), and sputum of subjects with severe, often glucocorticoid (GC)-dependent asthma, but the mechanisms behind this are not clear.1-3 Whether neutrophils function to worsen the disease or they are simply 'bystanders' in the process of severe asthma is not certain. However, recent studies suggest a possibly active role of neutrophils in the airway inflammation and potentially in the airway remodeling of asthma.4,5

  5. Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

    Science.gov (United States)

    McMahon, Fred; Banville, Nessa; Bergin, David A; Smedman, Christian; Paulie, Staffan; Reeves, Emer; Kavanagh, Kevin

    2016-02-01

    Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1β and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 μg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 μg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation.

  6. TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism.

    Science.gov (United States)

    Smuda, Craig; Wechsler, Joshua B; Bryce, Paul J

    2011-12-30

    Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8h post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses.

  7. Trace of antibody to myeloperoxidase with nanocrystal quantum dot labeled antibody recognizing activating neutrophils

    Science.gov (United States)

    Hoshino, Akiyoshi; Nagao, Tomokazu; Yamamoto, Kenji; Suzuki, Kazuo

    2006-02-01

    It is assumed that activated neutrophils contribute to the development of anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis due to the association of myelopeoxidase(MPO)-ANCA with MPO expressed on the surface of activated neutrophils. FITC-labeled antibody (Ab) used widely are not suitable for neutrophil examination because of the labile fluorescence emission of FITC. Therefore, it is necessary to develop specific fluorescent probes for MPO detection in neutrophils in vivo. Recently, fluorescent nanocrystal quantum dots (QDs) have been used for biotechnological and medical applications because of their greater and far longer fluorescence in. QDs have several advantages over organic fluorophores: high luminescence, far longer stability against photobleaching, and a range of fluorescence wavelengths from blue to infrared, depending on particle size. Thus, we examined the role of MPO and the Ab to MPO in the pathogenesis of glomerulonephritis associated with MPO-ANCA in experimental glomerulonephritis mice using QDs. We demonstrated the QD-conjugated anti-MPO Ab visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. In addition, QD immuno-conjugates with anti-recombinant murine MPO (rmMPO) Ab revealed the trafficking of MPO-ANCA in vivo. Deceleration of blood flow in kidney vessels occurred in model mice, in which serum proteins including anti-rmMPO Ab were leaked out from collapsed glomeruli into the proximal tubule. Thus, sustained MPO expression on the neutrophil surface was significantly related to glomerulonephritis. These results indicate that the expressed MPO on the activated neutrophils with anti-MPO Ab may coordinately play essential roles in the initial steps for the development of glomerulonephritis.

  8. Pharmacological inhibition of p38 mitogen-activated protein kinases affects KC/CXCL1-induced intraluminal crawling, transendothelial migration, and chemotaxis of neutrophils in vivo.

    Science.gov (United States)

    Xu, Najia; Hossain, Mokarram; Liu, Lixin

    2013-01-01

    p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

  9. Pharmacological Inhibition of p38 Mitogen-Activated Protein Kinases Affects KC/CXCL1-Induced Intraluminal Crawling, Transendothelial Migration, and Chemotaxis of Neutrophils In Vivo

    Directory of Open Access Journals (Sweden)

    Najia Xu

    2013-01-01

    Full Text Available p38 mitogen-activated protein kinase (MAPK signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1 gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

  10. Role for membrane fusion in the activation of the respiratory burst in human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Manara-Shediac, F.S.

    1986-01-01

    Components of the respiratory burst oxidase reside in intracellular membranes of the tertiary granules in resting cells, yet oxidase activity in the activated cells occurs at the neutrophil surface. The role of degranulation in activation of the neutrophil respiratory burst was therefore investigated. Surface labeling experiments were carried out on resting and activated neutrophils using three impermeant labeling methods. Activated neutrophils labeled with (/sup 35/S) diazobenzene sulfonic acid showed a fourfold higher specific radioactivity than resting neutrophils. Similar results were obtained with the pyridoxal phosphate/borotritide labeling method. On the other hand, little difference in labeling was seen using the periodate/borotritide method which detects the carbohydrate of glycoproteins. These results suggest that either a large amount of protein, or a highly reactive protein becomes exposed upon activation. Resting, activated, and enucleated cells were labeled using the (/sup 125/I) lactoperoxidase method, then subjected to polyacrylamide gel electrophoresis. Autoradiograms of these gels showed that two proteins of about 75 and 45 kD, are labeled at the external surface of enucleated and activated cells but not resting cells.

  11. Haptoglobin is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation

    DEFF Research Database (Denmark)

    Theilgaard-Mönch, Kim; Jacobsen, Lars C; Nielsen, Marianne J

    2006-01-01

    Haptoglobin (Hp) is a plasma protein synthesized primarily by hepatocytes. It exerts a broad range of anti-inflammatory activities and acts indirectly as a bacteriostatic agent and an antioxidant by virtue of its ability to bind free hemoglobin (Hb) and to facilitate its immediate clearance......, these findings demonstrate that Hp is stored in specific granules and is released by neutrophils in response to activation. Hence, neutrophil-derived Hp might reduce tissue damage and bacterial growth at sites of infection or injury by propagating anti-inflammatory activities and Hb clearance....... granules binds to Hb. Finally, the CCAAT enhancer binding protein-epsilon (C/EBPepsilon) induced Hp transcription in a myeloid cell line, suggesting that Hp expression in myeloid cells, as in hepatocytes, is at least partially regulated by members of the C/EBP transcription factor family. Collectively...

  12. The reported clinical utility of taurine in ischemic disorders may reflect a down-regulation of neutrophil activation and adhesion.

    Science.gov (United States)

    McCarty, M F

    1999-10-01

    The first publications regarding clinical use of taurine were Italian reports claiming therapeutic efficacy in angina, intermittent claudication and symptomatic cerebral arteriosclerosis. A down-regulation of neutrophil activation and endothelial adhesion might plausibly account for these observations. Endothelial platelet-activating factor (PAF) is a crucial stimulus to neutrophil adhesion and activation, whereas endothelial nitric oxide (NO) suppresses PAF production and acts in various other ways to antagonize binding and activation of neutrophils. Hypochlorous acid (HOCl), a neutrophil product which avidly oxidizes many sulfhydryl-dependent proteins, can be expected to inhibit NO synthase while up-regulating PAF generation; thus, a vicious circle can be postulated whereby HOCl released by marginating neutrophils acts on capillary or venular endothelium to promote further neutrophil adhesion and activation. Taurine is the natural detoxicant of HOCl, and thus has the potential to intervene in this vicious circle, promoting a less adhesive endothelium and restraining excessive neutrophil activation. Agents which inhibit the action of PAF on neutrophils, such as ginkgolides and pentoxifylline, have documented utility in ischemic disorders and presumably would complement the efficacy of taurine in this regard. Fish oil, which inhibits endothelial expression of various adhesion factors and probably PAF as well, and which suppresses neutrophil leukotriene production, may likewise be useful in ischemia. These agents may additionally constitute a non-toxic strategy for treating inflammatory disorders in which activated neutrophils play a prominent pathogenic role. Double-blind studies to confirm the efficacy of taurine in symptomatic chronic ischemia are needed.

  13. Activation of adherent vascular neutrophils in the lung during acute endotoxemia

    Directory of Open Access Journals (Sweden)

    Laskin Jeffrey D

    2002-08-01

    Full Text Available Abstract Background Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. Methods Rats were treated with 5 mg/kg lipopolysaccharide (i.v. to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. Results Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein. Conclusion These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells.

  14. Oral antibiotics increase blood neutrophil maturation and reduce bacteremia and necrotizing enterocolitis in the immediate postnatal period of preterm pigs.

    Science.gov (United States)

    Nguyen, Duc Ninh; Fuglsang, Eva; Jiang, Pingping; Birck, Malene M; Pan, Xiaoyu; Kamal, Shamrulazhar B S; Pors, Susanne E; Gammelgaard, Pernille L; Nielsen, Dennis S; Thymann, Thomas; Levy, Ofer; Frøkiær, Hanne; Sangild, Per T

    2016-01-01

    Immature immunity may predispose preterm neonates to infections and necrotizing enterocolitis (NEC). Intravenous antibiotics are frequently given to prevent and treat sepsis, while oral antibiotics are seldom used. We hypothesized that oral antibiotics promote maturation of systemic immunity and delay gut bacterial colonization and thereby protect preterm neonates against both NEC and bacteremia in the immediate postnatal period. Preterm pigs were given formula and administered saline (CON) or broad-spectrum antibiotics orally (ORA) or systemically (SYS) for 5 d after birth. Temporal changes in blood parameters and bacterial composition in the intestine, blood and immune organs were analyzed. Newborn preterm pigs had few blood neutrophils and a high frequency of progenitor cells. Neutrophils gradually matured after preterm birth with increasing CD14 and decreasing CD172a expressions. Preterm neutrophil and monocyte TLR2 expression and TLR2-mediated blood cytokine responses were low relative to adults. ORA pigs showed enhanced blood neutrophil maturation with reduced cell size and CD172a expression. Only ORA pigs, but not SYS pigs, were protected from a high density of gut Gram-positive bacteria, high gut permeability, Gram-positive bacteremia and NEC. Neonatal oral antibiotics may benefit mucosal and systemic immunity via delayed gut colonization and enhanced blood neutrophil maturation just after preterm birth.

  15. Estrogen effect on post-exercise skeletal muscle neutrophil infiltration and calpain activity.

    Science.gov (United States)

    Tiidus, P M; Holden, D; Bombardier, E; Zajchowski, S; Enns, D; Belcastro, A

    2001-05-01

    We hypothesized that estrogen administration would attenuate skeletal muscle neutrophil infiltration, indices of muscle membrane disruption, and muscle calpain activity shortly after the termination of exercise. Ovariectomized female rats were implanted with either an estogen pellet (25 mg beta-estradiol) or a placebo pellet. Two weeks postimplant, animals were killed either at rest or 1 h after running exercise (60 min at 21 m x min(-1), 12% grade). The 4 experimental groups (n = 12) used were: unexercised placebo (UP), unexercised estrogen (UE), exercised placebo (EP), and exercised estrogen (EE). Blood samples were analyzed for creatine kinase (CK) activity and estradiol content. Plantaris and gastrocnemius muscles were removed and histochemical determination of neutrophil content or biochemical determination of myeloperoxidase (MPO), glucose-6-phosphate dehydrogenase (G6PD), and calpain-like activity determined. Estrogen supplemented animals had 10-20-fold higher circulating estradiol levels than placebo animals. EP animals had significantly higher (P estrogen supplementation in ovariectomized rats attenuated 1-h post-exercise serum CK activities, muscle neutrophil infiltration, MPO activities, and calpain-like activities when compared with exercised, unsupplemented animals. This supports the possibility of a relationship between estrogen, calpain dependent production of neutrophil chemo-attractant peptides, and 1-h post-exercise skeletal muscle neutrophil infiltration.

  16. Involvement of BLT1 endocytosis and Yes kinase activation in leukotriene B4-induced neutrophil degranulation.

    Science.gov (United States)

    Gaudreault, Eric; Thompson, Charles; Stankova, Jana; Rola-Pleszczynski, Marek

    2005-03-15

    One of the important biological activities of human neutrophils is degranulation, which can be induced by leukotriene B4 (LTB4). Here we investigated the intracellular signaling events involved in neutrophil degranulation mediated by the high affinity LTB4 receptor, BLT1. Peripheral blood neutrophils as well as the promyeloid PLB-985 cell line, stably transfected with BLT1 cDNA and differentiated into a neutrophil-like cell phenotype, were used throughout this study. LTB4-induced enzyme release was inhibited by 50-80% when cells were pretreated with the pharmacological inhibitors of endocytosis sucrose, Con A and NH4Cl. In addition, transient transfection with a dominant negative form of dynamin (K44A) resulted in approximately 70% inhibition of ligand-induced degranulation. Pretreating neutrophils or BLT1-expressing PLB-985 cells with the Src family kinase inhibitor PP1 resulted in a 30-60% inhibition in BLT1-mediated degranulation. Yes kinase, but not c-Src, Fgr, Hck, or Lyn, was found to exhibit up-regulated kinase activity after LTB4 stimulation. Moreover, BLT1 endocytosis was found to be necessary for Yes kinase activation in neutrophils. LTB4-induced degranulation was also sensitive to inhibition of PI3K. In contrast, it was not affected by inhibition of the mitogen-activated protein kinase MEK kinase, the Janus kinases, or the receptor tyrosine kinase epidermal growth factor receptor or platelet-derived growth factor receptor. Taken together, our results suggest an essential role for BLT1 endocytosis and Yes kinase activation in LTB4-mediated degranulation of human neutrophils.

  17. Phagocytic and oxidative burst activity of neutrophils in the end stage of liver cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Anatol Panasiuk; Jolanta Wysocka; Elzbieta Maciorkowska; Bozena Panasiuk; Danuta Prokopowicz; Janusz Zak; Karol Radomski

    2005-01-01

    AIM: To evaluate the phagocytic activity and neutrophil oxidative burst in liver cirrhosis.METHODS: In 45 patients with advanced postalcoholic liver cirrhosis (aged 45±14 years) and in 25 healthy volunteers (aged 38±5 years), the percentage of phagocytizing cells after in vitro incubation with E. coli (Phagotest Kit), phagocytic activity (mean intensity of fluorescence, MIF) and the percentage of neutrophil oxidative burst (Bursttest Kit), and the level of free oxygen radical production (MIF of Rodamine 123)were analyzed by flow cytometry. The levels of soluble sICAM-1, sVCAM-1, sP-selectin, sE-selectin, sL-selectin,and TNF-α were determined in blood serum.RESULTS: The percentage of E. coli phagocytizing neutrophils in liver cirrhosis patients was comparable to that in healthy subjects. MIF of neutrophil - ingested E. coli was higher in patients with liver cirrhosis. The oxidative burst in E. coli phagocytizing neutrophils generated less amount of active oxygen compounds in liver cirrhosis patients (MIF of R123:24.7±7.1 and 29.7±6.6 in healthy,P<0.01). Phorbol myristate acetate (PMA) - stimulated neutrophilsproduced less reactive oxidants in liver cirrhosis patients than in healthy subjects (MIF of R123: 42.7±14.6 vs 50.2±13.3, P<0.01). A negative correlation was observed between oxidative burst MIF of PMA-stimulated neutrophils and ALT and AST levels (r -0.35, P<0.05;r-0.4, P<0.03). sVCAM-1, sICAM-1, sE-selectin concentrations correlated negatively with the oxygen free radical production (MIF of R123) in neutrophils after PMA stimulation in liver cirrhosis patients (r-0.45, P<0.05;r-0.41, P<0.05; r-0.39, P<0.05, respectively).CONCLUSION: Neutrophil metabolic activity diminishes together with the intensification of liver failure. The metabolic potential of phagocytizing neutrophils is significantly lower in liver cirrhosis patients, which can be one of the causes of immune mechanism damage. The evaluation of oxygen metabolism of E. coli

  18. Impact of neutrophil apoptosis on haemostatic activation in chronic liver disease patients.

    Science.gov (United States)

    Essawy, Faiza M; Bekheet, Iman W; Saleh, Abeya F; Madkour, Mona E; Bayoumi, Emad El-Din A

    2008-09-01

    Recent studies suggest the impact of apoptosis on the mechanisms leading to hypercoagulability. We aimed to clarify the potential role of neutrophil apoptosis in neutropenia and hypercoagulable state encountered in chronic liver disease patients. This study was conducted on 15 normal controls and 45 patients with chronic liver disease classified according to modified Child Pugh classification into, Child A, B and C groups (15 cases each). Haemostatic parameters studied include, prothrombin time, partial thromboplastin time, tissue factor, protein C antigen, protein S antigen, and markers of haemostatic activation [prothrombin fragment 1+2 (F1+2), thrombus precursor protein (TpP) and D-dimer]. Flowcytometric study was done for quantitative assay of neutrophil apoptotic subpopulations to detect the percentage of early and late apoptotic, and necrotic neutrophils using Annexin V-FITC/propidium iodide dye. Semiquantitative assay of apoptotic neutrophils showing DNA fragmentation was performed on neutrophil culture using terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling test. In addition to enzyme-linked immunosorbent assay for soluble Fas (APO-1/CD95) in culture supernatant. The results revealed a rise in the neutrophil apoptotic and necrotic markers with progression of the disease, and they were inversely correlated with the absolute neutrophil count. The apoptotic neutrophil cells showed a significant positive correlation with several haemostatic parameters (tissue factor, prothrombin fragment 1+2, thrombus precursor protein and D-dimer). Regression analysis proved that apoptotic parameters are independent determinants of prothrombotic markers, which further incriminate the apoptotic mechanisms in the hypercoagulable state encountered in this clinical setting.

  19. Statin Treatment Is Associated with Reduction in Serum Levels of Receptor Activator of NF-κB Ligand and Neutrophil Activation in Patients with Severe Carotid Stenosis

    Directory of Open Access Journals (Sweden)

    Sébastien Lenglet

    2014-01-01

    Full Text Available Systemic and intraplaque biomarkers have been widely investigated in clinical cohorts as promising surrogate parameters of cardiovascular vulnerability. In this pilot study, we investigated if systemic and intraplaque levels of calcification biomarkers were affected by treatment with a statin in a cohort of patients with severe carotid stenosis and being asymptomatic for ischemic stroke. Patients on statin therapy had reduced serum osteopontin (OPN, RANKL/osteoprotegerin (OPG ratio, and MMP-9/pro-MMP-9 activity as compared to untreated patients. Statin-treated patients exhibited increased levels of collagen and reduced neutrophil infiltration in downstream portions of carotid plaques as compared to untreated controls. In upstream plaque portions, OPG content was increased in statin-treated patients as compared to controls. Other histological parameters (such as lipid, macrophage, smooth muscle cell, and MMP-9 content as well as RANKL, RANK, and OPG mRNA levels did not differ between the two patient groups. Serum RANKL/OPG ratio positively correlated with serum levels of neutrophilic products, intraplaque neutrophil, and MMP-9 content within downstream portions of carotid plaques. In conclusion, statin treatment was associated with improvement in serum RANKL levels and reduced neutrophil activity both systemically and in atherosclerotic plaques.

  20. Rabbit neutrophil chemotactic protein (NCP) activates both CXCR1 and CXCR2 and is the functional homologue for human CXCL6.

    Science.gov (United States)

    Catusse, Julie; Struyf, Sofie; Wuyts, Anja; Weyler, Myke; Loos, Tamara; Gijsbers, Klara; Gouwy, Mieke; Proost, Paul; Van Damme, Jo

    2004-11-15

    Neutrophil chemotactic protein (NCP) is a rabbit CXC chemokine with activating and chemotactic properties on neutrophilic granulocytes. Although its selective activity on neutrophils is demonstrated, its interactions with specific chemokine receptors are not defined. For further functional characterization, NCP was chemically synthesized and was found to be equipotent as natural NCP in neutrophil chemotaxis. To identify its human homologue, we separately expressed two potential rabbit NCP receptors (CXCR1 and CXCR2) in Jurkat cells. Pure synthetic NCP was equally efficient to promote chemotaxis through either rabbit CXCR1 or CXCR2. Moreover, chemotaxis assays on rabbit CXCR1 and CXCR2 transfectants showed that NCP uses the same receptors as interleukin-8 (IL-8), a major rabbit CXC chemokine, but not rabbit GROalpha, which only recognized CXCR2. In addition, specific inhibitors for CXCR1 or CXCR2 reduced rabbit neutrophil chemotaxis induced by NCP and rabbit IL-8. Furthermore, NCP and the structurally related human CXCR1/CXCR2 agonist CXCL6/GCP-2 (granulocyte chemotactic protein-2) cross-desensitized each other in intracellular calcium release assays on human neutrophils, further indicating that both chemokines share the same receptors. The inflammatory role of NCP was also evidenced by its potent granulocytosis inducing capacity in rabbits upon systemic administration. This study provides in vitro and in vivo evidences that NCP is the functional rabbit homologue for human CXCL6/GCP-2 rather than the most related CXCR2 agonist CXCL5/ENA-78 (epithelial cell-derived neutrophil activating peptide-78). It is concluded that the rabbit is a better model to study human neutrophil activation compared to mice, which lack CXCL8/IL-8.

  1. Resuscitation of traumatic hemorrhagic shock patients with hypertonic saline-without dextran-inhibits neutrophil and endothelial cell activation.

    Science.gov (United States)

    Junger, Wolfgang G; Rhind, Shawn G; Rizoli, Sandro B; Cuschieri, Joseph; Shiu, Maria Y; Baker, Andrew J; Li, Linglin; Shek, Pang N; Hoyt, David B; Bulger, Eileen M

    2012-10-01

    Posttraumatic inflammation and excessive neutrophil activation cause multiple organ dysfunction syndrome (MODS), a major cause of death among hemorrhagic shock patients. Traditional resuscitation strategies may exacerbate inflammation; thus, novel fluid treatments are needed to reduce such posttraumatic complications. Hypertonic resuscitation fluids inhibit inflammation and reduce MODS in animal models. Here we studied the anti-inflammatory efficacy of hypertonic fluids in a controlled clinical trial. Trauma patients in hypovolemic shock were resuscitated in a prehospital setting with 250 mL of either 7.5% hypertonic saline (HS; n = 9), 7.5% hypertonic saline + 6% dextran 70 (HSD; n = 8), or 0.9% normal saline (NS; n = 17). Blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Multicolor flow cytometry was used to quantify neutrophil expression of cell-surface activation/adhesion (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers as well as oxidative burst activity. Circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVACM-1), P- and E-selectins, myeloperoxidase (MPO), and matrix metalloproteinase 9 (MMP-9) were assessed by immunoassay. Multiple organ dysfunction syndrome, leukocytosis, and mortality were lower in the HS and HSD groups than in the NS group. However, these differences were not statistically significant. Hypertonic saline prevented priming and activation and neutrophil oxidative burst and CD11b and CD66b expression. Hypertonic saline also reduced circulating markers of neutrophil degranulation (MPO and MMP-9) and endothelial cell activation (sICAM-1, sVCAM-1, soluble E-selectin, and soluble P-selectin). Hypertonic saline + 6% dextran 70 was less capable than HS of suppressing the upregulation of most of these activation markers. This study demonstrates that initial resuscitation with HS, but neither NS nor HSD, can attenuate

  2. Differential activation of RAGE by HMGB1 modulates neutrophil-associated NADPH oxidase activity and bacterial killing.

    Science.gov (United States)

    Tadié, Jean-Marc; Bae, Hong-Beom; Banerjee, Sami; Zmijewski, Jaroslaw W; Abraham, Edward

    2012-01-01

    The receptor for advanced glycation end products (RAGE) plays an important role in host defense against bacterial infection. In the present experiments, we investigated the mechanisms by which RAGE contributes to the ability of neutrophils to eradicate bacteria. Wild-type (RAGE(+/+)) neutrophils demonstrated significantly greater ability to kill Escherichia coli compared with RAGE(-/-) neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE(-/-) as compared with RAGE(+/+) mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and enhanced killing of E. coli, and intraperitoneal injection of AGE enhanced bacterial clearance during peritonitis. However, incubation of neutrophils with high mobility group box 1 protein (HMGB1), which also binds to RAGE, diminished E. coli-induced activation of NADPH oxidase in neutrophils and bacterial killing both in vitro and in vivo. Deletion of the COOH-terminal tail of HMGB1, a region necessary for binding to RAGE, abrogated the ability of HMGB1 to inhibit bacterial killing. Incubation of neutrophils with HMGB1 diminished bacterial or AGE-dependent activation of NADPH oxidase. The increase in phosphorylation of the p40(phox) subunit of NADPH oxidase that occurred after culture of neutrophils with E. coli was inhibited by exposure of the cells to HMGB1. These results showing that HMGB1, through RAGE-dependent mechanisms, diminishes bacterial killing by neutrophils as well as NADPH oxidase activation provide a novel mechanism by which HMGB1 can potentiate sepsis-associated organ dysfunction and mortality.

  3. L-Carnitine Supplementation Reduces Short-Term Neutrophil-Lymphocyte Ratio in Patients Undergoing Coronary Artery Bypass Grafting.

    Science.gov (United States)

    Aldemir, Mustafa; Pektaş, Mehmet Bilgehan; Parlar, Ali İhsan; Akcı, Önder; Emren, Sadık Volkan; Tecer, Evren; Adalı, Fahri; Yüksel, Şeref; Darçın, Osman Tansel

    2015-07-01

    This study aims to investigate whether preoperative L-carnitine supplementation affects the neutrophil-to-lymphocyte ratio (NLR) in patients undergoing coronary artery bypass grafting surgery. The neutrophil-to-lymphocyte ratio is an inflammatory marker that has proven usefulness for predicting postoperative complications in coronary artery bypass surgery. A lot of studies concerning the role of L-carnitine in the immune system have been performed, contradictory results have been reported on its effects on absolute numbers of WBC subtypes. This randomized, double-blinded, placebo-controlled study was conducted among patients scheduled for coronary artery bypass grafting surgery between June 2012 and December 2013 in our cardiovascular surgery clinic. A total of 60 consecutive patients were randomized and divided into 2 groups. The first group received 2 g of L-carnitine in 1000 mL of 0.9% saline solution infused over 24 hours for each of the 3 preoperative days (L-carnitine group, n = 30), or only 1000 mL of 0.9% saline solution for the same time period (placebo group, n = 30). The basal values of leukocyte, neutrophil, lymphocyte counts, and neutrophil to lymphocyte ratio were similar in the 2 groups. After L-carnitine supplementation (just before surgery), leukocyte and neutrophil counts of the L-carnitine group were significantly lower than those of the placebo group (7.7 ± 1.5 versus 9.7 ± 2.6, P carnitine group (1.1 ± 0.6 versus 0.8 ± 0.9, P carnitine group at postoperative day 1 (20.7 ± 13.8 versus 10.8 ± 4.1, P carnitine supplementation may reduce neutrophil-lymphocyte ratio during the early postoperative period of coronary artery bypass grafting surgery.

  4. RNA-seq reveals activation of both common and cytokine-specific pathways following neutrophil priming.

    Directory of Open Access Journals (Sweden)

    Helen L Wright

    Full Text Available Neutrophils are central to the pathology of inflammatory diseases, where they can damage host tissue through release of reactive oxygen metabolites and proteases, and drive inflammation via secretion of cytokines and chemokines. Many cytokines, such as those generated during inflammation, can induce a similar "primed" phenotype in neutrophils, but it is unknown if different cytokines utilise common or cytokine-specific pathways to induce these functional changes. Here, we describe the transcriptomic changes induced in control human neutrophils during priming in vitro with pro-inflammatory cytokines (TNF-α and GM-CSF using RNA-seq. Priming led to the rapid expression of a common set of transcripts for cytokines, chemokines and cell surface receptors (CXCL1, CXCL2, IL1A, IL1B, IL1RA, ICAM1. However, 580 genes were differentially regulated by TNF-α and GM-CSF treatment, and of these 58 were directly implicated in the control of apoptosis. While these two cytokines both delayed apoptosis, they induced changes in expression of different pro- and anti-apoptotic genes. Bioinformatics analysis predicted that these genes were regulated via differential activation of transcription factors by TNF-α and GM-CSF and these predictions were confirmed using functional assays: inhibition of NF-κB signalling abrogated the protective effect of TNF-α (but not that of GM-CSF on neutrophil apoptosis, whereas inhibition of JAK/STAT signalling abrogated the anti-apoptotic effect of GM-CSF, but not that of TNF-α (p<0.05. These data provide the first characterisation of the human neutrophil transcriptome following GM-CSF and TNF-α priming, and demonstrate the utility of this approach to define functional changes in neutrophils following cytokine exposure. This may provide an important, new approach to define the molecular properties of neutrophils after in vivo activation during inflammation.

  5. Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils.

    Science.gov (United States)

    McGovern, Naomi N; Cowburn, Andrew S; Porter, Linsey; Walmsley, Sarah R; Summers, Charlotte; Thompson, Alfred A R; Anwar, Sadia; Willcocks, Lisa C; Whyte, Moira K B; Condliffe, Alison M; Chilvers, Edwin R

    2011-01-01

    Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α-dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.

  6. Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro.

    Science.gov (United States)

    Kampen, Annette H; Tollersrud, Tore; Lund, Arve

    2005-03-01

    Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.

  7. Evaluation of endotoxin (LPS) activity in bovine blood using neutrophil dependent chemiluminescence

    Science.gov (United States)

    The purpose of this study was to evaluate the applicability of a neutrophil chemiluminescence-based assay for the measurement of LPS stimulatory activity in bovine whole blood. The assay is based on the capacity for LPS to trigger the respiratory oxidative burst activity (RBA) of autologous neutroph...

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

    Science.gov (United States)

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

    2014-04-17

    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 lung injury (ALI), which is associated with a high mortality rate in critically ill patients. NETs consist of decondensed chromatin decorated with granular and cytosolic proteins and they can trap extracellular pathogens. The prerequisite for NET formation is the activation of neutrophils and the release of their DNA. In a neutrophil- and platelet-dependent mouse model of ventilator-induced lung injury (VILI), NETs were found in the lung microvasculature, and circulating NET components increased in the plasma. In this model, blocking integrin-mediated outside-in or either GPCR-signaling or heteromerization of platelet chemokines decreased NET formation and lung injury. Targeting NET components by DNAse1 application or neutrophil elastase-deficient mice protected mice from ALI, whereas DNase1(-/-)/Trap1(m/m) mice had an aggravated ALI, suggesting that NETs directly influence the severity of ALI. These data suggest that NETs form in the lungs during VILI, contribute to the disease process, and thus may be a promising new direction for the treatment of ALI.

  9. Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease.

    Science.gov (United States)

    Leffler, Jonatan; Martin, Myriam; Gullstrand, Birgitta; Tydén, Helena; Lood, Christian; Truedsson, Lennart; Bengtsson, Anders A; Blom, Anna M

    2012-04-01

    Ongoing inflammation including activation of the complement system is a hallmark of systemic lupus erythematosus (SLE). Antimicrobial neutrophil extracellular traps (NETs) are composed of secreted chromatin that may act as a source of autoantigens typical for SLE. In this study, we investigated how complement interacts with NETs and how NET degradation is affected by complement in SLE patients. We found that sera from a subset of patients with active SLE had a reduced ability to degrade in vitro-generated NETs, which was mostly restored when these patients were in remission. Patients that failed to degrade NETs had a more active disease and they also displayed lower levels of complement proteins C4 and C3 in blood. We discovered that NETs activated complement in vitro and that deposited C1q inhibited NET degradation including a direct inhibition of DNase-I by C1q. Complement deposition on NETs may facilitate autoantibody production, and indeed, Abs against NETs and NET epitopes were more pronounced in patients with impaired ability to degrade NETs. NET-bound autoantibodies inhibited degradation but also further increased C1q deposition, potentially exacerbating the disease. Thus, NETs are a potent complement activator, and this interaction may play an important role in SLE. Targeting complement with inhibitors or by removing complement activators such as NETs could be beneficial for patients with SLE.

  10. Blockade of p-selectin reduces neutrophil infiltration into the murine testis after ischemia-reperfusion-injury.

    Science.gov (United States)

    Celebi, M; Paul, A G A

    2008-12-01

    Germ cell specific apoptosis after ischemia-reperfusion (I/R) induced testicular injury is dependent on neutrophil recruitment to the testis. Intravascular adhesion molecules like the P- and E- selectins play an important role in this recruitment.The purpose of this study was to inhibit neutrophil recruitment in I/R induced testicular injury by using a function-blocking monoclonal anti-mouse P-selectin antibody. Adult mice were subjected to a 2 h period of testicular torsion (ischemia) followed by detorsion (reperfusion).Ten minutes after the onset of reperfusion, mice received either 100 microg of a function-blocking monoclonal P-selectin antibody (FBMAB group) or isotype-matched control antibody (IMCA group). Separate groups of mice underwent sham-operation (SO group) or received 500 ng of TNFalpha (IF group) to induce inflammation. Mice were sacrificed 24 h after reperfusion and testiscular interstitial cells were isolated and analyzed for the presence of neutrophils by means of flow cytometry. The function-blocking monoclonal P-selectin antibody reduced neutrophil recruitment in I/R induced testicular injury significantly (FBMAB group as compared to the IMCA group 26 +/- 4 vs. 52 +/- 10% Gr-1 +CD11 b+ of total leucocytes; P < 0.001). Therefore, blocking P-selectin may be therapeutically beneficial to protect postischemic testis.

  11. Similar activation state of neutrophils in sputum of asthma patients irrespective of sputum eosinophilia.

    Science.gov (United States)

    Tak, T; Hilvering, B; Tesselaar, K; Koenderman, L

    2015-11-01

    Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13, eosinophilia were characterized by increased asthma control questionnaire (ACQ) scores and blood eosinophil counts. Both sputum neutrophils and eosinophils displayed an activated and degranulated phenotype compared to cells obtained from blood. Specifically, degranulation of all granule types was detected in sputum cells, combined with an increased expression of the activation markers (activated) Mac-1 (CD11b), programmed death ligand 1 (PD-L1) (CD274) and a decreased expression of CD62L. CD69 expression was only increased on sputum eosinophils. Surface marker expression of neutrophils was similar in the presence or absence of eosinophilia, either by single or multi-dimensional analysis. Sputum neutrophils were highly activated and degranulated irrespective of sputum eosinophilia. Therefore, we conclude that differences in granulocyte activation in sputum and/or blood are not associated with clinical differences in the two groups of asthma patients. The finding of PD-L1 expression on sputum granulocytes suggests an immunomodulatory role of these cells in the tissue. © 2015 British Society for Immunology.

  12. Biologic therapy improves psoriasis by decreasing the activity of monocytes and neutrophils.

    Science.gov (United States)

    Yamanaka, Keiichi; Umezawa, Yoshinori; Yamagiwa, Akisa; Saeki, Hidehisa; Kondo, Makoto; Gabazza, Esteban C; Nakagawa, Hidemi; Mizutani, Hitoshi

    2014-08-01

    Therapy with monoclonal antibodies to tumor necrosis factor (TNF)-α and the interleukin (IL)-12/23 p40 subunit has significantly improved the clinical outcome of patients with psoriasis. These antibodies inhibit the effects of the target cytokines and thus the major concern during their use is the induction of excessive immunosuppression. Recent studies evaluating the long-term efficacy and safety of biologic therapy in psoriasis have shown no significant appearance of serious adverse effects including infections and malignancies. However, the immunological consequence and the mechanism by which the blockade of a single cytokine by biologics can successfully control the activity of psoriasis remain unclear. In the current study, we investigated the effect of biologic therapy on cytokine production of various lymphocytes and on the activity of monocytes and neutrophils in psoriatic patients. Neutrophils, monocytes and T cells were purified from heparinized peripheral venous blood by Ficoll density gradient centrifugation, and γ-interferon, TNF-α and IL-17 production from lymphocytes was measured by flow cytometer. The activation maker of neutrophils and the activated subsets of monocytes were also analyzed. Biologic therapy induced no significant changes in the cytokine production by lymphocytes from the skin and gut-homing T cells. However, neutrophil activity and the ratio of activated monocyte population increased in severely psoriatic patients were normalized in psoriatic patients receiving biologic therapy. The present study showed that biologic therapy ameliorates clinical symptoms and controls the immune response in patients with psoriasis.

  13. Misregulation of the broad-range phospholipase C activity increases the susceptibility of Listeria monocytogenes to intracellular killing by neutrophils.

    Science.gov (United States)

    Blank, Bryant S; Abi Abdallah, Delbert S; Park, Justin J; Nazarova, Evgeniya V; Pavinski Bitar, Alan; Maurer, Kirk J; Marquis, Hélène

    2014-02-01

    Listeria monocytogenes is a facultative intracellular bacterial pathogen that tightly regulates the activities of various virulence factors during infection. A mutant strain (the plcBDpro mutant) that has lost the ability to control the activity of a phospholipase C (PC-PLC) is attenuated a hundred fold in mice. This attenuation is not due to a lack of bacterial fitness, but appears to result from a modified host response to infection. The transcriptomic pattern of immune-related genes indicated that PC-PLC did not enhance the innate immune response in infected macrophages. However, it partially protected the cells from bacteria-mediated mitochondrial fragmentation. In mice, the plcBDpro mutant transiently caused an increase in liver pathology, as judged by the size of neutrophil-filled micro-abscesses. Moreover, the plcBDpro mutant was more susceptible to intracellular killing by neutrophils than wild-type L. monocytogenes. Together, these data indicate that in vivo attenuation of the plcBDpro mutant results from its reduced ability to disrupt mitochondrial homeostasis and to resist intracellular killing by neutrophils.

  14. Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

    Science.gov (United States)

    Barrientos, Lorena; Bignon, Alexandre; Gueguen, Claire; de Chaisemartin, Luc; Gorges, Roseline; Sandré, Catherine; Mascarell, Laurent; Balabanian, Karl; Kerdine-Römer, Saadia; Pallardy, Marc; Marin-Esteban, Viviana; Chollet-Martin, Sylvie

    2014-12-01

    Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell-cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4(+) T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

  15. Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis.

    Science.gov (United States)

    Jarvis, James N; Petty, Howard R; Tang, Yuhong; Frank, Mark Barton; Tessier, Philippe A; Dozmorov, Igor; Jiang, Kaiyu; Kindzelski, Andrei; Chen, Yanmin; Cadwell, Craig; Turner, Mary; Szodoray, Peter; McGhee, Julie L; Centola, Michael

    2006-01-01

    Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8- and interferon-gamma (IFN-gamma)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-gamma and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-gamma) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and

  16. Effect of cardiopulmonary bypass on leukocyte activation : changes in membrane-bound elastase on neutrophils

    NARCIS (Netherlands)

    Tang, M; Gu, YJ; Wang, WJ; Xu, YP; Chen, CZ

    2004-01-01

    Background: Neutrophil elastase is known to be released from the activated leukocytes as a result of cardiopulmonary bypass (CPB). However, its biological effect on organ injury is questionable because it is quickly bound by natural proteinase inhibitors (PIs). Recently, membrane-bound elastase ( MB

  17. Two Polymorphisms in the Epithelial Cell-Derived Neutrophil-Activating Peptide (ENA-78 Gene

    Directory of Open Access Journals (Sweden)

    Mahsa M. Amoli

    2005-01-01

    Full Text Available Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78 has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing.

  18. Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1.

    Science.gov (United States)

    Yang, Shun-Chin; Chung, Pei-Jen; Ho, Chiu-Ming; Kuo, Chan-Yen; Hung, Min-Fa; Huang, Yin-Ting; Chang, Wen-Yi; Chang, Ya-Wen; Chan, Kwok-Hon; Hwang, Tsong-Long

    2013-06-15

    Neutrophils play a critical role in acute and chronic inflammatory processes, including myocardial ischemia/reperfusion injury, sepsis, and adult respiratory distress syndrome. Binding of formyl peptide receptor 1 (FPR1) by N-formyl peptides can activate neutrophils and may represent a new therapeutic target in either sterile or septic inflammation. Propofol, a widely used i.v. anesthetic, has been shown to modulate immunoinflammatory responses. However, the mechanism of propofol remains to be established. In this study, we showed that propofol significantly reduced superoxide generation, elastase release, and chemotaxis in human neutrophils activated by fMLF. Propofol did not alter superoxide generation or elastase release in a cell-free system. Neither inhibitors of γ-aminobutyric acid receptors nor an inhibitor of protein kinase A reversed the inhibitory effects of propofol. In addition, propofol showed less inhibitory effects in non-FPR1-induced cell responses. The signaling pathways downstream from FPR1, involving calcium, AKT, and ERK1/2, were also competitively inhibited by propofol. These results show that propofol selectively and competitively inhibits the FPR1-induced human neutrophil activation. Consistent with the hypothesis, propofol inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analog of fMLF, to FPR1 in human neutrophils, differentiated THP-1 cells, and FPR1-transfected human embryonic kidney-293 cells. To our knowledge, our results identify, for the first time, a novel anti-inflammatory mechanism of propofol by competitively blocking FPR1 in human neutrophils. Considering the importance of N-formyl peptides in inflammatory processes, our data indicate that propofol may have therapeutic potential to attenuate neutrophil-mediated inflammatory diseases by blocking FPR1.

  19. Oxidant-scavenging activities of ampicillin and sulbactam and their effects on neutrophil functions.

    Science.gov (United States)

    Gunther, M R; Mao, J; Cohen, M S

    1993-05-01

    Luminol-enhanced luminescence is a method used to measure formation of reactive oxygen intermediates important in the ability of neutrophils to kill microbes. Several studies have demonstrated that under some conditions of incubation, ampicillin can inhibit neutrophil-derived luminol-enhanced luminescence. We evaluated the mechanism(s) by which ampicillin inhibited the luminescent response of stimulated neutrophils. We also investigated sulbactam, a beta-lactamase inhibitor which has been given in combination with ampicillin and other beta-lactam antibiotics to increase their spectra, for possible similar effects. Both ampicillin and sulbactam attenuated luminol-enhanced luminescence by approximately 40%. Superoxide production was not prevented by added ampicillin, nor was superoxide scavenged by it. Myeloperoxidase reacts with H2O2 and Cl- to generate OCl-, which is believed to be the oxidizer of luminol that is primarily responsible for enhancement of neutrophil-derived luminescence. Hydroxyl radicals (HO.), which may also oxidize luminol, resulting in luminescence, can be formed from O2- and H2O2 via either myeloperoxidase-dependent (involving intermediate OCl-) or myeloperoxidase-independent (through a metal ion catalyst) reactions. Ampicillin scavenged H2O2 and OCl- and prevented 95% of Fenton reaction-generated HO. from reacting with 5,5-dimethyl-1-pyrroline-N-oxide. Sulbactam was found to scavenge OCl- and HO., but less avidly than ampicillin did. Neither ampicillin nor sulbactam inhibited myeloperoxidase activity. Sublethal concentrations of sulbactam had no significant effect on neutrophil killing of Staphylococcus aureus and Escherichia coli. Our results demonstrate a mechanism(s) by which ampicillin inhibits luminol-enhanced luminescence from stimulated neutrophils, namely, through scavenging of the oxidant(s) primarily responsible for the generation of luminescence.

  20. 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.

  1. Glutamine supplementation prevents exercise-induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression.

    Science.gov (United States)

    Lagranha, Claudia J; Hirabara, Sandro M; Curi, Rui; Pithon-Curi, Tania C

    2007-01-01

    We have previously shown that a single session of exercise induces DNA fragmentation, mitochondrial membrane depolarization, increases expression of pro-apoptotic genes (bax and bcl-xS) and decreases expression of anti-apoptotic genes (bcl-xL) in rat neutrophils. Glutamine supplementation had a protective effect in the apoptosis induced by a single session of exercise. The mechanism involved in the effect of single session of exercise to induce apoptosis was investigated by measuring expression of p53 and caspase 3 and phosphorylation of p38 mitogen-activated protein kinases (MAPK) and cJun NH(2)-terminal kinase (JNK) in neutrophils from rats supplemented or not with glutamine. Exercise was carried out on a treadmill for 1 h and the rats were killed by decapitation. Neutrophils were obtained by intraperitoneal (i.p.) lavage with PBS, 4 h after injection of oyster glycogen solution. Glutamine supplementation (1g per Kg b.w.) was given by gavage 1 h before the exercise session. Gene expression and protein phosphorylation were then analyzed by reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. A single session of exercise increased p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression. Glutamine supplementation partially prevented the increase in p38 MAPK and JNK phosphorylation and p53 expression, and fully abolished the increase in caspase 3 expression. Thus, neutrophil apoptosis induced by a single session of exercise is accompanied by increased p53 and caspase 3 expression and p38 MAPK and JNK phosphorylation. Glutamine supplementation prevents these effects of exercise and reduces apoptosis.

  2. An In Vitro Model for Studying Neutrophil Activation During Cardiopulmonary Bypass by Using a Polymerase Chain Reaction Thermocycler

    NARCIS (Netherlands)

    Tang, Min; Zhao, Xiao-Gang; Gu, Y. John; Chen, Chang-Zhi

    2010-01-01

    The accurate temperature control of a polymerase chain reaction (PCR) thermocycler was exploited in developing an in vitro model to study neutrophil activation during cardiopulmonary bypass. Neutrophils from 12 volunteers underwent temperature changes in a PCR thermocycler (37 degrees C for 30 minut

  3. An In Vitro Model for Studying Neutrophil Activation During Cardiopulmonary Bypass by Using a Polymerase Chain Reaction Thermocycler

    NARCIS (Netherlands)

    Tang, Min; Zhao, Xiao-Gang; Gu, Y. John; Chen, Chang-Zhi

    The accurate temperature control of a polymerase chain reaction (PCR) thermocycler was exploited in developing an in vitro model to study neutrophil activation during cardiopulmonary bypass. Neutrophils from 12 volunteers underwent temperature changes in a PCR thermocycler (37 degrees C for 30

  4. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  5. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  6. Periodontal Ligament Stem Cells Regulate Apoptosis of Neutrophils

    Science.gov (United States)

    Wang, Qing; Ding, Gang; Xu, Xin

    2017-01-01

    Abstract Periodontal ligament stem cells (PDLSCs) are promising cell resource for the cell-based therapy for periodontitis and regeneration of bio-root. In this study, we investigated the effect of PDLSCs on neutrophil, a critical constituent of innate immunity, and the underlying mechanisms. The effect of PDLSCs on the proliferation and apoptosis of resting neutrophils and IL-8 activated neutrophils was tested under cell-cell contact culture and Transwell culture, with or without anti-IL-6 neutralizing antibody. We found that PDLSCs could promote the proliferation and reduce the apoptosis of neutrophils whether under cell-cell contact or Transwell culture. Anti-IL-6 antibody reduced PDLSCs-mediated inhibition of neutrophil apoptosis. IL-6 at the concentration of 10ng/ml and 20ng/ml could inhibit neutrophil apoptosis statistically. Collectively, PDLSCs could reduce the apoptosis of neutrophils via IL-6.

  7. Human neutrophil migration and activation by BJcuL, a galactose binding lectin purified from Bothrops jararacussu venom

    Directory of Open Access Journals (Sweden)

    Fernandes Luiz

    2011-01-01

    Full Text Available Abstract Background Neutrophil migration to an inflamed site constitutes the first line of the innate immune response against invading microorganisms. Given the crucial role of endogenous lectins in neutrophil mobilization and activation, lectins from exogenous sources have often been considered as putative modulators of leukocyte function. Lectins purified from snake venom have been described as galactoside ligands that induce erythrocyte agglutination and platelet aggregation. This study evaluated human neutrophil migration and activation by C-type lectin BJcuL purified from Bothrops jararacussu venom. Results Utilizing fluorescence microscopy, we observed that biotinylated-BJcuL was evenly distributed on the neutrophil surface, selectively inhibited by D-galactose. Lectin was able to induce modification in the neutrophil morphology in a spherical shape for a polarized observed by optical microscopy and exposure to BJcuL in a Boyden chamber assay resulted in cell migration. After 30 minutes of incubation with BJcuL we found enhanced neutrophil functions, such as respiratory burst, zymozan phagocytosis and an increase in lissosomal volume. In addition, BJcuL delays late apoptosis neutrophils. Conclusion These results demonstrate that BJcuL can be implicated in a wide variety of immunological functions including first-line defense against pathogens, cell trafficking and induction of the innate immune response since lectin was capable of inducing potent neutrophil activation.

  8. The Association Between Neutrophil/Lymphocyte Ratio and Disease Activity in Rheumatoid Arthritis and Ankylosing Spondylitis.

    Science.gov (United States)

    Mercan, Ridvan; Bitik, Berivan; Tufan, Abdurrahman; Bozbulut, Utku Burak; Atas, Nuh; Ozturk, Mehmet Akif; Haznedaroglu, Seminur; Goker, Berna

    2016-09-01

    Elevated neutrophil count is associated with poor prognosis and increased mortality in many conditions. Neutrophil to lymphocyte ratio (NLR) has emerged as a marker of inflammation in neoplastic and cardiovascular disorders. Herein, we investigated utility of this simple tool in rheumatoid arthritis (RA) and ankylosing spondylitis (AS). The study consisted of 136 RA and 140 AS patients, along with 117 healthy control subjects. RA and AS activities were determined with Disease Activity Score (DAS) and Bath Ankylosing Spondylitis Disease Activity indices (BASDAI), respectively. The association between NLR and disease activity was analyzed. Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and neutrophil counts were significantly higher in RA and AS patients compared to healthy controls. Similarly, NLR was higher compared to control subjects, both in RA (2.53 ± 1.4 vs. 2.16 ± 1.0, P = 0.019) and AS (2.43 ± 1.4 vs. 2.16 ± 1.0, P = 0.077). NLR correlated well with ESR and CRP, both in RA and AS. Moreover, NLR increased across worsening DAS28 activity groups (2.1 ± 1.0 in patients with remission, 2.5 ± 1.0 in low-moderate, 3.8 ± 2.5 in high disease activity). However, no association was found between NLR and BASDAI. NLR is a cheap and readily available marker for the assessment of disease activity in RA. © 2015 Wiley Periodicals, Inc.

  9. Relationship of phosphatidylinositol bisphosphate hydrolysis to calcium mobilization and functional activation in fluoride-treated neutrophils.

    OpenAIRE

    English, D.; Debono, D J; Gabig, T G

    1987-01-01

    Sodium fluoride (20 mM) effected rapid hydrolysis of phosphatidylinositol bisphosphate (PIP2) in human neutrophils. Intracellular free Ca2+ levels increased after PIP2 hydrolysis but before respiratory burst activation. Both the increase in intracellular free Ca2+ levels and the extent of functional activation were dependent on the availability of extracellular Ca2+. The rate of F(-)-stimulated PIP2 hydrolysis, however, was not affected when the rise in cytosolic Ca2+ was severely limited by ...

  10. Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

    NARCIS (Netherlands)

    Lian, J.P. (Jian); Crossley, L. (Lisa); Zhan, Q. (Qian); Huang, R. (Riyun); Coffer, P.J.; Toker, A. (Alex); Robinson, D. (Dwight); Badwey, J.A. (John)

    2002-01-01

    Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of ~63 and 69 kDa (y- and a-Pak). Previous studies have shown that

  11. Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid.

    Science.gov (United States)

    Hinz, Christine; Aldrovandi, Maceler; Uhlson, Charis; Marnett, Lawrence J; Longhurst, Hilary J; Warner, Timothy D; Alam, Saydul; Slatter, David A; Lauder, Sarah N; Allen-Redpath, Keith; Collins, Peter W; Murphy, Robert C; Thomas, Christopher P; O'Donnell, Valerie B

    2016-06-24

    Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.

  12. Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid*

    Science.gov (United States)

    Hinz, Christine; Aldrovandi, Maceler; Uhlson, Charis; Marnett, Lawrence J.; Longhurst, Hilary J.; Warner, Timothy D.; Alam, Saydul; Slatter, David A.; Lauder, Sarah N.; Allen-Redpath, Keith; Collins, Peter W.; Murphy, Robert C.; Thomas, Christopher P.; O'Donnell, Valerie B.

    2016-01-01

    Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation. PMID:27129261

  13. Neutrophil Activation in Severe, Early-Onset COPD Patients versus Healthy Non-Smoker Subjects in vitro: Effects of Antioxidant Therapy

    National Research Council Canada - National Science Library

    Milara, Javier; Juan, Gustavo; Peiró, Teresa; Serrano, Adela; Cortijo, Julio

    2012-01-01

    ...) on neutrophil activation in vitro. Methods: Neutrophils were isolated from 15 severe, early-onset COPD patients and 15 age-matched healthy subjects and stimulated with N-formyl- Met-Leu-Phe (fMLP...

  14. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...... that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca2+ but, despite subsequent spikes in Ca2+ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes...

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

    Science.gov (United States)

    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

  16. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    LENUS (Irish Health Repository)

    Wakai, A

    2012-02-03

    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

  17. Human platelets utilize cycloxygenase-1 to generate dioxolane A3, a neutrophil activating eicosanoid

    OpenAIRE

    Hinz, Christine; Aldrovandi, MacEler; Alam, Saydul; Slatter, David; Lauder, Sarah Nicol; Allen-Redpath, Keith; Collins, Peter William; Thomas, Christopher P.; O'Donnell, Valerie Bridget

    2016-01-01

    Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lip...

  18. Neutrophil proteolytic activation cascades: a possible mechanistic link between chronic periodontitis and coronary heart disease.

    Science.gov (United States)

    Alfakry, Hatem; Malle, Ernst; Koyani, Chintan N; Pussinen, Pirkko J; Sorsa, Timo

    2016-01-01

    Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activate proteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD.

  19. (--Epigallocatechin-3-gallate Reduces Cigarette Smoke-Induced Airway Neutrophilic Inflammation and Mucin Hypersecretion in Rats

    Directory of Open Access Journals (Sweden)

    Yingmin Liang

    2017-09-01

    Full Text Available Background: Cigarette smoking is the leading cause of chronic obstructive pulmonary disease. (--Epigallocatechin-3-gallate (EGCG, the major catechins in Chinese green tea, has been studied for its anti-oxidative and anti-inflammatory properties in cell and animal models. In this study, we aimed to analyze the effects of EGCG on cigarette smoke (CS-induced airway inflammation and mucus secretion in the CS-exposed rat model.Methods: Male Sprague-Dawley rats were randomly divided into either sham air (SA or CS exposure. EGCG (50 mg/kg b.wt. was given by oral gavage every other day in both SA and CS-exposed animals. Oxidative stress and inflammatory markers were determined in serum and/or bronchoalveolar lavage fluid by biochemical assays or ELISA. Lung morphological changes were examined by Periodic Acid-Schiff, Masson’s Trichrome staining and immunohistochemical analysis. Western blot analysis was performed to explore the effects of EGCG on epidermal growth factor receptor (EGFR-mediated signaling pathway.Results: (--Epigallocatechin-3-gallate treatment attenuated CS-induced oxidative stress, lung cytokine-induced neutrophil chemoattractant-1 release and neutrophil recruitment. CS exposure caused an increase in the number of goblet cells in line with MUC5AC upregulation, and increased lung collagen deposition, which were alleviated in the presence of EGCG. In addition, CS-induced phosphorylation of EGFR in rat lung was abrogated by EGCG treatment.Conclusion: (--Epigallocatechin-3-gallate treatment ameliorated CS-induced oxidative stress and neutrophilic inflammation, as well as airway mucus production and collagen deposition in rats. The present findings suggest that EGCG has a therapeutic effect on chronic airway inflammation and abnormal airway mucus production probably via inhibition of EGFR signaling pathway.

  20. Helicobacter pylori neutrophil-activating protein: from molecular pathogenesis to clinical applications.

    Science.gov (United States)

    Fu, Hua-Wen

    2014-05-14

    Helicobacter pylori (H. pylori) neutrophil-activating protein (HP-NAP) was originally identified as a virulence factor of H. pylori for its ability to activate neutrophils to generate respiratory burst by releasing reactive oxygen species. Later on, HP-NAP was also found to be involved in the protection of H. pylori from DNA damage, supporting the survival of H. pylori under oxidative stress. This protein is highly conserved and expressed by virtually all clinical isolates of H. pylori. The majority of patients infected with H. pylori produced antibodies specific for HP-NAP, suggesting its important role in immunity. In addition to acting as a pathogenic factor by activating the innate immunity through a wide range of human leukocytes, including neutrophils, monocytes, and mast cells, HP-NAP also mediates adaptive immunity through the induction of T helper cell type I responses. The pro-inflammatory and immunomodulatory properties of HP-NAP not only make it play an important role in disease pathogenesis but also make it a potential candidate for clinical use. Even though there is no convincing evidence to link HP-NAP to a disease outcome, recent findings supporting the pathogenic role of HP-NAP will be reviewed. In addition, the potential clinical applications of HP-NAP in vaccine development, clinical diagnosis, and drug development will be discussed.

  1. Chronic Inflammation and Neutrophil Activation as Possible Causes of Joint Diseases in Ballet Dancers

    Science.gov (United States)

    Borges, Leandro da Silva; Santos, Vinicius Coneglian; de Moura, Nivaldo Ribeiro; Dermargos, Alexandre; Cury-Boaventura, Maria Fernanda; Gorjão, Renata; Pithon-Curi, Tania Cristina; Hatanaka, Elaine

    2014-01-01

    Herein, we investigated the effects of a ballet class on the kinetic profiles of creatine kinase (CK) and lactate dehydrogenase (LDH) activities, cytokines, complement component 3 (C3), and the concentrations of immunoglobulin (Ig), IgA and IgM, in ballerinas. We also verified neutrophil death and ROS release. Blood samples were taken from 13 dancers before, immediately after, and 18 hours after a ballet class. The ballet class increased the plasma activities of CK-total (2.0-fold) immediately after class, while the activities of CK-cardiac muscle (1.0-fold) and LDH (3.0-fold) were observed to increase 18 hours after the class. Levels of the TNF-α, IL-1β, IgG, and IgA were not affected under the study conditions. The exercise was found to induce neutrophil apoptosis (6.0-fold) 18 hours after the ballet class. Additionally, immediately after the ballet class, the neutrophils from the ballerinas were found to be less responsive to PMA stimulus. Conclusion. Ballet class was found to result in inflammation in dancers. The inflammation caused by the ballet class remained for 18 hours after the exercise. These findings are important in preventing the development of chronic lesions that are commonly observed in dancers, such as those with arthritis and synovitis. PMID:24701035

  2. Chronic Inflammation and Neutrophil Activation as Possible Causes of Joint Diseases in Ballet Dancers

    Directory of Open Access Journals (Sweden)

    Leandro da Silva Borges

    2014-01-01

    Full Text Available Herein, we investigated the effects of a ballet class on the kinetic profiles of creatine kinase (CK and lactate dehydrogenase (LDH activities, cytokines, complement component 3 (C3, and the concentrations of immunoglobulin (Ig, IgA and IgM, in ballerinas. We also verified neutrophil death and ROS release. Blood samples were taken from 13 dancers before, immediately after, and 18 hours after a ballet class. The ballet class increased the plasma activities of CK-total (2.0-fold immediately after class, while the activities of CK-cardiac muscle (1.0-fold and LDH (3.0-fold were observed to increase 18 hours after the class. Levels of the TNF-α, IL-1β, IgG, and IgA were not affected under the study conditions. The exercise was found to induce neutrophil apoptosis (6.0-fold 18 hours after the ballet class. Additionally, immediately after the ballet class, the neutrophils from the ballerinas were found to be less responsive to PMA stimulus. Conclusion. Ballet class was found to result in inflammation in dancers. The inflammation caused by the ballet class remained for 18 hours after the exercise. These findings are important in preventing the development of chronic lesions that are commonly observed in dancers, such as those with arthritis and synovitis.

  3. Relationship of phosphatidylinositol bisphosphate hydrolysis to calcium mobilization and functional activation in fluoride-treated neutrophils.

    Science.gov (United States)

    English, D; Debono, D J; Gabig, T G

    1987-07-01

    Sodium fluoride (20 mM) effected rapid hydrolysis of phosphatidylinositol bisphosphate (PIP2) in human neutrophils. Intracellular free Ca2+ levels increased after PIP2 hydrolysis but before respiratory burst activation. Both the increase in intracellular free Ca2+ levels and the extent of functional activation were dependent on the availability of extracellular Ca2+. The rate of F(-)-stimulated PIP2 hydrolysis, however, was not affected when the rise in cytosolic Ca2+ was severely limited by depletion of extracellular Ca2+. Fluoride caused the specific hydrolysis of PIP2 in isolated neutrophil plasma membranes. This effect occurred in the presence of low levels of available Ca2+ and was accompanied by the release of inositol phosphates. We conclude that PIP2 hydrolysis is an early event in the response of neutrophils to F-. This response is not Ca2+-regulated but may lead to an influx of Ca2+ from the extracellular medium. Activation of a PIP2-specific phospholipase independent of a change in cytosolic free Ca2+ levels may be the initial event in the stimulus-response pathway triggered by fluoride.

  4. Neutrophil activation by Candida glabrata but not Candida albicans promotes fungal uptake by monocytes.

    Science.gov (United States)

    Duggan, Seána; Essig, Fabian; Hünniger, Kerstin; Mokhtari, Zeinab; Bauer, Laura; Lehnert, Teresa; Brandes, Susanne; Häder, Antje; Jacobsen, Ilse D; Martin, Ronny; Figge, Marc Thilo; Kurzai, Oliver

    2015-09-01

    Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes.

  5. Indirect evidence for a role of a subpopulation of activated neutrophils in the remodelling process after percutaneous coronary intervention

    NARCIS (Netherlands)

    Costa, MA; de Wit, LEA; de Valk, V.; Serrano, P; Wardeh, AJ; Serruys, PW; Sluiter, W

    2001-01-01

    Aim Leukocytes have been implicated in restenosis following percutaneous transluminal coronary angioplasty. We investigated the link between the activated status of circulating neutrophils and restenosis after angioplasty. Methods and Results The population of 108 patients with single, de novo lesio

  6. CHEMILUMINESCENT ACTIVITY OF NEUTROPHILIC GRANULOCYTES IN PROGRESSION OF OBSTRUCTIVE JAUNDICE DEPENDING ON ITS ORIGIN AND BILIRUBIN LEVELS

    Directory of Open Access Journals (Sweden)

    O. V. Smirnova

    2016-01-01

    Full Text Available Mechanical jaundice (MJ is a severe pathological condition, caused by obstruction of the bile ducts, requiring immediate surgical intervention. Etiologically, MJ can be of benign (60-80% of the cases, or malignant origin. MJ progression depends on the underlying pathology, and, moreover, on bilirubin levels. Focal inflammation in affected area represents a significant mechanism of the MJ progression. Neutrophilic granulocytes, are primarily involved into the immune response, i.e., pathogen elimination. Hence, the MJ progression may depend on their functional activity. In this context, the aim of our study was to investigate chemiluminescent activity of neutrophil granulocytes in progression of MJ, depending on the bilirubin levels and origin of the jaundice. All the MJ patients showed altered chemiluminescent activity of granulocytes. Both spontaneous and induced chemiluminescence (CL intensity was decreased in the patients with gallstoneassociated MJ. Meanwhile, the CL intensity did not change in MJ caused by benign tumors (BTP. An increased activation index reflected higher induced activity of phagocytes. In patients with MJ of malignant origin, the largest number of changes was found, i.e., an increase in the induced luminescence intensity and higher activation indexes were revealed. The MJ progression depends on blood bilirubin levels. All the patients with gallstone-related MJ exhibited a decrease in spontaneous and induced CL activity of the neutrophils. In cases of BTP-caused MJ, the indexes of spontaneous and induced CL decreased at the bilirubin levels of 60 to 200 mmol/L, with increased activation index suggesting elevated induced activity over its spontaneous levels. In patients with MJ and bilirubin levels >200 mmol/L, distinct unidirectional changes in the granulocyte activation were observed, with increased spontaneous and induced CL intensity. In patients with MJ caused by malignancies with bilirubin levels <60

  7. Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction.

    Science.gov (United States)

    English, D; Schell, M; Siakotos, A; Gabig, T G

    1986-07-01

    gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma

  8. Neutrophils, from marrow to microbes

    DEFF Research Database (Denmark)

    Borregaard, Niels

    2010-01-01

    . Neutrophils circulate in the blood as dormant cells. At sites of infection, endothelial cells capture bypassing neutrophils and guide them through the endothelial cell lining whereby the neutrophils are activated and tuned for the subsequent interaction with microbes. Once in tissues, neutrophils kill...... microorganisms by microbicidal agents liberated from granules or generated by metabolic activation. As a final act, neutrophils can extrude stands of DNA with bactericidal proteins attached that act as extracellular traps for microorganisms....

  9. BIIL 284 reduces neutrophils numbers but increases P. aeruginosa bacteraemia and inflammation in mouse lungs

    Science.gov (United States)

    Döring, Gerd; Bragonzi, Alessandra; Paroni, Moira; Aktürk, Firdevs-Fatma; Cigana, Cristina; Schmidt, Annika; Gilpin, Deirdre; Heyder, Susanne; Born, Torsten; Smaczny, Christina; Kohlhäufl, Martin; Wagner, Thomas O. F.; Loebinger, Michael R.; Bilton, Diana; Tunney, Michael M.; Elborn, J. Stuart; Pier, Gerald B.; Konstan, Michael W.; Ulrich, Martina

    2014-01-01

    Background A clinical study to investigate the leukotriene B4 (LTB4)-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients. Methods P. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar beads murine model of Pseudomonas aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs. Result Most CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals. Conclusions Decreased airway neutrophils induced lung proliferation and severe bacteraemia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections. PMID:24183915

  10. Neutrophils at work

    DEFF Research Database (Denmark)

    Nauseef, William M; Borregaard, Niels

    2014-01-01

    blood to tissues in models of blood-borne infections versus bacterial invasion through epithelial linings. We examine data on novel aspects of the activation of NADPH oxidase and the heterogeneity of phagosomes and, finally, consider the importance of two neutrophil-derived biological agents: neutrophil......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...

  11. Hypoxia Selectively Inhibits Respiratory Burst Activity and Killing of Staphylococcus aureus in Human Neutrophils

    OpenAIRE

    McGovern, Naomi N.; Cowburn, Andrew S.; Porter, Linsey; Walmsley, Sarah R.; Summers, Charlotte; Thompson, Alfred A. R.; Anwar, Sadia; Willcocks, Lisa C.; Moira K B Whyte; Condliffe, Alison M; Chilvers, Edwin R.

    2010-01-01

    Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils...

  12. North American ginseng protects against muscle damage and reduces neutrophil infiltration after an acute bout of downhill running in rats.

    Science.gov (United States)

    Estaki, Mehrbod; Noble, Earl G

    2015-02-01

    Eccentric muscle contractions such as those experienced during downhill running are associated with inflammation, delayed-onset of muscle soreness, myofiber damage, and various functional deficits. North American ginseng (Panax quinquefolius L.) has been reported to possess anti-inflammatory properties and thus may offset some of this exercise-induced damage. Hence, we tested the hypothesis that intervention with North American ginseng would reduce eccentric exercise-induced muscle damage and inflammation. Male Wistar rats were fed (300 mg/(kg·day)(-1)) of either an alcohol (AL) or aqueous (AQ) extract of North American ginseng for 14 days before a single bout of downhill running and were compared with matching nonexercised (C) groups. Plasma creatine kinase levels were significantly reduced in both ginseng treated groups compared with the C group that received a water placebo (p damage (hemotoxylin and eosin) as well as reduced levels of infiltrating neutrophils (HIS48) in the soleus muscle (p muscle damage and inflammation.

  13. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Vlasova, Irina I., E-mail: irina.vlasova@yahoo.com [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Vakhrusheva, Tatyana V. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Sokolov, Alexey V.; Kostevich, Valeria A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Research Institute for Experimental Medicine, Russian Academy of Medical Science, Saint Petersburg (Russian Federation); Gusev, Alexandr A.; Gusev, Sergey A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Melnikova, Viktoriya I. [Institute of Developmental Biology, Russian Academy of Science, Moscow (Russian Federation); Lobach, Anatolii S. [Institute of Problems of Chemical Physics, Russian Academy of Science, Chernogolovka (Russian Federation)

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H{sub 2}O{sub 2} system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. -- Highlights: ► Myeloperoxidase (MPO) product hypochlorous acid is able to degrade CNTs. ► PEGylated SWCNTs stimulate isolated neutrophils to produce hypochlorous acid. ► SWCNTs are capable of activating neutrophils in blood samples. ► Activation of

  14. Extracellular acidosis induces neutrophil activation by a mechanism dependent on activation of phosphatidylinositol 3-kinase/Akt and ERK pathways.

    Science.gov (United States)

    Martínez, Diego; Vermeulen, Mónica; Trevani, Analía; Ceballos, Ana; Sabatté, Juan; Gamberale, Romina; Alvarez, María Eugenia; Salamone, Gabriela; Tanos, Tamara; Coso, Omar A; Geffner, Jorge

    2006-01-15

    Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IkappaB was observed, supporting the hypothesis that NF-kappaB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.

  15. Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

    Science.gov (United States)

    Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

    2015-01-01

    Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

  16. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

    Science.gov (United States)

    Vlasova, Irina I; Vakhrusheva, Tatyana V; Sokolov, Alexey V; Kostevich, Valeria A; Gusev, Alexandr A; Gusev, Sergey A; Melnikova, Viktoriya I; Lobach, Anatolii S

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H(2)O(2) system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes.

  17. Nuclear factor of activated T cells regulates neutrophil recruitment, systemic inflammation, and T-cell dysfunction in abdominal sepsis.

    Science.gov (United States)

    Zhang, Su; Luo, Lingtao; Wang, Yongzhi; Gomez, Maria F; Thorlacius, Henrik

    2014-08-01

    The signaling mechanisms regulating neutrophil recruitment, systemic inflammation, and T-cell dysfunction in polymicrobial sepsis are not clear. This study explored the potential involvement of the calcium/calcineurin-dependent transcription factor, nuclear factor of activated T cells (NFAT), in abdominal sepsis. Cecal ligation and puncture (CLP) triggered NFAT-dependent transcriptional activity in the lung, spleen, liver, and aorta in NFAT-luciferase reporter mice. Treatment with the NFAT inhibitor A-285222 prior to CLP completely prevented sepsis-induced NFAT activation in all these organs. Inhibition of NFAT activity reduced sepsis-induced formation of CXCL1, CXCL2, and CXCL5 chemokines and edema as well as neutrophil infiltration in the lung. Notably, NFAT inhibition efficiently reduced the CLP-evoked increases in HMBG1, interleukin 6 (IL-6), and CXCL5 levels in plasma. Moreover, administration of A-285222 restored sepsis-induced T-cell dysfunction, as evidenced by markedly decreased apoptosis and restored proliferative capacity of CD4 T cells. Along these lines, treatment with A-285222 restored gamma interferon (IFN-γ) and IL-4 levels in the spleen, which were markedly reduced in septic mice. CLP-induced formation of regulatory T cells (CD4(+) CD25(+) Foxp3(+)) in the spleen was also abolished in A-285222-treated animals. All together, these novel findings suggest that NFAT is a powerful regulator of pathological inflammation and T-cell immune dysfunction in abdominal sepsis. Thus, our data suggest that NFAT signaling might be a useful target to protect against respiratory failure and immunosuppression in patients with sepsis.

  18. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases.

    Directory of Open Access Journals (Sweden)

    Andrea Seper

    Full Text Available The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

  19. Age-related differences of neutrophil activation in a skeletal muscle ischemia-reperfusion model.

    Science.gov (United States)

    Mowlavi, Arian; Reynolds, Christopher; Neumeister, Michael W; Wilhelmi, Bradon J; Song, Yao-Hua; Naffziger, Ryan; Glatz, Frank R; Russell, Robert C

    2003-04-01

    Free tissue transfers and replantation of amputated limbs are better tolerated by young adolescents than mature adults. The authors hypothesized that this observation may be, in part, because of an attenuated ischemia-reperfusion (IR) injury in younger patients. Because neutrophils have been identified as a critical cell line responsible for IR injury, the authors investigated the effects of animal age on the degree of neutrophil activation in a rat model. Activation was evaluated by monitoring expression of integrin surface markers (mean fluorescence intensity [MFI] of CD11b) and oxidative burst potential (MFI of dihydrorhodamine [DHR] oxidation) by flow cytometry in neutrophils analyzed after 4 hours of ischemia and 1, 4, and 16 hours of reperfusion in a gracilis muscle flap model in mature adult and young adolescent rats. Neutrophil activation was also evaluated in control sham-operated animals, which underwent elevation of gracilis muscle flaps without exposure to an ischemic insult. Muscle edema, determined by wet-to-dry muscle weight ratio, and muscle viability, determined by nitro blue tetrazolium (NBT) staining, were completed for gracilis muscles exposed to ischemia after 24 hours of reperfusion for each of the groups. Integrin expression, assessed by MFI of CD11b, was increased significantly in ischemic muscles of mature adult rats at 4 hours of reperfusion (71.10+/-3.53 MFI vs. 54.88+/-12.73 MFI, p=0.025). Neutrophil oxidative potential, assessed by MFI of DHR oxidation, was increased significantly in ischemic muscles of mature adult rats compared with young adolescent rats at 1 hour of reperfusion (78.10+/-9.53 MFI vs. 51.78+/-16.91 MFI, p=0.035) and 4 hours of reperfusion (83.69+/-15.29 MFI vs. 46.55+/-8.09 MFI, p=0.005). Increased edema formation was observed in the ischemic muscles of mature adult rats when compared with young adolescent rats (1.25+/-0.04 vs. 1.12+/-0.05, p=0.031) after 24 hours of reperfusion. A trend toward decreased muscle

  20. 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...... 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...

  1. Elevated glucose concentrations promote receptor-independent activation of adherent human neutrophils: an experimental and computational approach

    DEFF Research Database (Denmark)

    Kummer, Ursula; Zobeley, Jürgen; Brasen, Jens Christian;

    2007-01-01

    these dynamic metabolic changes, mathematical simulations were performed. A model for glycolysis in neutrophils was created. The results indicated that the frequency change in NAD(P)H oscillations can result from the activation of the hexose monophosphate shunt, which competes with glycolysis for glucose-6......-phosphate. Experimental confirmation of these simulations was performed by measuring the effect of glucose concentrations on flavoprotein autofluorescence, an indicator of the rate of mitochondrial electron transport. Moreover, after prolonged exposure to elevated glucose levels, neutrophils return......Neutrophil activation plays integral roles in host tissue damage and resistance to infectious diseases. As glucose uptake and NADPH availability are required for reactive oxygen metabolite production by neutrophils, we tested the hypothesis that pathological glucose levels (>or=12 m...

  2. Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction

    Energy Technology Data Exchange (ETDEWEB)

    English, D.; Schell, M.; Siakotos, A.; Gabig, T.G.

    1986-07-01

    ..gamma..-hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with /sup 32/P to 4 x 10/sup -4/ M ..gamma..-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, ..gamma..-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10/sup 6/ cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messenger for activation of the NADPH-dependent O/sub 2//sup -/-generating system, neutrophil responses to ..gamma..-hexachlorocyclohexane were compared with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, ..gamma..-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of ..gamma..-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that n intact cells indicated that concurrent degranulation was not required for sustained O/sub 2//sup -/ generation in response to this agent.

  3. Signalling through neutrophil Fc gamma RIII, Fc gamma RII, and CD59 is not impaired in active rheumatoid arthritis.

    OpenAIRE

    1996-01-01

    OBJECTIVE: To compare neutrophil Fc receptor (Fc gamma R) and CD59 signalling responses in normal healthy subjects and patients with active rheumatoid arthritis (RA). METHODS: Intracellular free calcium concentrations were measured in neutrophils loaded with the fluorescent calcium indicator fura-2, using a spectrofluorimeter. RESULTS: Basal intracellular calcium ion concentrations were similar in both groups when no primary antibody, CD59, or CD32 (Fc gamma RIII) antibody was added. When CD1...

  4. Platelets augment respiratory burst in neutrophils activated by selected species of gram-positive or gram-negative bacteria.

    Directory of Open Access Journals (Sweden)

    Kamila Pytel

    2008-12-01

    Full Text Available Neutrophils and platelets circulate in blood system and play important physiological roles as part of immunological system. Neutrophils are the first line of host defense against various intruders, and platelets are satellite cells cooperating with other components of defense system. Recent studies report about the cooperation among these types of cells. We analyzed the effect of platelets on oxygen burst in neutrophils triggered by Staphylococcus aureus and Escherichia coli bacteria in vitro. The effect of platelets on oxygen burst in neutrophils was measured by luminol enhanced chemiluminescence. Opsonized and non-opsonized bacteria were used as activators. Activation of neutrophils with live non-opsonized and opsonized bacteria in the presence of platelets increased the oxygen burst as compared to the same system without platelets. The gram-positive bacteria (Staphylococcus aureus were causing higher activation than gram-negative bacteria (Escherichia coli. This work demonstrate that platelets potentate the response of neutrophils augmenting their respiratory burst in vitro when triggered by bacteria.

  5. Gene expression during the generation and activation of mouse neutrophils: implication of novel functional and regulatory pathways.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Ericson

    Full Text Available As part of the Immunological Genome Project (ImmGen, gene expression was determined in unstimulated (circulating mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes, thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the

  6. Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase.

    Science.gov (United States)

    Bissonnette, Sarah A; Glazier, Christina M; Stewart, Mary Q; Brown, Glenn E; Ellson, Chris D; Yaffe, Michael B

    2008-01-25

    In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state

  7. Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways.

    Science.gov (United States)

    Lin, Y; Jia, R; Liu, Y; Gao, Y; Zeng, X; Kou, J; Yu, B

    2014-12-01

    Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.

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

    Directory of Open Access Journals (Sweden)

    Orion D Weiner

    2006-02-01

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

  9. Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy.

    Science.gov (United States)

    Mitra, Soumya; Modi, Kshitij D; Foster, Thomas H

    2013-10-01

    We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population.

  10. One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Kuo-Shun Shih

    Full Text Available Helicobacter pylori neutrophil-activating protein (HP-NAP, a major virulence factor of Helicobacter pylori (H. pylori, is capable of activating human neutrophils to produce reactive oxygen species (ROS and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis. This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.

  11. Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.

    Science.gov (United States)

    Christy, Alison L; Walker, Margaret E; Hessner, Martin J; Brown, Melissa A

    2013-05-01

    The meninges are often considered inert tissues that house the CSF and provide protection for the brain and spinal cord. Yet emerging data demonstrates that they are also active sites of immune responses. Furthermore, the blood-CSF barrier surrounding meningeal blood vessels, together with the blood-brain barrier (BBB), is postulated to serve as a gateway for the pathological infiltration of immune cells into the CNS in multiple sclerosis (MS). Our previous studies using mast cell-deficient (Kit(W/Wv)) mice demonstrated that mast cells resident in the dura mater and pia mater exacerbate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by facilitating CNS inflammatory cell influx. Here we examined the underlying mechanisms that mediate these effects. We demonstrate that there are dramatic alterations in immune associated gene expression in the meninges in pre-clinical disease, including those associated with mast cell and neutrophil function. Meningeal mast cells are activated within 24 h of disease induction, but do not directly compromise CNS vascular integrity. Rather, through production of TNF, mast cells elicit an early influx of neutrophils, cells known to alter vascular permeability, into the meninges. These data add to the growing evidence that inflammation in the meninges precedes CNS immune cell infiltration and establish that mast cells are among the earliest participants in these disease-initiating events. We hypothesize that mast cell-dependent neutrophil recruitment and activation in the meninges promotes early breakdown of the local BBB and CSF-blood barrier allowing initial immune cell access to the CNS.

  12. Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration.

    Science.gov (United States)

    Heineke, Marieke H; van der Steen, Lydia P E; Korthouwer, Rianne M; Hage, J Joris; Langedijk, Johannes P M; Benschop, Joris J; Bakema, Jantine E; Slootstra, Jerry W; van Egmond, Marjolein

    2017-07-24

    The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. In vitro killing of oral Capnocytophaga by granule fractions of human neutrophils is associated with cathepsin G activity.

    Science.gov (United States)

    Miyasaki, K T; Bodeau, A L

    1991-05-01

    The Capnocytophaga are inhabitants of the hypoxic human gingival crevice that are normally prevented by neutrophils from causing periodontal and systemic infection. To identify potential nonoxidative bactericidal mechanisms against Capnocytophaga within human neutrophils, gel filtration chromatography was used to fractionate neutrophil granule extracts. Seven granule fractions, designated A through G, were obtained. The Capnocytophaga were most sensitive to killing by fraction D. Fraction D exhibited substantial bactericidal activity under aerobic and anaerobic conditions. The bactericidal activity associated with ion-exchange subfractions D8-D11, which contained primarily cathepsin G as assessed by enzymatic activity, amino acid composition, and NH2-terminal sequence. Heat-inactivation, diisopropylfluorophosphate, PMSF, and N-benzyloxycarbonylglycylleucylphenylalanyl-chloromethyl ketone inhibited bactericidal activity against Capnocytophaga sputigena but not Escherichia coli. We conclude that (a) human neutrophil cathepsin G is an important antimicrobial system against the Capnocytophaga, (b) the bactericidal activity of cathepsin G against Capnocytophaga is oxygen independent, and (c) an intact enzyme active site is involved in the killing of C. sputigena but not E. coli. We suggest that human neutrophil cathepsin G is an important antimicrobial system against certain oral bacteria and that cathepsin G kills bacteria by two distinct mechanisms.

  14. Glibenclamide reduces pro-inflammatory cytokine production by neutrophils of diabetes patients in response to bacterial infection

    Science.gov (United States)

    Kewcharoenwong, Chidchamai; Rinchai, Darawan; Utispan, Kusumawadee; Suwannasaen, Duangchan; Bancroft, Gregory J.; Ato, Manabu; Lertmemongkolchai, Ganjana

    2013-11-01

    Type 2 diabetes mellitus is a major risk factor for melioidosis, which is caused by Burkholderia pseudomallei. Our previous study has shown that polymorphonuclear neutrophils (PMNs) from diabetic subjects exhibited decreased functions in response to B. pseudomallei. Here we investigated the mechanisms regulating cytokine secretion of PMNs from diabetic patients which might contribute to patient susceptibility to bacterial infections. Purified PMNs from diabetic patients who had been treated with glibenclamide (an ATP-sensitive potassium channel blocker for anti-diabetes therapy), showed reduction of interleukin (IL)-1β and IL-8 secretion when exposed to B. pseudomallei. Additionally, reduction of these pro-inflammatory cytokines occurred when PMNs from diabetic patients were treated in vitro with glibenclamide. These findings suggest that glibenclamide might be responsible for the increased susceptibility of diabetic patients, with poor glycemic control, to bacterial infections as a result of its effect on reducing IL-1β production by PMNs.

  15. Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst.

    Science.gov (United States)

    Akard, L P; English, D; Gabig, T G

    1988-07-01

    The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N-ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N-ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N-ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.

  16. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    Science.gov (United States)

    Hermann, M; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-09-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.

  17. Isolation of Mouse Neutrophils.

    Science.gov (United States)

    Swamydas, Muthulekha; Luo, Yi; Dorf, Martin E; Lionakis, Michail S

    2015-08-03

    Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments.

  18. Killing activity of neutrophils is mediated through activation of proteases by K+ flux

    NARCIS (Netherlands)

    Reeves, EP; Lu, H; Lortat-Jacob, H; Messina, CGM; Bolsover, S; Gabella, G; Potma, EO; Warley, A; Roes, J; Segal, AW

    2002-01-01

    According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served a

  19. The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1

    Science.gov (United States)

    de la Luz Sierra, Maria; Sakakibara, Shuhei; Gasperini, Paola; Salvucci, Ombretta; Jiang, Kan; McCormick, Peter J.; Segarra, Marta; Stone, Jim; Maric, Dragan; Zhu, Jinfang; Qian, Xiaolan; Lowy, Douglas R.

    2010-01-01

    The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen–activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis. PMID:20203268

  20. Inflammatory mechanisms and treatment of obstructive airway diseases with neutrophilic bronchitis.

    Science.gov (United States)

    Simpson, Jodie L; Phipps, Simon; Gibson, Peter G

    2009-10-01

    Obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are major global health issues. Although considered as distinct diseases, airway inflammation is a key underlying pathophysiological process in asthma, COPD and bronchiectasis. Persistent neutrophilic airway inflammation (neutrophilic bronchitis) occurs with innate immune activation and is a feature of each of these airway diseases. Little is known about the mechanisms leading to neutrophilic bronchitis and few treatments are effective in reducing neutrophil accumulation in the airways. There is a similar pattern of inflammatory mediator release and toll like receptor 2 expression in asthma, COPD and bronchiectasis. We propose the existence of an active amplification mechanism, an effector arm of the innate immune system, involving toll like receptor 2, operating in persistent neutrophilic bronchitis. Neutrophil persistence in the airways can occur through a number of mechanisms such as impaired apoptosis, efferocytosis and mucus hypersecretion, all of which are impaired in airways disease. Impairment of neutrophil clearance results in a reduced ability to respond to bacterial infection. Persistent activation of airway neutrophils may result in the persistent activation of the innate immune system resulting in further airway insult. Current therapies are limited for the treatment of neutrophilic bronchitis; possible treatments being investigated include theophylline, statins, antagonists of pro-inflammatory cytokines and macrolide antibiotics. Macrolides have shown great promise in their ability to reduce airway inflammation, and can reduce airway neutrophils, levels of CXCL8 and neutrophil proteases in the airways. Studies also show improvements in quality of life and exacerbation rates in airways diseases.

  1. Quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions.

    Science.gov (United States)

    Wilhelmsen, Kevin; Farrar, Katherine; Hellman, Judith

    2013-08-23

    The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

  2. [Physiology and pathology of bactericidal activity in human polymorphonuclear neutrophils (author's transl)].

    Science.gov (United States)

    Hakim, J

    1980-09-13

    Blood polymorphonuclear neutrophils defend man against aggressions from pathogens. Under the combined influence of granulocytic and non-granulocytic factors, the defensive process develops by steps: the neutrophil moves towards the pathogenic organism which, one reached, is engulfed and killed. The killing systems of the cell are either oxygen-dependent or independent. the oxygen-dependent system is triggered off by stimulation of the cell membrane and involves various reactions, including cyanide-resistant oxygen consumption, production of activated oxygen, oxygen peroxide and halogenisation of the pathogen membrane. Impairment of the killing activity requires quantitative assessment of its various components in the presence of autologous or control serum with the view of: determining the origin (granulocytic or non-granulocytic) of the impairment, and identifying the step in oxygen metabolism that is affected. In the vast majority of non-granulocytic insufficiencies the cause lies in defective opsonins. In granulocytic insufficiencies, global failure of the system indicates chromic granulomatous disease, a syndrome that is now being dismembered. Defective halogenisation should lead to testing for deficiency of myeloperoxidase or abnormal degranulation. The non oxygen-independent bactericidal system, although highly effective in vitro, appears to be less important in vivo than the oxygen-dependent system. Little is known of its pathology.

  3. An experimental evaluation of in vitro immunomodulatory activity of isolated compound of Ricinus communis on human neutrophils

    Directory of Open Access Journals (Sweden)

    Arvind Kumar

    2011-01-01

    Full Text Available In the present study, the in vitro immunomodulatory activity of Ricinus communis Linn (Euphorbiaceae was determined on human neutrophils. The isolated compound (tannin of R. communis leaves was screened for its possible immunomodulatory activity by carrying out nitroblue tetrazolium test, phagocytosis of killed Candida albicans, neutrophil locomotion and chemotaxis. The isolated compound was tested at concentrations, viz. 10 μg/ml, 20 μg/ml, 40 μg/ml, 100 μg/ml and 1000 μg/ml. The isolated compound of R. communis showed predominantly significant activity on human neutrophils in all the parameters tested, which was comparable to the standard and control at different concentrations, indicating the possible immunostimulating effect.

  4. The possible link between elevated serum levels of epithelial cell-derived neutrophil- activating peptide-78 (ENA-78/CXCL5) and autoimmunity in autistic children

    OpenAIRE

    Mostafa, Gehan Ahmed; AL-Ayadhi, Laila Yousef

    2015-01-01

    Background In autoimmune disorders, the underlying pathogenic mechanism is the formation of antigen-antibody complexes which trigger an inflammatory response by inducing the infiltration of neutrophils. Epithelial cell-derived neutrophil-activating peptide-78 (ENA-78) is a chemokine that recruits and activates neutrophils, thus it could play a pathogenic role in inflammation and autoimmune disorders. Some autistic children have elevated levels of brain specific auto-antibodies. We are the fir...

  5. Are Neutrophil Extracellular Traps Playing a Role in the Parasite Control in Active American Tegumentary Leishmaniasis Lesions?

    Science.gov (United States)

    Morgado, Fernanda Nazaré; Nascimento, Michelle T C; Saraiva, Elvira M; de Oliveira-Ribeiro, Carla; Madeira, Maria de Fátima; da Costa-Santos, Marcela; Vasconcellos, Erica C F; Pimentel, Maria Ines F; Rosandiski Lyra, Marcelo; Schubach, Armando de Oliveira; Conceição-Silva, Fátima

    2015-01-01

    Neutrophil extracellular traps (NETs) have been described as a network of extracellular fibers composed by DNA, histones and various proteins/enzymes. Studies have demonstrated that NETs could be responsible for the trapping and elimination of a variety of infectious agents. In order to verify the presence of NETs in American tegumentary leishmaniasis (ATL) and their relationship with the presence of amastigotes we evaluated active cutaneous lesions of 35 patients before treatment by the detection of parasites, neutrophils (neutrophil elastase) and histones through immunohistochemistry and confocal immunofluorescence. Intact neutrophils could be detected in all ATL lesions. NETs were present in 27 patients (median 1.1; range from 0.1 to 23.5/mm2) with lesion duration ranging from one to seven months. NETs were in close proximity with neutrophils (r = 0.586; p = 0.0001) and amastigotes (r = 0.710; p = 0.0001). Two patterns of NET formation were detected: small homogeneously distributed networks observed in all lesions; and large structures that could be visualized at a lower magnification in lesions presenting at least 20% of neutrophils. Lesions presenting the larger NET formation showed high parasite detection. A correlation between NET size and the number of intact amastigotes was observed (p=0.02). As we detected an association between NET and amastigotes, our results suggest that neutrophil migration and NET formation could be stimulated and maintained by stimuli derived from the parasite burden/parasite antigen in the extracellular environment. The observation of areas containing only antigens not intermingled with NETs (elastase and histone) suggests that the involvement of these structures in the control of parasite burden is a dynamic process in which the formation of NETs is exhausted with the destruction of the parasites. Since NETs were also associated with granulomas, this trapping would favor the activity of macrophages in order to control the parasite

  6. Paediatric Crohn disease patients with stricturing behaviour exhibit ileal granulocyte–macrophage colony-stimulating factor (GM-CSF) autoantibody production and reduced neutrophil bacterial killing and GM-CSF bioactivity

    Science.gov (United States)

    Jurickova, I; Collins, M H; Chalk, C; Seese, A; Bezold, R; Lake, K; Allmen, D; Frischer, J S; Falcone, R A; Trapnell, B C; Denson, L A

    2013-01-01

    Granulocyte–macrophage colony-stimulating factor (GM-CSF) autoantibodies are associated with stricturing behaviour in Crohn disease (CD). We hypothesized that CD ileal lamina propria mononuclear cells (LPMC) would produce GM-CSF autoantibodies and peripheral blood (PB) samples would contain GM-CSF neutralizing capacity (NC). Paediatric CD and control PBMC and ileal biopsies or LPMC were isolated and cultured and GM-CSF, immunoglobulin (Ig)G and GM-CSF autoantibodies production were measured by enzyme-linked immunosorbent assay (ELISA). Basal and GM-CSF-primed neutrophil bacterial killing and signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation (pSTAT5) were measured by flow cytometry. GM-CSF autoantibodies were enriched within total IgG for LPMC isolated from CD ileal strictures and proximal margins compared to control ileum. Neutrophil bacterial killing was reduced in CD patients compared to controls. Within CD, neutrophil GM-CSF-dependent STAT5 activation and bacterial killing were reduced as GM-CSF autoantibodies increased. GM-CSF stimulation of pSTAT5 did not vary between controls and CD patients in washed PB granulocytes in which serum was removed. However, GM-CSF stimulation of pSTAT5 was reduced in whole PB samples from CD patients. These data were used to calculate the GM-CSF NC. CD patients with GM-CSF NC greater than 25% exhibited a fourfold higher rate of stricturing behaviour and surgery. The likelihood ratio (95% confidence interval) for stricturing behaviour for patients with elevation in both GM-CSF autoantibodies and GM-CSF NC was equal to 5 (2, 11). GM-CSF autoantibodies are produced by LPMC isolated from CD ileal resection specimens and are associated with reduced neutrophil bacterial killing. CD peripheral blood contains GM-CSF NC, which is associated with increased rates of stricturing behaviour. PMID:23600834

  7. Intravenous immunoglobulin enhances the killing activity and autophagy of neutrophils isolated from immunocompromised patients against multidrug-resistant bacteria.

    Science.gov (United States)

    Matsuo, Hidemasa; Itoh, Hiroshi; Kitamura, Naoko; Kamikubo, Yasuhiko; Higuchi, Takeshi; Shiga, Shuichi; Ichiyama, Satoshi; Kondo, Tadakazu; Takaori-Kondo, Akifumi; Adachi, Souichi

    2015-08-14

    Intravenous immunoglobulin (IVIG) is periodically administered to immunocompromised patients together with antimicrobial agents. The evidence that supports the effectiveness of IVIG is mostly based on data from randomized clinical trials; the underlying mechanisms are poorly understood. A recent study revealed that killing of multidrug-resistant bacteria and drug-sensitive strains by neutrophils isolated from healthy donors is enhanced by an IVIG preparation. However, the effectiveness of IVIG in immunocompromised patients remains unclear. The present study found that IVIG increased both killing activity and O2(-) release by neutrophils isolated from six patients receiving immune-suppressive drugs after hematopoietic stem cell transplantation (HSCT); these neutrophils killed both multidrug-resistant extended-spectrum β-lactamase-producing Escherichia coli (E. coli) and multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa). Moreover, IVIG increased the autophagy of the neutrophils, which is known to play an important role in innate immunity. These results suggest that IVIG promotes both the killing activity and autophagy of neutrophils isolated from immunocompromised patients against multidrug-resistant bacteria.

  8. Mucosal adaptation to aspirin induced gastric damage in humans. Studies on blood flow, gastric mucosal growth, and neutrophil activation.

    Science.gov (United States)

    Konturek, J W; Dembinski, A; Stoll, R; Domschke, W; Konturek, S J

    1994-01-01

    The gastropathy associated with the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin is a common side effect of this class of drugs, but the precise mechanisms by which they cause mucosal damage have not been fully explained. During continued use of an injurious substance, such as aspirin, the extent of gastric mucosal damage decreases and this phenomenon is named gastric adaptation. To assess the extent of mucosal damage by aspirin and subsequent adaptation the effects of 14 days of continuous, oral administration of aspirin (2 g per day) to eight healthy male volunteers was studied. To estimate the rate of mucosal damage, gastroscopy was performed before (day 0) and at days 3, 7, 14 of aspirin treatment. Gastric microbleeding and gastric mucosal blood flow were measured using laser Doppler flowmeter and mucosal biopsy specimens were taken for the estimation of tissue DNA synthesis and RNA and DNA concentration. In addition, the activation of neutrophils in peripheral blood was assessed by measuring their ability to associate with platelets. Aspirin induced acute damage mainly in gastric corpus, reaching at day 3 about 3.5 on the endoscopic Lanza score but lessened to about 1.5 at day 14 pointing to the occurrence of gastric adaptation. Mucosal blood flow increased at day 3 by about 50% in the gastric corpus and by 88% in the antrum. The in vitro DNA synthesis and RNA concentration, an index of mucosal growth, were reduced at day 3 but then increased to reach about 150% of initial value at the end of aspirin treatment. It is concluded that the treatment with aspirin in humans induces gastric adaptation to this agent, which entails the increase in mucosal blood flow, the rise in neutrophil activation, and the enhancement in mucosal growth. PMID:7959223

  9. Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia.

    Directory of Open Access Journals (Sweden)

    Alexandra Birle

    Full Text Available Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS under spinal anaesthesia (mepivacaine, sufentanil, epidural anaesthesia (ropivacaine or bupivacaine, sufentanil or general anaesthesia (ketamine, thiopental, succinylcholine. Chemokinesis was higher (p=0.008 in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02. After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.

  10. Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide.

    Science.gov (United States)

    Parker, Heather; Albrett, Amelia M; Kettle, Anthony J; Winterbourn, Christine C

    2012-03-01

    A variety of inflammatory stimuli induces NETs. These structures consist of a network of chromatin strands associated with predominately granule proteins, including MPO. NETs exhibit antimicrobial activity, which is proposed to augment the more-established mechanism of phagosomal killing. They may also be detrimental to the host in situations such as chronic inflammation or severe sepsis. The objective of this study was to establish whether MPO associated with NETs is active and able to kill bacteria. Neutrophils were stimulated with PMA to release NETs. Peroxidase activity measurements were performed and showed that enzymatically active MPO was released from the neutrophils, 2-4 h after stimulation, concomitant with NET formation. Approximately 30% of the total cellular MPO was released, with the majority bound to the NETs. The bound enzyme retained its activity. Staphylococcus aureus were not killed when added to preformed NETs under our assay conditions. However, addition of H(2)O(2) to the bacteria in the presence of NETs resulted in MPO-dependent killing, which was observed with NETs in situ and with NETs when they were removed from the neutrophils by limited DNase digestion. Our results show that the enzymatic activity of MPO on NETs could contribute to antimicrobial activity or tissue injury when NETs are released from neutrophils at sites of infection or inflammation.

  11. Myeloperoxidase Stimulates Neutrophil Degranulation.

    Science.gov (United States)

    Grigorieva, D V; Gorudko, I V; Sokolov, A V; Kostevich, V A; Vasilyev, V B; Cherenkevich, S N; Panasenko, O M

    2016-08-01

    Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

  12. Cellular responses of Candida albicans to phagocytosis and the extracellular activities of neutrophils are critical to counteract carbohydrate starvation, oxidative and nitrosative stress.

    Science.gov (United States)

    Miramón, Pedro; Dunker, Christine; Windecker, Hanna; Bohovych, Iryna M; Brown, Alistair J P; Kurzai, Oliver; Hube, Bernhard

    2012-01-01

    Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all

  13. The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 °C

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    Kim Brügger

    2007-01-01

    Full Text Available Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H2S. Two large clusters of regularly interspaced repeats (CRISPRs are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment.

  14. Neutrophils, from marrow to microbes

    DEFF Research Database (Denmark)

    Borregaard, Niels

    2010-01-01

    . Neutrophils circulate in the blood as dormant cells. At sites of infection, endothelial cells capture bypassing neutrophils and guide them through the endothelial cell lining whereby the neutrophils are activated and tuned for the subsequent interaction with microbes. Once in tissues, neutrophils kill......Neutrophils are produced in the bone marrow from stem cells that proliferate and differentiate to mature neutrophils fully equipped with an armory of granules. These contain proteins that enable the neutrophil to deliver lethal hits against microorganisms, but also to cause great tissue damage...... microorganisms by microbicidal agents liberated from granules or generated by metabolic activation. As a final act, neutrophils can extrude stands of DNA with bactericidal proteins attached that act as extracellular traps for microorganisms....

  15. Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

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    Paino I.M.M.

    2005-01-01

    Full Text Available The release of reactive oxygen specie (ROS by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM, indomethacin (12 µM, naproxen (160 µM, piroxicam (13 µM, and tenoxicam (30 µM were incubated at 37ºC in PBS (10 mM, pH 7.4, for 30 min with rat neutrophils (1 x 10(6 cells/ml stimulated by phorbol-12-myristate-13-acetate (100 nM. The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6. For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6. Using the myeloperoxidase (MPO/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%, indomethacin (97 ± 2, 100 ± 1%, naproxen (56 ± 8, 76 ± 3%, piroxicam (77 ± 5, 99 ± 1%, and tenoxicam (90 ± 2, 100 ± 1%, respectively (N = 3. These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

  16. Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

    Institute of Scientific and Technical Information of China (English)

    Qiao-Zhen Kang; Guang-Cai Duan; Qing-Tang Fan; Yuan-Lin Xi

    2005-01-01

    AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

  17. Active ammonia oxidizers in an acidic soil are phylogenetically closely related to neutrophilic archaeon.

    Science.gov (United States)

    Wang, Baozhan; Zheng, Yan; Huang, Rong; Zhou, Xue; Wang, Dongmei; He, Yuanqiu; Jia, Zhongjun

    2014-03-01

    All cultivated ammonia-oxidizing archaea (AOA) within the Nitrososphaera cluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence of Nitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth of Nitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis of amoA genes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the "heavy" DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that (13)CO2 assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both (13)C-labeled amoA and 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strains Nitrososphaera viennensis EN76 and JG1 within the Nitrososphaera cluster. Our results provide strong evidence for the adaptive growth of Nitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

  18. In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

    Science.gov (United States)

    Miyasaki, K T; Bodeau, A L; Ganz, T; Selsted, M E; Lehrer, R I

    1990-12-01

    Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.

  19. Activity of neutrophil β-glucuronidase in diabetic and nondiabetic patients with chronic generalized periodontitis and healthy subjects.

    Science.gov (United States)

    Surna, Algimantas; Sakalauskienė, Jurgina; Gleiznys, Alvydas; Ivanauskienė, Eglė; Saferis, Viktoras

    2011-01-01

    OBJECTIVE. The aim of the study was to establish the dynamics of β-glucuronidase activity in subjects suffering from type 1 diabetes and chronic untreated generalized periodontitis, subjects suffering from chronic untreated generalized periodontitis only, and control subjects not suffering from generic diseases with healthy periodontal tissue. MATERIAL AND METHODS. The study involved 165 19-50-year-old subjects who were divided into three groups: healthy subjects (n=55), subjects with chronic untreated generalized periodontitis (n=55), and subjects with type 1 diabetes and chronic untreated generalized periodontitis (n=55). Neutrophilic leukocytes of peripheral venous blood were exposed to bacterial stimuli: opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan. The activity of β-glucuronidase was determined by the spectrofluorimetry method. RESULTS. The diagnostic value of changes in β-glucuronidase activity of neutrophilic leukocytes markedly increased in all study groups after stimulation of neutrophilic leukocytes by opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan as compared to control media not exposed to any stimulus (Pperiodontal pathology markers and the activity of β-glucuronidase of neutrophilic leukocytes in incubated media in patients with type 1 diabetes mellitus and periodontitis was found under the effect of nonopsonized Staphylococcus aureus. CONCLUSIONS. If periodontal impairment is severe, diabetes mellitus possibly causes a faster destruction of the periodontal tissue and presents a higher risk of periodontitis for patients with diabetes.

  20. NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

    DEFF Research Database (Denmark)

    Perera, Natascha C; Wiesmüller, Karl-Heinz; Larsen, Maria Torp

    2013-01-01

    Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4...

  1. Bubbles, microparticles, and neutrophil activation: changes with exercise level and breathing gas during open-water SCUBA diving.

    Science.gov (United States)

    Thom, Stephen R; Milovanova, Tatyana N; Bogush, Marina; Yang, Ming; Bhopale, Veena M; Pollock, Neal W; Ljubkovic, Marko; Denoble, Petar; Madden, Dennis; Lozo, Mislav; Dujic, Zeljko

    2013-05-15

    The study goal was to evaluate responses in humans following decompression from open-water SCUBA diving with the hypothesis that exertion underwater and use of a breathing mixture containing more oxygen and less nitrogen (enriched air nitrox) would alter annexin V-positive microparticle (MP) production and size changes and neutrophil activation, as well as their relationships to intravascular bubble formation. Twenty-four divers followed a uniform dive profile to 18 m of sea water breathing air or 22.5 m breathing 32% oxygen/68% nitrogen for 47 min, either swimming with moderately heavy exertion underwater or remaining stationary at depth. Blood was obtained pre- and at 15 and 120 min postdive. Intravascular bubbles were quantified by transthoracic echocardiography postdive at 20-min intervals for 2 h. There were no significant differences in maximum bubble scores among the dives. MP number increased 2.7-fold, on average, within 15 min after each dive; only the air-exertion dive resulted in a significant further increase to 5-fold over baseline at 2 h postdive. Neutrophil activation occurred after all dives. For the enriched air nitrox stationary at depth dive, but not for other conditions, the numbers of postdive annexin V-positive particles above 1 μm in diameter were correlated with intravascular bubble scores (correlation coefficients ∼0.9, P bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation appear to exist, but more study is required to improve confidence in the associations.

  2. High fidelity processing and activation of the human α-defensin HNP1 precursor by neutrophil elastase and proteinase 3.

    Directory of Open Access Journals (Sweden)

    Prasad Tongaonkar

    Full Text Available The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1-4. HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages ("folded proHNP1" and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1. Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE, proteinase 3 (PR3 or cathepsin G (CG, serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.

  3. NITRIC OXIDE ACTIVITY OF NEUTROPHIL IN BLOOD AND CEREBROSPINAL FLUID OF THE CHILDREN WITH BACTERIAL AND VIRAL MENINGITIS

    Directory of Open Access Journals (Sweden)

    V. P. Molochniy

    2014-01-01

    Full Text Available The article presents the results of study of nitric oxide activity of neutrophil leucocytic and freeradical processes in blood and cerebrospinal fluid of the children with bacterial and viral meningitison the acute period diseases. The peculiarities or activity of freeradical processes and nitric oxide of cerebrospinal fluid with bacterial meningitis in acute period diseases and activities of studies of ferments with the health children. 

  4. Comparison of disease activity measures for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis

    Science.gov (United States)

    Merkel, PA; Cuthbertson, DD; Hellmich, B; Hoffman, GS; Jayne, DRW; Kallenberg, CGM; Krischer, JP; Luqmani, R; Mahr, AD; Matteson, EL; Specks, U; Stone, JH

    2011-01-01

    Aim Currently, several different instruments are used to measure disease activity and extent in clinical trials of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, leading to division among investigative groups and difficulty comparing study results. An exercise comparing six different vasculitis instruments was performed. Methods A total of 10 experienced vasculitis investigators from 5 countries scored 20 cases in the literature of Wegener granulomatosis or microscopic polyangiitis using 6 disease assessment tools: the Birmingham Vasculitis Activity Score (BVAS), The BVAS for Wegener granulomatosis (BVAS/WG), BVAS 2003, a Physician Global Assessment (PGA), the Disease Extent Index (DEI) and the Five Factor Score (FFS). Five cases were rescored by all raters. Results Reliability of the measures was extremely high (intraclass correlations for the six measures all=0.98). Within each instrument, there were no significant differences or outliers among the scores from the 10 investigators. Test/retest reliability was high for each measure: range=0.77 to 0.95. The scores of the five acute activity measures correlated extremely well with one another. Conclusions Currently available tools for measuring disease extent and activity in ANCA-associated vasculitis are highly correlated and reliable. These results provide investigators with confidence to compare different clinical trial data and helps form common ground as international research groups develop new, improved and universally accepted vasculitis disease assessment instruments. PMID:18664546

  5. The Fc Receptor Polymorphisms and Expression of Neutrophil Activation Markers in Patients with Sickle Cell Disease from Western India

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    Harshada K. Kangne

    2013-01-01

    Full Text Available Objective. Sickle cell disease has variable clinical manifestations. Activation of neutrophils plays an important role in the initiation and propagation of vaso occlusive crises which can be analysed by determining the expression of neutrophil antigens such as CD16, CD32, and CD62L. The common FcγR polymorphisms (FcγRIIA and FcγRIIIB are considered to influence clinical presentation. This study focuses on distribution of FcγR polymorphisms and their association with neutrophil activity among the patients from western India. Methods. In this paper 127 sickle cell anemia patients and 58 patients with sickle-β-thalassemia (median age 12±8.58 years with variable clinical phenotypes along with 175 normals were investigated. FcγRs polymorphisms were analysed by RFLP and AS-PCR. Activation of neutrophils was measured by flow cytometry. Results. The genotypic frequency of the H/R genotype of FcγRIIA and the NA1/NA1 genotype of FcγRIIIB was significantly decreased in patients compared to normals (P-0.0074, P-0.0471, resp.. We found a significant difference in the expression of CD32 and CD62L among the patients as against normals. A significantly higher expression of CD32 was seen in the milder patients with the H/H genotype (P-0.0231, whereas the expression of CD16 was higher in severe patients with the NA2/NA2 genotype (P-0.0312. Conclusion. The two FcγR polymorphisms had significant association with variable phenotypes of sickle cell disease. The expression of CD62L decreased in our patients indicating activation of neutrophils.

  6. Rho-kinase regulates adhesive and mechanical mechanisms of pulmonary recruitment of neutrophils in abdominal sepsis.

    Science.gov (United States)

    Palani, Karzan; Rahman, Milladur; Hasan, Zirak; Zhang, Su; Qi, Zhongquan; Jeppsson, Bengt; Thorlacius, Henrik

    2012-05-05

    We hypothesized that Rho-kinase signaling plays a role in mechanical and adhesive mechanisms of neutrophil accumulation in lung. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 prior to cecal ligation and puncture (CLP). Lung levels of myeloperoxidase (MPO) and histological tissue damage were determined 6h and 24h after CLP. Expression of Mac-1 and F-actin formation in neutrophils were quantified by using flow cytometry 6h after CLP. Mac-1 expression and F-actin formation were also determined in isolated neutrophils up to 3h after stimulation with CXCL2. Labeled and activated neutrophils co-incubated with Y-27632, an anti-Mac-1 antibody and cytochalasin B were adoptively transferred to CLP mice. Y-27632 reduced the CLP-induced pulmonary injury and MPO activity as well as Mac-1 on neutrophils. Neutrophil F-actin formation peaked at 6h and returned to baseline levels 24h after CLP induction. Rho-kinase inhibition decreased CLP-provoked F-actin formation in neutrophils. CXCL2 rapidly increased Mac-1 expression and F-actin formation in neutrophils. Co-incubation with Y-27632 abolished CXCL2-induced Mac-1 up-regulation and formation of F-actin in neutrophils. Notably, co-incubation with cytochalasin B inhibited formation of F-actin but did not reduce Mac-1 expression on activated neutrophils. Adoptive transfer experiments revealed that co-incubation of neutrophils with the anti-Mac-1 antibody or cytochalasin B significantly decreased pulmonary accumulation of neutrophils in septic mice. Our data show that targeting Rho-kinase effectively reduces neutrophil recruitment and tissue damage in abdominal sepsis. Moreover, these findings demonstrate that Rho-kinase-dependent neutrophil accumulation in septic lung injury is regulated by both adhesive and mechanical mechanisms.

  7. Evaluation of the antioxidant activity of passion fruit (Passiflora edulis and Passiflora alata) extracts on stimulated neutrophils and myeloperoxidase activity assays.

    Science.gov (United States)

    Zeraik, Maria Luiza; Serteyn, Didier; Deby-Dupont, Ginette; Wauters, Jean-Nöel; Tits, Monique; Yariwake, Janete H; Angenot, Luc; Franck, Thierry

    2011-09-15

    The antioxidant activity of methanol extracts from Passiflora edulis and Passiflora alata pulp, and P. edulis rinds, healthy or infected with the passion fruit woodiness virus (PWV), was investigated using the oxidant activities of the neutrophil and the neutrophil granule enzyme myeloperoxidase (MPO), both playing key roles in inflammation. The reactive oxygen species produced by stimulated neutrophils were evaluated by lucigenin-enhanced chemiluminescence (CL) and the activity of purified MPO was measured by SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection), a technique for studying the direct interaction of a compound with the enzyme. The rind extracts of P. edulis possessed higher and dose-dependent inhibitory effects on CL response and on the peroxidase activity of MPO than total pulp extracts from both passion fruit species. The quantification of isoorientin in the extracts showed a correlation with their antioxidant activity, suggesting the potential of P. edulis rinds as functional food or as a possible source of natural flavonoids.

  8. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein.

    Science.gov (United States)

    Shenkar, R; Abraham, E

    1999-07-15

    Acute inflammatory lung injury occurs frequently in the setting of severe infection or blood loss. Accumulation of activated neutrophils in the lungs and increased pulmonary proinflammatory cytokine levels are major characteristics of acute lung injury. In the present experiments, we examined mechanisms leading to neutrophil accumulation and activation in the lungs after endotoxemia or hemorrhage. Levels of IL-1 beta, TNF-alpha, and macrophage inflammatory protein-2 mRNA were increased in lung neutrophils from endotoxemic or hemorrhaged mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic, hemorrhaged, or control mice. The transcriptional regulatory factors NF-kappa B and cAMP response element binding protein were activated in lung but not blood neutrophils after hemorrhage or endotoxemia. Xanthine oxidase inhibition, achieved by feeding allopurinol or tungsten-containing diets, did not affect neutrophil trafficking to the lungs after hemorrhage or endotoxemia. Xanthine oxidase inhibition did prevent hemorrhage- but not endotoxemia-induced increases in proinflammatory cytokine expression among lung neutrophils. Hemorrhage- or endotoxemia-associated activation of NF-kappa B in lung neutrophils was not affected by inhibition of xanthine oxidase. cAMP response element binding protein activation was increased after hemorrhage, but not endotoxemia, in mice fed xanthine oxidase-inhibiting diets. Our results indicate that xanthine oxidase modulates cAMP response element binding protein activation and proinflammatory cytokine expression in lung neutrophils after hemorrhage, but not endotoxemia. These findings suggest that the mechanisms leading to acute inflammatory lung injury after hemorrhage differ from those associated with endotoxemia.

  9. Gβ1 is required for neutrophil migration in zebrafish.

    Science.gov (United States)

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

    2017-08-01

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

  10. Endothelium and the effect of activated neutrophils on arterial smooth muscle

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    Bauer Viktor

    2015-03-01

    Full Text Available The aim of the study was to analyze the involvement of the endothelium in the effects of neutrophils (PMNL on phenylephrine-pre-contracted isolated rings of the rat thoracic aorta and to compare their effects with those of peroxynitrite (ONOO− and hypochlorous acid (HOCl. Activated PMNL-induced contraction of the precontracted aorta was prevented by the blockade of NO-synthase and by endothelium removal. In the endothelium-free preparations, the effect of PMNL reappeared in the presence of sodium nitroprusside. The effect of ONOO− and HOCl significantly differed from that of activated PMNL both in the presence and absence of the endothelium. It is therefore likely that neither ONOO− nor HOCl generated by transformation of superoxide anion radical (O2•− produced by PMNL is involved in their action. Reduction of the relaxant effect of nitric oxide derived from the endothelium by O2•− seems to be the keystone mechanism in generation of PMNL-induced contraction.

  11. Iron-chelating agent, deferasirox, inhibits neutrophil activation and extracellular trap formation.

    Science.gov (United States)

    Kono, Mari; Saigo, Katsuyasu; Yamamoto, Shiori; Shirai, Kohei; Iwamoto, Shuta; Uematsu, Tomoko; Takahashi, Takayuki; Imoto, Shion; Hashimoto, Makoto; Minami, Yosuke; Wada, Atsushi; Takenokuchi, Mariko; Kawano, Seiji

    2016-10-01

    Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

  12. Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

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    André Holdfeldt

    2016-09-01

    Full Text Available The data described here is related to the research article titled (Gabl et al., 2016 [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8 and CXCR4 (ATI-2341, respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe6-NH2 (both are FPR2 specific inhibitors, but not to the FPR1 specific inhibitor cyclosporine H.

  13. Intramedullary nailing of the femur and the systemic activation of monocytes and neutrophils

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    Koenderman Leo

    2011-10-01

    Full Text Available Abstract Background Trauma such as found patients with femur fractures, induces a systemic inflammatory response, which ranges from mild SIRS to ARDS. Neutrophils (i.e. PMN play an important role in the pathogenesis of this inflammatory condition. Additional activation of PMNs during intramedullary nailing (IMN is thought to act as a second immunological hit. Damage control orthopedics has been developed to limit this putative exacerbation of the inflammatory response. The hypothesis is tested that IMN exacerbates systemic inflammation, thereby increasing the risk for ARDS. Methods Thirty-eight trauma patients who required IMN for femur fracture were included. The development of SIRS and ARDS was recorded. Blood samples were taken prior and 18 hours after IMN. Inflammatory response was analyzed by changes in plasma IL-6 levels, monocyte (HLA-DR and PMN phenotype (MAC-1 and responsiveness for the innate immune stimulus fMLP in the context of active FcγRII. Results Plasma IL-6 was significantly enhanced in severely injured patients compared to patients with isolated femur fractures and matched controls (P = 0.005; P = 0.018. This enhanced inflammatory tone was associated with a lower percentage HLA-DR positive monocytes (P = 0.002. The systemic PMN compartment was activated, characterized by an increased MAC-1 expression and a significantly decreased sensitivity for the innate stimulus fMLP Interestingly the PMN compartment was not affected by IMN. Conclusions Multitrauma patients were characterized by a marked activation of the systemic inflammatory response, associated with a systemic activation of the monocyte and PMN compartments. IMN particularly affected the monocyte arm of the systemic innate immune system.

  14. Modulation of neutrophil function by the tripeptide feG

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    Davison Joseph S

    2003-03-01

    Full Text Available Abstract Background Neutrophils are critical in the defense against potentially harmful microorganisms, but their excessive and inappropriate activation can contribute significantly to tissue damage and a worsening pathology. Through the release of endocrine factors submandibular glands contribute to achieving a balance in neutrophil function by modulating the state of activation and migratory potential of circulating neutrophils. A putative hormonal candidate for these effects on neutrophils was identified as a heptapeptide named submandibular gland peptide T (SGP-T; sequence = TDIFEGG. Since the tripeptide FEG, derived from SGP-T, and its D-amino acid analogue feG had similar inhibitory effects on inflammatory reactions, we investigated the effects of feG on human and rat neutrophil function. Results With human neutrophils feG had no discernible effect on oxidative burst or phagocytosis, but in picomolar amounts it reduced PAF-induced neutrophil movement and adhesion, and the binding of CD11b by 34% and that of CD16b close to control values. In the rat feG (10-11M reduced the binding of CD11b and CD16 antibodies to PAF-stimulated circulating neutrophils by 35% and 43%, respectively, and at 100 micrograms/kilograms intraperitoneally feG reduced neutrophil in vivo migration by 40%. With ovalbumin-sensitized rats that were challenged with antigen, feG inhibited binding of antibodies against CD16b but not CD11b, on peritoneal leukocytes. Conclusions The inhibitory effect of feG on neutrophil movement may be mediated by alterations in the co-stimulatory molecules CD11b and CD16.

  15. Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure

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    Shirong Cai

    2016-01-01

    Full Text Available This study evaluates the ability of Lactobacillus rhamnosus GG (LGG to activate DC and neutrophils and modulate T cell activation and the impact of bacterial dose on these responses. Murine bone marrow derived DC or neutrophils were stimulated with LGG at ratios of 5 : 1, 10 : 1, and 100 : 1 (LGG : cells and DC maturation (CD40, CD80, CD86, CD83, and MHC class II and cytokine production (IL-10, TNF-α, and IL-12p70 were examined after 2 h and 18 h coculture and compared to the ability of BCG (the present immunotherapeutic agent for bladder cancer to stimulate these cells. A 2 h exposure to 100 : 1 (high dose or an 18 h exposure to 5 : 1 or 10 : 1 (low dose, LGG : cells, induced the highest production of IL-12 and upregulation of CD40, CD80, CD86, and MHC II on DC. In DCs stimulated with LGG activated neutrophils IL-12 production decreased with increasing dose. LGG induced 10-fold greater IL-12 production than BCG. T cell IFNγ and IL-2 production was significantly greater when stimulated with DC activated with low dose LGG. In conclusion, DC or DC activated with neutrophils exposed to low dose LGG induced greater Th1 polarization in T cells and this could potentially exert stronger antitumor effects. Thus the dose of LGG used for immunotherapy could determine treatment efficacy.

  16. 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.

  17. Involvement of neutrophil hyporesponse and the role of Toll-like receptors in human immunodeficiency virus 1 protection.

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    Juan C Hernandez

    Full Text Available Neutrophils contribute to pathogen clearance through pattern recognition receptors (PRRs activation. However, the role of PRRs in neutrophils in both HIV-1-infected [HIV-1(+] and HIV-1-exposed seronegative individuals (HESN is unknown. Here, a study was carried out to evaluate the level of PRR mRNAs and cytokines produced after activation of neutrophils from HIV-1(+, HESN and healthy donors.The neutrophils were stimulated with specific agonists for TLR2, TLR4 and TLR9 in the presence of HIV-1 particles. Pro-inflammatory cytokine production, expression of neutrophil activation markers and reactive oxygen species (ROS production were analyzed in neutrophils from HESN, HIV-1(+ and healthy donors (controls.We found that neutrophils from HESN presented reduced expression of PRR mRNAs (TLR4, TLR9, NOD1, NOD2, NLRC4 and RIG-I and reduced expression of cytokine mRNAs (IL-1β, IL-6, IL-18, TNF-α and TGF-β. Moreover, neutrophils from HESN were less sensitive to stimulation through TLR4. Furthermore, neutrophils from HESN challenged with HIV-1 and stimulated with TLR2 and TLR4 agonists, produced significantly lower levels of reactive oxygen species, versus HIV-1(+.A differential pattern of PRR expression and release of innate immune factors in neutrophils from HESN is evident. Our results suggest that lower neutrophil activation can be involved in protection against HIV-1 infection.

  18. Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1.

    Science.gov (United States)

    Winther, Malene; Holdfeldt, André; Gabl, Michael; Wang, Ji Ming; Forsman, Huamei; Dahlgren, Claes

    2016-01-01

    Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits.

  19. Apoptosis is essential for neutrophil functional shutdown and determines tissue damage in experimental pneumococcal meningitis.

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    Uwe Koedel

    2009-05-01

    Full Text Available During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1beta and G-CSF as well as reduced levels of anti-inflammatory TGF-beta. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils.

  20. Ability of Staphylococcus aureus coagulase genotypes to resist neutrophil bactericidal activity and phagocytosis

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Scott, N. L.; Sordillo, L. M.

    1994-01-01

    genotype. The interaction between bacteria and neutrophils was measured by phagocytosis and bactericidal effect. The average percent killing of bacteria was lowest (40.0%) with strains belonging to the most common genotype, medium (50%) with strains belonging to the intermediate type, and highest (64......; rare type, 10.5/cell). These findings suggest that one of the reasons for the variation in prevalence of different genotypes of S. aureus in the mammary gland is due to the superior ability of some types to resist phagocytosis and/or killing by bovine neutrophils......This study investigated the functional capabilities of neutrophils against different Staphylococcus aureus genotypes isolated from cows with mastitis. Six strains of S. aureus were chosen for use in the study, two with a common genotype, two with an intermediate genotype, and two with a rare...

  1. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis.

    Science.gov (United States)

    Knäuper, V; Docherty, A J; Smith, B; Tschesche, H; Murphy, G

    1997-03-17

    Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P1' position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala239-Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247 --> Ala (P1 of the minor cleavage site Pro247-Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly242-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro247 sequence motif to Gly242-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro258 to Pro247-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme.

  2. Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

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    Olena Kamenyeva

    2015-04-01

    Full Text Available Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

  3. Outer membrane mutants of Salmonella typhimurium LT2 have lipopolysaccharide-dependent resistance to the bactericidal activity of anaerobic human neutrophils.

    Science.gov (United States)

    Okamura, N; Spitznagel, J K

    1982-06-01

    The capacity of neutrophil polymorphonuclear granulocytes (PMNs) to phagocytize bacteria under anaerobic as well as aerobic conditions afforded the opportunity to compare the bactericidal activities of oxygen-independent and oxygen-dependent antimicrobial mechanisms in human PMNs challenged with Salmonella typhimurium LT2 and its lipopolysaccharide mutants (outer membrane mutants). Anaerobic human PMNs challenged with either opsonized LT2 or serum-treated zymosan failed to produce detectable superoxide anion (O2-) or to reduce nitroblue tetrazolium, although aerobic PMNs readily produced O2- in response to such challenge. Anaerobic PMNs killed these bacteria in an ordered fashion that appeared to be dependent on their lipopolysaccharide chemotype. As the carbohydrate content of the mutant lipopolysaccharide decreased, the bacteria became less resistant to the oxygen-independent bactericidal activity. The results resembled the ordered resistance to oxygen-independent killing observed with LT2 and its mutants in PMN-free systems with PMN granule proteins. Studies on the kinetics of killing showed these to be less rapid in anaerobic as compared with aerobic conditions. Opsonization increased the rate of phagocytosis, but such factors as opsonization and the rate of phagocytosis did not appear to affect intraleukocytic bactericidal capacity in that the resultant proportion of bacteria remaining viable after ingestion was similar regardless of which serum was used (normal serum, C6-deficient serum, C8-deficient serum, or no serum at all). The results are consistent with an active and substantial participation by oxygen-independent systems in the antimicrobial effects of neutrophils.

  4. Complement-mediated neutrophil activation in sepsis- and trauma-related adult respiratory distress syndrome. Clarification with radioaerosol lung scans

    Energy Technology Data Exchange (ETDEWEB)

    Tennenberg, S.D.; Jacobs, M.P.; Solomkin, J.S.

    1987-01-01

    Complement-mediated neutrophil activation (CMNA) has been proposed as an important pathogenic mechanism causing acute microvascular lung injury in the adult respiratory distress syndrome (ARDS). To clarify the relationship between CMNA and evolving lung injury, we studied 26 patients with multiple trauma and sepsis within 24 hours of risk establishment for ARDS. Pulmonary alveolar-capillary permeability (PACP) was quantified as the clearance rate of a particulate radioaerosol. Seventeen patients (65%) had increased PACP (six developed ARDS) while nine (35%) had normal PACP (none developed ARDS; clearance rates of 3.4%/min and 1.5%/min, respectively). These patients, regardless of evidence of early lung injury, had elevated plasma C3adesArg levels and neutrophil chemotactic desensitization to C5a/C5adesArg. Plasma C3adesArg levels correlated weakly, but significantly, with PACP. Thus, CMNA may be a necessary, but not a sufficient, pathogenic mechanism in the evolution of ARDS.

  5. Ability of Staphylococcus aureus coagulase genotypes to resist neutrophil bactericidal activity and phagocytosis

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Scott, N. L.; Sordillo, L. M.

    1994-01-01

    This study investigated the functional capabilities of neutrophils against different Staphylococcus aureus genotypes isolated from cows with mastitis. Six strains of S. aureus were chosen for use in the study, two with a common genotype, two with an intermediate genotype, and two with a rare......; rare type, 10.5/cell). These findings suggest that one of the reasons for the variation in prevalence of different genotypes of S. aureus in the mammary gland is due to the superior ability of some types to resist phagocytosis and/or killing by bovine neutrophils...

  6. Pranlukast reduces neutrophil but not macrophage/microglial accumulation in brain after focal cerebral ischemia in mice

    Institute of Scientific and Technical Information of China (English)

    Li-sheng CHU; Er-qing WEI; Guo-liang YU; San-hua FANG; Yu ZHOU; Meng-ling WANG; Wei-ping ZHANG

    2006-01-01

    Aim:To determine whether pranlukast.a cysteinyl leukotriene receptor-1 antagonist,exerts an anti-inflammatory effect on focal cerebral ischemia in mice.Methods:Focal cerebral ischemia in mice was induced by permanent middle cerebral artery occlusion(MCAO).In addition to neurological deficits,infarct volume,degenerated neurons and endogenous IgG exudation,we detected accumulation of neutrophils and macrophage/microglia in the ischemic brain tissue 72 h after MCAO.Pranlukast was iP injected 30 min before and after MCAO.Results:Pranlukast significantly attenuated neurological deficits,infarct volume,neuron degeneration and IgG exudation.Importantly,pranlukast(0.01 and 0.1 mg/kg) inhibited myeloperoxidase-positive neutrophil,but not CDllb-positive macrophage/microglial accumulation in the ischemic cortical tissue.Conclusion:Pranlukast exerts an anti-inflammatory effect on focal cerebral ischemia in the subacute phase that is limited to neutrophil recruitment through the disrupted blood-brain barrier.

  7. Influence of gut microbiota-derived ellagitannins' metabolites urolithins on pro-inflammatory activities of human neutrophils.

    Science.gov (United States)

    Piwowarski, Jakub P; Granica, Sebastian; Kiss, Anna K

    2014-07-01

    Ellagitannin-rich products exhibit beneficial influence in the case of inflammation-associated diseases. Urolithins, metabolites of ellagitannins produced by gut microbiota, in contrary to high molecular weight hydrophilic parental polyphenols, possess well established bioavailability. Because of the important role of neutrophils in progression of inflammation, the influence of urolithins on their pro-inflammatory functions was tested. Urolithin B at a concentration of 20 µM showed significant inhibition of interleukin 8 and extracellular matrix-degrading enzyme MMP-9 production. It was also significantly active in prevention of cytochalasin A/formyl-met-leu-phenylalanine-triggered selectin CD62L shedding. Urolithin C was the only active compound towards inhibition of elastase release from cytochalasin A/formyl-met-leu-phenylalanine-stimulated neutrophils with 39.0 ± 15.9% inhibition at a concentration of 5 µM. Myeloperoxidase release was inhibited by urolithins A and C (at 20 µM by 46.7 ± 16.1 and 63.8 ± 8.6%, respectively). Urolithin A was the most potent reactive oxygen species release inhibitor both in formyl-met-leu-phenylalanine and 4β-phorbol-12β-myristate-R13-acetate-stimulated neutrophils. At the concentration of 1 µM, it caused reactive oxygen species level decrease by 42.6 ± 26.6 and 53.7 ± 16.0%, respectively. Urolithins can specifically modulate inflammatory functions of neutrophils, and thus could contribute to the beneficial health effects of ellagitannin-rich medicinal plant materials and food products.

  8. Neutrophil biology

    OpenAIRE

    Kobayashi, Yoshiro

    2015-01-01

    Neutrophil extracellular traps (NETs) are involved in bacterial killing as well as autoimmunity, because NETs contain proteases, bactericidal peptides, DNA and ribonucleoprotein. NETs are formed via a novel type of cell death called NETosis. NETosis is distinct from apoptosis, but it resembles necrosis in that both membranes are not intact so that they allow intracellular proteins to leak outside of the cells. Removal of NETs and neutrophils undergoing NETosis by phagocytes and its subsequent...

  9. Neutrophil-activating protein (HP-NAP) versus ferritin (Pfr): comparison of synthesis in Helicobacter pylori.

    Science.gov (United States)

    Dundon, W G; Polenghi, A; Del Guidice, G; Rappuoli, R; Montecucco, C

    2001-05-15

    We recently reported that the neutrophil-activating protein (HP-NAP) of Helicobacter pylori is capable of binding iron in vitro. To more fully understand the relationship between iron and HP-NAP the synthesis of HP-NAP was compared to that of Pfr, another iron-binding protein of H. pylori. Synthesis of HP-NAP and Pfr in growing cultures of H. pylori was analysed under iron depletion and iron, copper, nickel and zinc overload. The synthesis of HP-NAP and Pfr in H. pylori was also analysed under conditions of varying pH and oxidative stress. In addition, recombinant HP-NAP and Pfr were produced in Escherichia coli to assess the contribution of the two proteins to increased survival of E. coli under heavy metal overload. Our data reveal that both HP-NAP and Pfr accumulate in the stationary phase of growth. HP-NAP synthesis is not regulated by iron depletion or overload or by the presence of copper, nickel or zinc in liquid medium and it does not confer resistance to these metals when produced in E. coli. Except for an increase in the synthesis of Pfr at pH 5.7 neither the pH or oxidative stress conditions investigated had an affect on the synthesis of either protein. An increase in Pfr synthesis was observed under iron overload and a decrease was observed under conditions of copper, nickel and zinc overload confirming previous reports. Recombinant Pfr, as well as conferring resistance to iron and copper as previously reported, also conferred resistance to zinc overload when produced in E. coli.

  10. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

    Institute of Scientific and Technical Information of China (English)

    Bo Sun; Zhao-Shen Li; Zhen-Xing Tu; Guo-Ming Xu; Yi-Qi Du

    2006-01-01

    AIM: To construct a live attenuated Salmonella typhimurium (S.typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity.METHODS: By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments.RESULTS: A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recompinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylor(i) whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response.CONCLUSION: The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.

  11. Inhibition of platelet-activating factor- and zymosan-activated serum-induced chemotaxis of human neutrophils by nedocromil sodium, BN 52021 and sodium cromoglycate.

    Science.gov (United States)

    Bruijnzeel, P. L.; Warringa, R. A.; Kok, P. T.

    1989-01-01

    1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma since they infiltrate into the lung tissue. These cells are mobilized by lipid-like and protein-like chemotactic factors. As illustrative examples of both groups, platelet-activating-factor (Paf) and zymosan-activated-serum (ZAS) were used in this study. The inhibitory effects of nedocromil sodium, the Paf antagonist BN 52021 and sodium cromoglycate on Paf- and ZAS-induced neutrophil chemotaxis were evaluated. 2. All tested drugs inhibited Paf-induced neutrophil chemotaxis with approximately the same potency (IC50 approximately 1 nM). 3. Nedocromil sodium and sodium cromoglycate were equally potent in inhibiting ZAS-induced neutrophil chemotaxis (IC50 = 0.1-1 microM), whereas BN 52021 was considerably less potent (IC30 = 10 microM). 4. To find out whether the drugs tested could inhibit early events in cell activation, their capacity to inhibit Paf- and ZAS-induced cytosolic free Ca2+-mobilization was investigated. BN 52021, at a concentration of 100 microM, completely inhibited Paf-induced Ca2+-mobilization and inhibited ZAS-induced Ca2+-mobilization by about 50%. Nedocromil sodium and sodium cromoglycate were ineffective. PMID:2551444

  12. Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation

    NARCIS (Netherlands)

    Koffel, R.; Meshcheryakova, A.; Warszawska, J.; Hennig, A.; Wagner, K.; Jorgl, A.; Gubi, D.; Moser, D.; Hladik, A.; Hoffmann, U.; Fischer, M.B.; Berg, W.B. van den; Koenders, M.I.; Scheinecker, C.; Gesslbauer, B.; Knapp, S.; Strobl, H.

    2014-01-01

    During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell s

  13. Neutrophils are immuno-modulatory in rhinovirus infections

    NARCIS (Netherlands)

    Tang, Francesca; Hansbro, Philip; Burgess, Janette; Baines, Katherine; Oliver, Brian

    2015-01-01

    Background: Neutrophils are important in controlling bacterial infections however; their role in viral infections remains unclear. Previously, we found that neutrophils respond to viral mimetics but not replication competent rhinovirus (RV). Aim: To investigate if neutrophils are activated when expo

  14. Neutrophils are immuno-modulatory in rhinovirus infections

    NARCIS (Netherlands)

    Tang, Francesca; Hansbro, Philip; Burgess, Janette; Baines, Katherine; Oliver, Brian

    2015-01-01

    Background: Neutrophils are important in controlling bacterial infections however; their role in viral infections remains unclear. Previously, we found that neutrophils respond to viral mimetics but not replication competent rhinovirus (RV). Aim: To investigate if neutrophils are activated when

  15. Novel pathways for glucocorticoid effects on neutrophils in chronic inflammation.

    Science.gov (United States)

    Goulding, N J; Euzger, H S; Butt, S K; Perretti, M

    1998-10-01

    Neutrophils have been implicated in mediating much of the tissue damage associated with chronic inflammatory diseases such as rheumatoid arthritis, where they are involved in destruction of both cartilage and bone. Glucocorticoids are powerful anti-inflammatory agents, often used in the treatment of this autoimmune disease. They exert significant inhibitory effects on neutrophil activation and functions, such as chemotaxis, adhesion, transmigration, apoptosis, oxidative burst, and phagocytosis. The mechanisms by which glucocorticoids exert these effects on neutrophils are unclear. Evidence from studies of inflammation in human subjects and animal models suggests that annexin-I an endogenous, glucocorticoid-induced protein also known as lipocortin-1, has a pivotal role in modulating neutrophil activation, transmigratory, and phagocytic functions. Furthermore, we present evidence for altered neutrophil functions in rheumatoid arthritis that correspond to a significantly reduced capacity of these cells to bind annexin-I. A proposed novel pathway for glucocorticoid actions on neutrophils involving annexin-I could explain the development of chronic neutrophil activation in diseases such as rheumatoid arthritis.

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

    Directory of Open Access Journals (Sweden)

    Pablo Alarcón

    2017-08-01

    Full Text Available Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(− lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(− lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(− lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET production (NETosis in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(− lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(− lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1. d(− lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(− lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(− lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

  17. Functional neutrophils from human ES cells

    OpenAIRE

    Sweeney, Colin L; Malech, Harry L.

    2009-01-01

    In this issue of Blood, Yokoyama and colleagues demonstrate in vitro differentiation of hESCs into mature neutrophils with functional capabilities (chemotaxis, phagocytosis, microbicidal oxidase activity, and bacterial killing) approaching or equal to that of normal peripheral blood neutrophils.

  18. Polyphenol Content and Modulatory Activities of Some Tropical Dietary Plant Extracts on the Oxidant Activities of Neutrophils and Myeloperoxidase

    Directory of Open Access Journals (Sweden)

    Thierry Franck

    2012-01-01

    Full Text Available Young leaves of Manihot esculenta Crantz (Euphorbiaceae, Abelmoschus esculentus (Malvaceae, Hibiscus acetosella (Malvaceae and Pteridium aquilinum (Dennstaedtiaceae are currently consumed as green vegetables by peoples in sub-Saharan Africa, Latin America, Asia and their migrants living in Western Europe. Sub-Saharan peoples use Manihot, Abelmoschus and Hibiscus also in the folk medicine to alleviate fever and pain, in the treatment of conjunctivitis, rheumatism, hemorrhoid, abscesses, ... The present study investigates the effects of aqueous extracts of those plants on the production of reactive oxygen species (ROS and the release of myeloperoxidase (MPO by equine neutrophils activated with phorbol 12-myristate 13-acetate (PMA. The ROS production was measured by lucigenin-enhanced chemiluminescence (CL, and the release of total MPO by an ELISA method. The study also investigates the effect of the extracts on the activity of MPO by studying its nitration activity on tyrosine and by using a new technique called SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection that allows studying the direct interaction of compounds with the enzyme. In all experiments, the aqueous extracts of the plants developed concentration-dependent inhibitory effects. A moderate heat treatment did not significantly modify the inhibitory capacity of the extracts in comparison to not heated ones. Total polyphenol and flavonoid contents were determined with an HPLC-UV/DAD analysis and a spectroscopic method using Folin-Ciocalteu reagent. Some polyphenols with well-known antioxidant activities (caffeic acid, chlorogenic acid, hyperoside, rosmarinic acid and rutin were found in the extracts and may partly explain the inhibitory activities observed. The role of those dietary and medicinal plants in the treatment of ROS-dependent inflammatory diseases could have new considerations for health.

  19. Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab.

    Science.gov (United States)

    Golay, Josée; Da Roit, Fabio; Bologna, Luca; Ferrara, Claudia; Leusen, Jeanette H; Rambaldi, Alessandro; Klein, Christian; Introna, Martino

    2013-11-14

    Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcγR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab')2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B.

  20. The Level of Oxidative Neutrophil Response When Determining Endotoxin Activity Assay: A New Biomarker for Defining the Indications and Effectiveness of Intensive Care in Patients with Sepsis

    Directory of Open Access Journals (Sweden)

    Michael Yaroustovsky

    2017-01-01

    Full Text Available Background. To analyse the clinical informativity of the neutrophil oxidative response level (“Response” during an Endotoxin Activity Assay (EAA as a new biomarker defining the indications and effectiveness of intensive care in cardiac surgical patients with septic complications. Methods. Blood samples were taken from 198 adult patients who were admitted to the ICU after cardiac surgery (SIRS: 34, MODS: 36, and sepsis: 128. The composite of laboratory studies included CRP, PCT, EAA with “Response” level, and presepsin. Results. 83% of patients had a “normal” neutrophil response, 12% of patients had a low neutrophil response, and 5% of patients had a critically low neutrophil response. Patients with critically low responses had the lowest values of the EAA and the highest concentrations of PSP and D-dimer (p 0.5 has a negative predictive value; the EAA < 0.6 at “Response” < 0.5 may indicate a high level of endotoxaemia.

  1. Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-κB pathway and mitochondrial stability.

    Science.gov (United States)

    Barcellos-de-Souza, Pedro; Canetti, Cláudio; Barja-Fidalgo, Christina; Arruda, Maria Augusta

    2012-10-01

    Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. The mDial formin is required for neutrophil polarization, migration, and activation of the LARG/RhoA/ROCK signaling axis during chemotaxis.

    Science.gov (United States)

    Shi, Yongquan; Zhang, Jinyi; Mullin, Michael; Dong, Baoxia; Alberts, Arthur S; Siminovitch, Katherine A

    2009-03-15

    Neutrophil chemotaxis depends on actin dynamics, but the roles for specific cytoskeleton regulators in this response remain unclear. By analysis of mammalian diaphanous-related formin 1 (mDia1)-deficient mice, we have identified an essential role for this actin nucleator in neutrophil chemotaxis. Lack of mDia1 was associated with defects in chemoattractant-induced neutrophil actin polymerization, polarization, and directional migration, and also with impaired activation of RhoA, its downstream target p160-Rho-associated coil-containing protein kinase (ROCK), and the leukemia-associated RhoA guanine nucleotide exchange factor (LARG). Our data also revealed mDia1 to be associated with another cytoskeletal regulator, Wiskott-Aldrich syndrome protein (WASp), at the leading edge of chemotaxing neutrophils and revealed polarized morphology and chemotaxis to be more mildly impaired in WAS(-/-) than in mDia1(-/-) neutrophils, but essentially abrogated by combined mDia1/WASp deficiency. Thus, mDia1 roles in neutrophil chemotaxis appear to be subserved in concert with WASp and are realized at least in part by activation of the LARG/RhoA/ROCK signaling pathway.

  3. MPLA inhibits release of cytotoxic mediators from human neutrophils while preserving efficient bacterial killing.

    Science.gov (United States)

    Ruchaud-Sparagano, Marie-Hélène; Mills, Ross; Scott, Jonathan; Simpson, A John

    2014-10-01

    Monophosphoryl lipid A (MPLA) is a lipopolysaccharides (LPS) derivative associated with neutrophil-dependent anti-inflammatory outcomes in animal models of sepsis. Little is known about the effect of MPLA on neutrophil function. This study sought to test the hypothesis that MPLA would reduce release of cytotoxic mediators from neutrophils without impairing bacterial clearance. Neutrophils were isolated from whole blood of healthy volunteers. The effects of MPLA and LPS on autologous serum-opsonised Pseudomonas aeruginosa killing by neutrophils and phagocytosis of autologous serum-opsonised zymosan were examined. Neutrophil oxidative burst, chemotaxis, enzyme and cytokine release as well as Toll-like receptor 4 (TLR4) expression were assessed following exposure to LPS or MPLA. LPS, but not MPLA, induced significant release of superoxide and myeloperoxidase from neutrophils. However, MPLA did not impair neutrophil capacity to ingest microbial particles and kill P. aeruginosa efficiently. MPLA was directly chemotactic for neutrophils, involving TLR4, p38 mitogen-activated protein kinase and tyrosine and alkaline phosphatases. LPS, but not MPLA, impaired N-formyl-methionyl-leucyl phenylalanine-directed migration of neutrophils, increased surface expression of TLR4, increased interleukin-8 release and strongly activated the myeloid differentiation primary response 88 pathway. Phosphoinositide 3-kinase inhibition significantly augmented IL-8 release from MPLA-treated neutrophils. The addition of MPLA to LPS-preincubated neutrophils led to a significant reduction in LPS-mediated superoxide release and TLR4 surface expression. Collectively, these findings suggest that MPLA directs efficient chemotaxis and bacterial killing in human neutrophils without inducing extracellular release of cytotoxic mediators and suggest that MPLA warrants further attention as a potential therapeutic in human sepsis.

  4. The transcriptional activation program of human neutrophils in skin lesions supports their important role in wound healing

    DEFF Research Database (Denmark)

    Theilgaard-Monch, K.; Knudsen, Steen; Follin, P.

    2004-01-01

    receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing...... and function, and promotes wound healing.......To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin...

  5. Anti-Pseudomonas aeruginosa IgY antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils

    DEFF Research Database (Denmark)

    Thomsen, Kim; Christophersen, Lars; Jensen, Peter Østrup

    2016-01-01

    Moderation of polymorphonuclear neutrophils (PMNs) as part of a critical defense against invading pathogens may offer a promising therapeutic approach to supplement the antibiotic eradication of Pseudomonas aeruginosa infection in non-chronically infected cystic fibrosis (CF) patients. We have...... observed that egg yolk antibodies (IgY) harvested from White leghorn chickens that target P. aeruginosa opsonize the pathogen and enhance the PMN-mediated respiratory burst and subsequent bacterial killing in vitro. The effects on PMN phagocytic activity were observed in different Pseudomonas aeruginosa...

  6. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  7. Neutrophil function and metabolism in individuals with diabetes mellitus

    Directory of Open Access Journals (Sweden)

    T.C. Alba-Loureiro

    2007-08-01

    Full Text Available Neutrophils act as first-line-of-defense cells and the reduction of their functional activity contributes to the high susceptibilityto and severity of infections in diabetes mellitus. Clinical investigations in diabetic patients and experimental studies in diabetic rats and mice clearly demonstrated consistent defects of neutrophil chemotactic, phagocytic and microbicidal activities. Other alterations that have been reported to occur during inflammation in diabetes mellitus include: decreased microvascular responses to inflammatory mediators such as histamine and bradykinin, reduced protein leakage and edema formation, reduced mast cell degranulation, impairment of neutrophil adhesionto the endothelium and migration to the site of inflammation, production of reactive oxygen species and reduced release of cytokines and prostaglandin by neutrophils, increased leukocyte apoptosis, and reduction in lymph node retention capacity. Since neutrophil function requires energy, metabolic changes (i.e., glycolytic and glutaminolytic pathways may be involved in the reduction of neutrophil function observed in diabetic states. Metabolic routes by which hyperglycemia is linked to neutrophil dysfunction include the advanced protein glycosylation reaction, the polyol pathway, oxygen-free radical formation, the nitric oxide-cyclic guanosine-3'-5'monophosphate pathway, and the glycolytic and glutaminolytic pathways. Lowering of blood glucose levels by insulin treatment of diabetic patients or experimental animals has been reported to have significant correlation with improvement of neutrophil functional activity. Therefore, changes might be primarily linked to a continuing insulin deficiency or to secondary hyperglycemia occurring in the diabetic individual. Accordingly, effective control with insulin treatment is likely to be relevant during infection in diabetic patients.

  8. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    Science.gov (United States)

    Simon, R H; DeHart, P D; Todd, R F

    1986-11-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing.

  9. Oral antibiotics increase blood neutrophil maturation and reduce bacteremia and necrotizing enterocolitis in the immediate postnatal period of preterm pigs

    DEFF Research Database (Denmark)

    Nguyen, Duc Ninh; Fuglsang, Eva; Jiang, Pingping

    2016-01-01

    Immature immunity may predispose preterm neonates to infections and necrotizing enterocolitis (NEC). Intravenous antibiotics are frequently given to prevent and treat sepsis, while oral antibiotics are seldom used. We hypothesized that oral antibiotics promote maturation of systemic immunity...... and delay gut bacterial colonization and thereby protect preterm neonates against both NEC and bacteremia in the immediate postnatal period. Preterm pigs were given formula and administered saline (CON) or broad-spectrum antibiotics orally (ORA) or systemically (SYS) for 5 d after birth. Temporal changes......, high gut permeability, Gram-positive bacteremia and NEC. Neonatal oral antibiotics may benefit mucosal and systemic immunity via delayed gut colonization and enhanced blood neutrophil maturation just after preterm birth....

  10. The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 degrees C

    DEFF Research Database (Denmark)

    Brügger, Kim; Chen, Lanming; Stark, Markus

    2007-01-01

    Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up...... to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated...... clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated...

  11. Response of neutrophils to stimulus infusion: differential sensitivity of cytoskeletal activation and oxidant production

    OpenAIRE

    1988-01-01

    The response of human neutrophils to N-formyl peptides were studied under conditions where ligand binding was controlled by infusing a cell suspension with the peptide over a time period comparable to the normal half-time for binding. Receptor occupancy was measured in real time with a fluorescently labeled peptide using flow cytometry. This binding was approximated by a simple reversible model using typical on (7 X 10(8) M- min-1) and off (0.35/min) rate constants and the infusion rates (0.0...

  12. Neutrophil function in healthy aged horses and horses with pituitary dysfunction.

    Science.gov (United States)

    McFarlane, Dianne; Hill, Kim; Anton, Jason

    2015-06-15

    Immunosuppression leading to opportunist bacterial infection is a well-recognized sequela of equine pituitary pars intermedia dysfunction (PPID). The mechanisms responsible for immune dysfunction in PPID however, are as of yet poorly characterized. Horses with PPID have high concentrations of hormones known to impact immune function including α-melanocyte stimulating hormone (α-MSH) and insulin. α-MSH and related melanocortins have been shown in rodents and people to impair neutrophil function by decreasing superoxide production (known as oxidative burst activity), migration and adhesion. The goal of this study was to determine if neutrophil function is impaired in horses with PPID and, if so, to determine if plasma α-MSH or insulin concentration correlated with the severity of neutrophil dysfunction. Specifically, neutrophil phagocytosis, oxidative burst activity, chemotaxis and adhesion were assessed. Results of this study indicate that horses with PPID have reduced neutrophil function, characterized by decreased oxidative burst activity and adhesion. In addition, chemotaxis was greater in healthy aged horses than in young horses or aged horses with PPID. Plasma insulin: α-MSH ratio, but not individual hormone concentration was correlated to neutrophil oxidative burst activity. In summary, neutrophil function is impaired in horses with PPID, likely due to altered hormone concentrations and may contribute to increased risk of opportunistic infections. Whether regulation of hormone concentration profiles in horses with PPID using therapeutic intervention improves neutrophil function and reduces infections needs to be explored.

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

    Science.gov (United States)

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

    2011-07-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  15. Neutrophil Migration in the Activation of the Innate Immune Response to Different Flavobacterium psychrophilum Vaccines in Zebrafish (Danio rerio

    Directory of Open Access Journals (Sweden)

    Camila J. Solís

    2015-01-01

    Full Text Available Flavobacterium psychrophilum is a Gram-negative bacterium, responsible for the bacterial cold-water disease and the rainbow trout fry syndrome in freshwater salmonid fish. At present, there is only one commercial vaccine in Chile, made with two Chilean F. psychrophilum isolates and another licensed in Europe. The present study analyzed neutrophil migration, as a marker of innate immune activation, in zebrafish (Danio rerio in response to different F. psychrophilum bath vaccines, which is the first step in evaluating vaccine effectiveness and efficiency in fish. Results indicated that bacterins of the LM-02-Fp isolate were more immunogenic than those from the LM-13-Fp isolate. However, no differences were observed between the same bacteria inactivated by either formaldehyde or heat. Importantly, the same vaccine formulation without an adjuvant only triggered a mild neutrophil migration compared to the complete vaccine. Observations also found that, after a year of storage at 4°C, the activation of the innate immune system by the different vaccines was considerably decreased. Finally, new vaccine formulations prepared with heat and formaldehyde inactivated LM-02-Fp were significantly more efficient than the available commercial vaccine in regard to stimulating the innate immune system.

  16. Cigarette smoke-induced damage-associated molecular pattern release from necrotic neutrophils triggers proinflammatory mediator release.

    Science.gov (United States)

    Heijink, Irene H; Pouwels, Simon D; Leijendekker, Carin; de Bruin, Harold G; Zijlstra, G Jan; van der Vaart, Hester; ten Hacken, Nick H T; van Oosterhout, Antoon J M; Nawijn, Martijn C; van der Toorn, Marco

    2015-05-01

    Cigarette smoking, the major causative factor for the development of chronic obstructive pulmonary disease, is associated with neutrophilic airway inflammation. Cigarette smoke (CS) exposure can induce a switch from apoptotic to necrotic cell death in airway epithelium. Therefore, we hypothesized that CS promotes neutrophil necrosis with subsequent release of damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), alarming the innate immune system. We studied the effect of smoking two cigarettes on sputum neutrophils in healthy individuals and of 5-day CS or air exposure on neutrophil counts, myeloperoxidase, and HMGB1 levels in bronchoalveolar lavage fluid of BALB/c mice. In human peripheral blood neutrophils, mitochondrial membrane potential, apoptosis/necrosis markers, caspase activity, and DAMP release were studied after CS exposure. Finally, we assessed the effect of neutrophil-derived supernatants on the release of chemoattractant CXCL8 in normal human bronchial epithelial cells. Cigarette smoking caused a significant decrease in sputum neutrophil numbers after 3 hours. In mice, neutrophil counts were significantly increased 16 hours after repeated CS exposure but reduced 2 hours after an additional exposure. In vitro, CS induced necrotic neutrophil cell death, as indicated by mitochondrial dysfunction, inhibition of apoptosis, and DAMP release. Supernatants from CS-treated neutrophils significantly increased the release of CXCL8 in normal human bronchial epithelial cells. Together, these observations show, for the first time, that CS exposure induces neutrophil necrosis, leading to DAMP release, which may amplify CS-induced airway inflammation by promoting airway epithelial proinflammatory responses.

  17. Impaired bactericidal but not fungicidal activity of polymorphonuclear neutrophils in patients with chronic lymphocytic leukemia.

    Science.gov (United States)

    Kontoyiannis, Dimitrios P; Georgiadou, Sarah P; Wierda, William G; Wright, Susan; Albert, Nathaniel D; Ferrajoli, Alessandra; Keating, Michael; Lewis, Russell E

    2013-08-01

    We examined the qualitative polymorphonuclear neutrophil (PMN)-associated immune impairment in patients with chronic lymphocytic leukemia (CLL) by characterizing phagocytic killing of key non-opsonized bacterial (Staphylococcus aureus and Pseudomonas aeruginosa) and fungal (Candida albicans and Aspergillus fumigatus) pathogens. Neutrophils were collected from 47 non-neutropenic patients with CLL (PMN count > 1000/mm(3)) and age-matched and young healthy controls (five each). A subset of patients (13%) had prior or subsequent infections. We found that the patients with CLL had diminished PMN microbicidal response against bacteria but not against fungi compared with the controls. Compared to patients with effective PMN responses, we did not identify differences of basal PMN pathogen-associated molecular pattern receptor gene expression, soluble pathogen-associated molecular pattern gene expression or inflammatory cytokine signatures in patients with impaired PMN responses when PMNs were analyzed in multiplex real-time polymerase chain reaction assays. However, differences in PMN microbicidal response against A. fumigatus in patients with CLL were associated with the degree of hypogammaglobulinemia.

  18. Fcγ and Complement Receptors and Complement Proteins in Neutrophil Activation in Rheumatoid Arthritis: Contribution to Pathogenesis and Progression and Modulation by Natural Products

    Directory of Open Access Journals (Sweden)

    Adriana Balbina Paoliello-Paschoalato

    2015-01-01

    Full Text Available Rheumatoid arthritis (RA is a highly disabling disease that affects all structures of the joint and significantly impacts on morbidity and mortality in RA patients. RA is characterized by persistent inflammation of the synovial membrane lining the joint associated with infiltration of immune cells. Eighty to 90% of the leukocytes infiltrating the synovia are neutrophils. The specific role that neutrophils play in the onset of RA is not clear, but recent studies have evidenced that they have an important participation in joint damage and disease progression through the release of proteolytic enzymes, reactive oxygen species (ROS, cytokines, and neutrophil extracellular traps, in particular during frustrated phagocytosis of immune complexes (ICs. In addition, the local and systemic activation of the complement system contributes to the pathogenesis of RA and other IC-mediated diseases. This review discusses (i the participation of Fcγ and complement receptors in mediating the effector functions of neutrophils in RA; (ii the contribution of the complement system and ROS-dependent and ROS-independent mechanisms to joint damage in RA; and (iii the use of plant extracts, dietary compounds, and isolated natural compounds in the treatment of RA, focusing on modulation of the effector functions of neutrophils and the complement system activity and/or activation.

  19. AMP-Activated Protein Kinase α2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1α and a Network of Proteins Affecting Metabolism and Apoptosis

    Science.gov (United States)

    Abdel Malik, Randa; Zippel, Nina; Frömel, Timo; Heidler, Juliana; Zukunft, Sven; Walzog, Barbara; Ansari, Nariman; Pampaloni, Francesco; Wingert, Susanne; Rieger, Michael A.; Wittig, Ilka; Fisslthaler, Beate

    2017-01-01

    Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. Objective: To determine the role of the AMPKα2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2−/− versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice. Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia. PMID:27777247

  20. Structures and Metal-Binding Properties of Helicobacter pylori Neutrophil-Activating Protein with a Di-Nuclear Ferroxidase Center

    Directory of Open Access Journals (Sweden)

    Hideshi Yokoyama

    2014-06-01

    Full Text Available Helicobacter pylori causes severe diseases, such as chronic gastritis, peptic ulcers, and stomach cancers. H. pylori neutrophil-activating protein (HP-NAP is an iron storage protein that forms a dodecameric shell, promotes the adhesion of neutrophils to endothelial cells, and induces the production of reactive oxygen radicals. HP-NAP belongs to the DNA-protecting proteins under starved conditions (Dps family, which has significant structural similarities to the dodecameric ferritin family. The crystal structures of the apo form and metal-ion bound forms, such as iron, zinc, and cadmium, of HP-NAP have been determined. This review focused on the structures and metal-binding properties of HP-NAP. These metal ions bind at the di-nuclear ferroxidase center (FOC by different coordinating patterns. In comparison with the apo structure, metal loading causes a series of conformational changes in conserved residues among HP-NAP and Dps proteins (Trp26, Asp52, and Glu56 at the FOC. HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of pores. Metal ions have been identified around one of the pores; therefore, the negatively-charged pore is suitable for the passage of metal ions.

  1. Human Neutrophils Kill Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  2. Human neutrophils kill Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Anne Mayer-Scholl

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  3. Human neutrophils kill Bacillus anthracis.

    Science.gov (United States)

    Mayer-Scholl, Anne; Hurwitz, Robert; Brinkmann, Volker; Schmid, Monika; Jungblut, Peter; Weinrauch, Yvette; Zychlinsky, Arturo

    2005-11-01

    Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  4. Structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a 3 formylmethionyl group

    DEFF Research Database (Denmark)

    Forsman, Huamei; Winther, Malene; Gabl, Michael

    2015-01-01

    Pathogenic Staphylococcus aureus strains produce N-formylmethionyl containing peptides, of which the tetrapeptide fMIFL is a potent activator of the neutrophil formyl peptide receptor 1 (FPR1) and the PSMα2 peptide is a potent activator of the closely related FPR2. Variants derived from these two...

  5. Activation of toll-like receptor 4 is necessary for trauma hemorrhagic shock-induced gut injury and polymorphonuclear neutrophil priming.

    Science.gov (United States)

    Reino, Diego C; Palange, David; Feketeova, Elenora; Bonitz, Robert P; Xu, Da Zhong; Lu, Qi; Sheth, Sharvil U; Peña, Geber; Ulloa, Luis; De Maio, Antonio; Feinman, Rena; Deitch, Edwin A

    2012-07-01

    Interactions of toll-like receptors (TLRs) with nonmicrobial factors play a major role in the pathogenesis of early trauma-hemorrhagic shock (T/HS)-induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4 mut) mice would be more resistant to T/HS-induced gut injury and polymorphonuclear neutrophil (PMN) priming than their wild-type littermates and found that both were significantly reduced in the TLR4 mut mice. In addition, the in vivo and ex vivo PMN priming effect of T/HS intestinal lymph observed in the wild-type mice was abrogated in TLR4 mut mice as well the TRIF mut-deficient mice and partially attenuated in Myd88 mice, suggesting that TRIF activation played a more predominant role than MyD88 in T/HS lymph-induced PMN priming. Polymorphonuclear neutrophil depletion studies showed that T/HS lymph-induced acute lung injury was PMN dependent, because lung injury was totally abrogated in PMN-depleted animals. Because the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of T/HS lymph was related to endogenous nonmicrobial TLR4 ligands. High-mobility group box 1 protein 1, heat shock protein 70, heat shock protein 27, and hyaluronic acid all have been implicated in ischemia-reperfusion-induced tissue injury. None of these "danger" proteins appeared to be involved, because their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in T/HS-induced gut injury and in T/HS lymph-induced PMN priming and lung injury. However, the T/HS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the T/HS lymph-associated effects of TLR4 on PMN priming.

  6. Mixed species biofilms of Fusobacterium necrophorum and Porphyromonas levii impair the oxidative response of bovine neutrophils in vitro.

    Science.gov (United States)

    Lockhart, Joey S; Buret, Andre G; Ceri, Howard; Storey, Douglas G; Anderson, Stefanie J; Morck, Douglas W

    2017-10-01

    Biofilms composed of anaerobic bacteria can result in persistent infections and chronic inflammation. Host immune cells have difficulties clearing biofilm-related infections and this can result in tissue damage. Neutrophils are a vital component of the innate immune system and help clear biofilms. The comparative neutrophilic response to biofilms versus planktonic bacteria remains incompletely understood, particularly in the context of mixed infections. The objective of this study was to generate mixed species anaerobic bacterial biofilms composed of two opportunistic pathogens, Fusobacterium necrophorum and Porphyromonas levii, and evaluate neutrophil responses to extracellular fractions from both biofilms and planktonic cell co-cultures of the same bacteria. Purified bovine neutrophils exposed to culture supernatants from mixed species planktonic bacteria showed elevated oxidative activity compared to neutrophils exposed to biofilms composed of the same bacteria. Bacterial lipopolysaccharide plays a significant role in the stimulation of neutrophils; biofilms produced substantially more lipopolysaccharide than planktonic bacteria under these experimental conditions. Removal of lipopolysaccharide significantly reduced neutrophil oxidative response to culture supernatants of planktonic bacteria. Oxidative responses to LPS-removed biofilm supernatants and LPS-removed planktonic cell supernatants were similar. The limited neutrophil response to biofilm bacteria observed in this study supports the reduced ability of the innate immune system to eradicate biofilm-associated infections. Lipopolysaccharide is likely important in neutrophil response; however, the presence of other extracellular, immune modifying molecules in the bacterial media also appears to be important in altering neutrophil function. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. MET is required for the recruitment of anti-tumoural neutrophils.

    Science.gov (United States)

    Finisguerra, Veronica; Di Conza, Giusy; Di Matteo, Mario; Serneels, Jens; Costa, Sandra; Thompson, A A Roger; Wauters, Els; Walmsley, Sarah; Prenen, Hans; Granot, Zvi; Casazza, Andrea; Mazzone, Massimiliano

    2015-06-18

    Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

  8. In vitro killing of oral Capnocytophaga by granule fractions of human neutrophils is associated with cathepsin G activity.

    OpenAIRE

    Miyasaki, K T; Bodeau, A L

    1991-01-01

    The Capnocytophaga are inhabitants of the hypoxic human gingival crevice that are normally prevented by neutrophils from causing periodontal and systemic infection. To identify potential nonoxidative bactericidal mechanisms against Capnocytophaga within human neutrophils, gel filtration chromatography was used to fractionate neutrophil granule extracts. Seven granule fractions, designated A through G, were obtained. The Capnocytophaga were most sensitive to killing by fraction D. Fraction D e...

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

    Science.gov (United States)

    2006-04-21

    vitro interaction of human neutrophils with MARV and EBOV. We report that although productive filovirus replication was not observed in human neutrophils...supernatants. Where alphaviruses such as Venezuelan equine encephalitis virus (VEEV) were used as nonfilovirus controls, the viruses to be inactivated...viral replication in human neutrophils exposed to live MARV or EBOV (MOI 1), as measured by plaque assay of supernatants 1, 6, 24, or 48 h

  10. Enhancement of neutrophil autophagy by an IVIG preparation against multidrug-resistant bacteria as well as drug-sensitive strains.

    Science.gov (United States)

    Itoh, Hiroshi; Matsuo, Hidemasa; Kitamura, Naoko; Yamamoto, Sho; Higuchi, Takeshi; Takematsu, Hiromu; Kamikubo, Yasuhiko; Kondo, Tadakazu; Yamashita, Kouhei; Sasada, Masataka; Takaori-Kondo, Akifumi; Adachi, Souichi

    2015-07-01

    Autophagy occurs in human neutrophils after the phagocytosis of multidrug-resistant bacteria and drug-sensitive strains, including Escherichia coli and Pseudomonas aeruginosa. The present study detected autophagy by immunoblot analysis of LC3B conversion, by confocal scanning microscopic examination of LC3B aggregate formation and by transmission electron microscopic examination of bacteria-containing autophagosomes. Patients with severe bacterial infections are often treated with IVIG alongside antimicrobial agents. Here, we showed that IVIG induced neutrophil-mediated phagocytosis of multidrug-resistant strains. Compared with untreated neutrophils, neutrophils exposed to IVIG showed increased levels of bacterial cell killing, phagocytosis, O(2)(-) release, MPO release, and NET formation. IVIG also increased autophagy in these cells. Inhibiting the late phase of autophagy (fusion of lysosomes with autophagosomes) with bafilomycin A1-reduced, neutrophil-mediated bactericidal activity. These findings indicate that autophagy plays a critical role in the bactericidal activity mediated by human neutrophils. Furthermore, the autophagosomes within the neutrophils contained bacteria only and their organelles only, or both bacteria and their organelles, a previously undocumented observation. Taken together, these results suggest that the contents of neutrophil autophagosomes may be derived from specific autophagic systems, which provide the neutrophil with an advantage. Thus, IVIG promotes the neutrophil-mediated killing of multidrug-resistant bacteria as well as drug-sensitive strains.

  11. Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Bjoern F Kraemer

    2011-11-01

    Full Text Available Human β-defensins (hBD are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus, forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET formation by target polymorphonuclear leukocytes (PMNs, which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.

  12. The transcriptional activation program of human neutrophils in skin lesions supports their important role in wound healing

    DEFF Research Database (Denmark)

    Theilgaard-Monch, K.; Knudsen, Steen; Follin, P.

    2004-01-01

    receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing......To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin...... lesions. After migration to skin lesions, PMNs demonstrated a significant transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming...

  13. Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Min Long; Jun Luo; Yan Li; Fang-Yin Zeng; Ming Li

    2009-01-01

    AIM: To detect and evaluate the antibodies against Helicobacter pylori ( H pylori) neutrophil-activating protein (HP-NAP) in patients with gastric cancer and other gastroduodenal diseases.METHODS: Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coli. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 (IL-8) and growth-related oncogene (GROα) cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected. RESULTS: The serum positivity and mean absorbance value of HP-NAP-specific antibodies in the gastric cancer group (97.7% and 1.01 ± 0.24) were significantly higher than those in the chronic gastritis group (85.7% and 0.89 ± 0.14, P < 0.005) and healthy control group (27.7% and 0.65 ± 0.18, P < 0.001). The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly upregulate IL-8 production in gastric epithelial cell lines but had no effect on GROα production. CONCLUSION: Infection with virulent H pylori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma. rHP-NAPbased ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.

  14. Hawthorn extract inhibits human isolated neutrophil functions.

    Science.gov (United States)

    Dalli, Ernesto; Milara, Javier; Cortijo, Julio; Morcillo, Esteban J; Cosín-Sales, Juan; Sotillo, José Francisco

    2008-06-01

    Hawthorn extract is a popular herbal medicine given as adjunctive treatment for chronic heart failure. In contrast to the cardiac properties of hawthorn extract, its anti-inflammatory effect has been scarcely investigated. This study examines the effects of a dry extract of leaves and flowers of Crataegus laevigata on various functional outputs of human neutrophils in vitro. Incubation of human neutrophils obtained from peripheral blood of healthy donors with C. laevigata extract (0.75-250 microg/ml) inhibited N-formyl-Met-Leu-Phe (FMLP)-induced superoxide anion generation, elastase release and chemotactic migration with potency values of 43.6, 21.9, and 31.6 microg/ml, respectively. By contrast, serum-opsonized zymosan-induced phagocytosis was unaltered by plant extract. C. laevigata extract (125 microg/ml) reduced FMLP-induced leukotriene B(4) production and lipopolysaccharide-induced generation of tumour necrosis factor-alpha and interleukin-8. Extract inhibited FMLP-induced intracellular calcium signal with potency of 17.4 microg/ml. Extract also markedly inhibited the extracellular calcium entry into calcium-depleted neutrophils, and the thapsigargin-induced intracellular calcium response. In conclusion, C. laevigata extract inhibited various functional outputs of activated human neutrophils which may be relevant to the pathophysiology of cardiac failure.

  15. Are urinary levels of high mobility group box 1 markers of active nephritis in anti-neutrophil cytoplasmic antibody-associated vasculitis?

    NARCIS (Netherlands)

    de Souza, A. W. S.; Abdulahad, W. H.; Sosicka, P.; Bijzet, J.; Limburg, P. C.; Stegeman, C. A.; Bijl, M.; Westra, J.; Kallenberg, C. G. M.

    2014-01-01

    The objective of this study is to evaluate urinary high mobility group box 1 (HMGB1) levels as markers for active nephritis in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in comparison with urinary CD4(+) effector memory T cells and urinary monocyte chemoatt

  16. Distinct Kinetics Features of LFA-1 and Mac-1 in Neutrophil Activation

    Institute of Scientific and Technical Information of China (English)

    LI Ning; MAO De-bin; GONG Yi-xin; LV Shou-qin; ZHANG Yan; LONG Mian

    2015-01-01

    LFA-1 and Mac-1, two β2 integrin members constitutively expressed on neutrophils, mediate leukocyte recruitment cascade by binding to the same ligand of ICAM-1The slow rolling and firm adhesion of leukocytes rely on LFA-1 while the cell crawling is dependent on Mac-1We hypothesized that their distinct roles are likely attributed to the differences in the binding kinetics or in the diverse responses of outside-in and inside-out signalingIn this study, we compared the ICAM-1 binding features between soluble or membrane-expressed LFA-1 and Mac-1 with different affinity conformation using optical trap techniqueOur data indicated that the affinity up-regulation from wide type (WT) to high affinity (HA) is off-rate dependent for LFA-1 but on-rate dependent for Mac-1The structural bases of this new finding were found to be consistent with our previous simulationsThese results furthered our understanding in their function differences under shear flow.

  17. Differential neutrophil activation in viral infections: Enhanced TLR-7/8-mediated CXCL8 release in asthma

    NARCIS (Netherlands)

    Tang, Francesca S.M.; Van Ly, David; Spann, Kirsten; Reading, Patrick C.; Burgess, Janette K.; Hartl, Dominik; Baines, Katherine J.; Oliver, Brian G.

    2016-01-01

    Background and objective Respiratory viral infections are a major cause of asthma exacerbations. Neutrophils accumulate in the airways and the mechanisms that link neutrophilic inflammation, viral infections and exacerbations are unclear. This study aims to investigate anti-viral responses in neutro

  18. Serum IL8 and mRNA level of CD11b in circulating neutrophils are increased in clinically amyopathic dermatomyositis with active interstitial lung disease.

    Science.gov (United States)

    Zou, Jing; Chen, Jie; Yan, Qingran; Guo, Qiang; Bao, Chunde

    2016-01-01

    The objective of this study is to assess serum IL8 and the potential activity of circulating neutrophils on relative messenger RNA (mRNA) levels and their relationship with disease activity in clinically amyopathic dermatomyositis (CADM) associated with interstitial lung disease (ILD). We studied 18 CADM patients and compared them with 18 classic dermatomyositis (DM) patients and 18 healthy control subjects. Serum IL8 level and mRNA expressions of neutrophils (chemokine (C-X-C motif) receptor 1 (CXCR1), cluster of differentiation molecule 11b (CD11b), cluster of differentiation 64 (CD64), myeloid cell leukemia 1 (MCL1), interleukin-18 (IL18)) were detected. The overproduction of serum IL8 level was most significant in the CADM group with active period. The mRNA expressions of CD11b, IL18, and MCL1 were greatly increased in the neutrophils in patients with CADM compared with DM or healthy controls. Up-expressions of CD11b, IL18, and MCL1 were detected in the neutrophils in CADM patients of active period compared with remission period. A positive correlation was found between CD11b mRNA level and high-resolution computed tomography (HRCT) score, in CADM associated with ILD. Serum IL8 level and mRNA levels of CD11b, MCL1, and IL18 in circulating neutrophils are related with the disease activity of CADM-ILD. The mRNA level of CD11b is positively correlated with HRCT score in CADM-ILD.

  19. 3,4-methylenedioxymethamphetamine (MDMA--Ecstasy) decreases neutrophil activity through the glucocorticoid pathway and impairs host resistance to Listeria monocytogenes infection in mice.

    Science.gov (United States)

    Ferraz-de-Paula, V; Ribeiro, A; Souza-Queiroz, J; Pinheiro, M L; Vecina, J F; Souza, D P M; Quinteiro-Filho, W M; Moreau, R L M; Queiroz, M L S; Palermo-Neto, J

    2014-12-01

    Ecstasy is the popular name of the abuse drug 3,4-methylenedioxymethamphetamine (MDMA) that decreases immunity in animals. The mechanisms that generate such alterations are still controversial. Seven independent pharmacological approaches were performed in mice to identify the possible mechanisms underlying the decrease of neutrophil activity induced by MDMA and the possible effects of MDMA on host resistance to Listeria monocytogenes. Our data showed that MDMA (10 mg kg(-1)) administration decreases NFκB expression in circulating neutrophils. Metyrapone or RU-486 administration prior to MDMA treatment abrogated MDMA effects on neutrophil activity and NFκB expression, while 6-OHDA or ICI-118,551 administration did not. As MDMA treatment increased the plasmatic levels of adrenaline and noradrenaline, propranolol pre-treatment effects were also evaluated. Propranolol suppressed both MDMA-induced increase in corticosterone serum levels and its effects on neutrophil activity. In a L. monocytogenes experimental infection context, we showed that MDMA: induced myelosuppression by decreasing granulocyte-macrophage hematopoietic progenitors (CFU-GM) in the bone marrow but increased CFU-GM in the spleen; decreased circulating leukocytes and bone marrow cellularity and increased spleen cellularity; decreased pro-inflammatory cytokine (IL-12p70, TNF, IFN-γ, IL-6) and chemokine (MCP-1) production 24 h after the infection; increased the production of pro-inflammatory cytokines and chemokines 72 h after infection and decreased IL-10 levels at all time points analyzed. It was proposed that MDMA immunosuppressive effects on neutrophil activity and host resistance to L monocytogenes rely on NFκB signaling, being mediated by HPA axis activity and corticosterone.

  20. Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites.

    Science.gov (United States)

    Tavares, Natália; Afonso, Lilian; Suarez, Martha; Ampuero, Mariana; Prates, Deboraci Brito; Araújo-Santos, Théo; Barral-Netto, Manoel; DosReis, George A; Borges, Valéria Matos; Brodskyn, Cláudia

    2016-02-15

    Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

  1. Imaging Inflammation in Asthma: Real Time, Differential Tracking of Human Neutrophil and Eosinophil Migration in Allergen Challenged, Atopic Asthmatics in Vivo

    Directory of Open Access Journals (Sweden)

    Joanna J. Lukawska

    2014-12-01

    Conclusions: Isolation and radiolabelling significantly increased activation of eosinophils (CD69 and completely activated neutrophils (CD11bHigh in all asthma groups. Net lung neutrophil efflux was significantly slower than that of eosinophils in all asthma study groups. There was a trend for pre-treatment with systemic corticosteroids to reduce lung retention of eosinophils following allergen challenge.

  2. Active structures to reduce torsional vibrations

    Science.gov (United States)

    Matthias, M.; Schlote, D.; Atzrodt, H.

    2013-03-01

    This paper describes the development of different active measures to reduce torsional vibrations in power trains. The measures are based on concepts developed for active mounts to reduce the transmission of structure-borne sound. To show the potential of these active measures and investigate their mode of operation to influence torsional vibrations, numerical simulations of powertrains with different active measures were done. First experimental results from tests on an experimental (reduced size) power train were used to align the numerical models. The work was done within the project 'LOEWE-Zentrum AdRIA: Adaptronik - Research, Innovation, Application' funded by the German federal state of Hessen, and the Project AKTos: 'Active control of torsional vibrations by coupling elements' placed in the research Framework program 'Navigation and Maritime Technology for the 21st Century' funded by the German Federal Ministry of Economics and Technology.

  3. Neutrophils with protumor potential could efficiently suppress tumor growth after cytokine priming and in presence of normal NK cells.

    Science.gov (United States)

    Sun, Rui; Luo, Jing; Li, Dong; Shu, Yu; Luo, Chao; Wang, Shan-Shan; Qin, Jian; Zhang, Gui-Mei; Feng, Zuo-Hua

    2014-12-30

    In tumor-bearing state, the function of neutrophils is converted from tumor-suppressing to tumor-promoting. Here we report that priming with IFN-γ and TNF-α could convert the potential of neutrophils from tumor-promoting to tumor-suppressing. The neutrophils with protumor potential have not lost their responsiveness to IFN-γ and TNF-α. After priming with IFN-γ and TNF-α, the potential of the neutrophils to express Bv8 and Mmp9 genes was reduced. Conversely, the tumor-promotional neutrophils recovered the expression of Rab27a and Trail, resumed the activation levels of PI3K and p38 MAPK pathways in response to stimuli, and expressed higher levels of IL-18 and NK-activating ligands such as RAE-1, MULT-1, and H60. Therefore, the anti-tumor function of the neutrophils was augmented, including the cytotoxicity to tumor cells, the capability of degranulation, and the capacity to activate NK cells. Since the function of NK cells is impaired in tumor-bearing state, the administration of normal NK cells could significantly augment the efficiency of tumor therapy based on neutrophil priming. These findings highlight the reversibility of neutrophil function in tumor-bearing state, and suggest that neutrophil priming by IFN-γ/TNF-α might be a potential approach to eliminate residual tumor cells in comprehensive strategy for tumor therapy.

  4. LPS responsiveness and neutrophil chemotaxis in vivo require PMN MMP-8 activity.

    Directory of Open Access Journals (Sweden)

    Angus M Tester

    Full Text Available We identify matrix metalloproteinase (MMP-8, the polymorphonuclear (PMN leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX at Ser(4-Val(5 and Lys(79-Arg(80. LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4 approximately 5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg(5-Ser(6 and at Val(7-Leu(8 in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.

  5. Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7.

    Science.gov (United States)

    Hmoteh, Jutharat; Syed Musthafa, Khadar; Pomwised, Rattanaruji; Voravuthikunchai, Supayang Piyawan

    2016-05-27

    Enterohaemorrhagic Escherichia coli (E. coli) O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125-250 µg/mL was 58.48%-50.28% and 69.13%-35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

  6. Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7

    Directory of Open Access Journals (Sweden)

    Jutharat Hmoteh

    2016-05-01

    Full Text Available Enterohaemorrhagic Escherichia coli (E. coli O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125–250 µg/mL was 58.48%–50.28% and 69.13%–35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

  7. p38MAPK, ERK and PI3K signaling pathways are involved in C5a-primed neutrophils for ANCA-mediated activation.

    Directory of Open Access Journals (Sweden)

    Jian Hao

    Full Text Available BACKGROUND: The complement system is one of the important contributing factors in the development of antineutrophil cytoplasmic antibody (ANCA-associated vasculitis (AAV. C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA-mediated neutrophil recruitment and activation. The current study further investigated the signaling pathways of C5a-mediated priming of human neutrophils for ANCA-induced neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: The effects of the p38 mitogen-activated protein kinase (p38MAPK inhibitor (SB202190, extracellular signal-regulated kinase (ERK inhibitor (PD98059, c-Jun N-terminal kinase (JNK inhibitor (6o and phosphoinositol 3-kinase (PI3K inhibitor (LY294002 were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of membrane-bound PR3 (mPR3 on neutrophils. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI value was 254.8±67.1, which decreased to 203.6±60.3, 204.4±36.7, 202.4±49.9 and 188±47.9 upon pre-incubation with SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.05, respectively. For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with above-mentioned inhibitors. The lactoferrin concentration increased in C5a-primed neutrophils induced by MPO or PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with above-mentioned three inhibitors. mPR3 expression increased from 923.3±182.4 in untreated cells to 1278.3±299.3 after C5a treatment and decreased to 1069.9±188.9, 1100±238.2, 1092.3±231.8 and 1053.9±200.3 by SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0

  8. A pro-inflammatory role of C5L2 in C5a-primed neutrophils for ANCA-induced activation.

    Directory of Open Access Journals (Sweden)

    Jian Hao

    Full Text Available BACKGROUND: The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA-associated vasculitis (AAV. In particular, C5a and its receptor on neutrophils, CD88, play a central role. The functional role of the second receptor of C5a, C5L2, remains unclear. In the current study, we investigated the role of C5L2 in C5a-primed neutrophils for ANCA-induced activation. METHODS: The effect of blocking C5L2 by anti-human C5L2 blocking antibody were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on membrane-bound proteinase 3 (mPR3 and concentration of myeloperoxidase (MPO in supernatant of C5a-primed neutrophils. An antagonist for CD88 was also employed. RESULTS: Blocking C5L2 resulted in a significantly decreased MPO concentration in the supernatant of C5a-primed neutrophils. mPR3 expression increased from 209.0±43.0 in untreated cells to 444.3±60.8 after C5a treatment (P<0.001, and decreased to 375.8±65.44, 342.2±54.3 and 313.7±43.6 by pre-incubating blocking C5L2 antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-priming group, P<0.001, P<0.001, and P<0.001, respectively. In C5a-primed neutrophils, subsequently activating with MPO-ANCA-positive IgG, the MFI value was 425.8±160.6, which decreased to 292.8±141.2, 289.7±130.0 and 280.3±136.4 upon pre-incubation with mouse anti-human C5L2 blocking antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-primed neutrophils, for MPO-ANCA-positive IgG-induced activation, P<0.05, P<0.05, and P<0.05, respectively. Blocking C5L2 also resulted in significantly decreased C5a-primed neutrophils for PR3-ANCA-positive IgG-induced activation. Moreover, the lactoferrin concentration in the supernant significantly decreased in pre-incubation with anti-human C5L2 blocking antibody, compared with C5a-primed neutrophils induced by PR3- or MPO-ANCA-positive IgG. CONCLUSIONS: C5L2 may be implicated in

  9. Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

    OpenAIRE

    Qureshi, Asfia; Grey, Angus; Rose, Kristie L; Schey, Kevin L.; Del Poeta, Maurizio

    2011-01-01

    We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in...

  10. A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.

    Science.gov (United States)

    Gabig, T G; Eklund, E A; Potter, G B; Dykes, J R

    1990-08-01

    The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.

  11. Baicalin promotes the bacteriostatic activity of lysozyme on S. aureus in mammary glands and neutrophilic granulocytes in mice.

    Science.gov (United States)

    Gao, Xuejiao; Guo, Mengyao; Zhang, Zecai; Shen, Peng; Yang, Zhengtao; Zhang, Naisheng

    2017-03-21

    Staphylococcus aureus causes mastitis as a result of community-acquired or nosocomial infections. Lysozyme (LYSO) is an enzyme that is upregulated in many organisms during the innate immune response against infection by bacterial pathogens. Baicalin is a bioactive flavonoid that can bind to enzymes, often to potentiate their effect. Here we tested the effects of baicalin on the activity of LYSO using the S. aureus mastitis mouse model and neutrophilic granulocyte model of S. aureus infection. In our experiments, S. aureus counts decreased with increasing baicalin concentration. Furthermore, qPCR and western blot analyses showed that LYSO expression was unaffected by baicalin, while fluorescence quenching and UV fluorescence spectral analyses showed that baicalin binds to LYSO. To test whether this binding increased LYSO activity, we assessed LYSO-induced bacteriostasis in the presence of baicalin. Our results showed that LYSO-induced S. aureus bacteriostasis increased with increasing concentrations of baicalin, and that baicalin binding to LYSO synergistically increased the antibacterial activity of LYSO. These results demonstrate that baicalin enhances LYSO-induced bacteriostasis during the innate immune response to S. aureus. They suggest baicalin is a potentially useful therapeutic agent for the treatment of bacterial infections.

  12. [Microbiological investigations and studies of phagocytic activities of peripheral neutrophils during the treatment of parodontitis by Unimag].

    Science.gov (United States)

    Saralidze, M G; Dzhashi, L M; Tskitishvili, T G; Gogebashvili, N N; Surguladze, B V

    2005-11-01

    During the treatment by Unimag (UN), quantity of microbes in the mouth cavity of patients with periodontitis (PD), significantly decreases in comparison with the patients treated by traditional scheme. That is due to direct and indirect influence of UN on the pathogenic microorganisms. During the treatment of patients with PD by UN, quantity of Gram-negative microbes gradually decreases and their substitution by Gram-positive microbes, typical for mouth cavity, takes place. On the background of the treatment by UN, phagocytic activity (PA) of polynuclear cells (PC) increases. In comparison with the patients treated by traditional scheme, increases both phagocytic number and number of active neutrophils. On 14-15 days after beginning of treatment of patients with PD by traditional scheme, PA of PC does not change significantly. Reduction of the microbial number in the mouth cavity and the active substitution of Gram-negative microbes by Gram-positive microorganisms during the treatment of patients with PD by UN, have prognostic importance and together with the reinforcement of PA of PC indicate to the improvement of the therapeutic effect and shortening of the duration of the treatment.

  13. BIRM 270: a novel inhibitor of arachidonate release that blocks leukotriene B4 and platelet-activating factor biosynthesis in human neutrophils.

    Science.gov (United States)

    Farina, P R; Graham, A G; Hoffman, A F; Watrous, J M; Borgeat, P; Nadeau, M; Hansen, G; Dinallo, R M; Adams, J; Miao, C K

    1994-12-01

    (S)-N-[2-Cyclohexyl-1-(2-pyridinyl)ethyl]-5-methyl-2-benzoxazolamine+ ++ (BIRM 270) was identified as a potent and enantiomerically selective inhibitor of calcium ionophore A23187-stimulated leukotriene B4 biosynthesis in human neutrophils. The (S)- and (R)-enantiomers exhibited IC50 values of 1 nM and 40 nM, respectively. BIRM 270 did not inhibit 5-lipoxygenase activity in a cell-free assay. In addition, the compound did not interfere with the conversion of exogenous 5-lipoxygenase substrate (15S)-hydroperoxyeicosatetraenoic acid to (5S, 15S)-dihydroxyeicosatetraenoic acid in intact, ionophore-stimulated neutrophils. Under the same experimental conditions, BIRM 270 inhibited the production of 5-lipoxygenase products from endogenous substrate, suggesting that the compound affected arachidonate availability rather than metabolism. Consistent with this concept, the inhibition of leukotriene B4 biosynthesis by BIRM 270 was overcome by the addition of exogenous arachidonic acid to the leukocyte preparation. Direct measurement of free arachidonate by gas chromatography-mass spectrometry confirmed that BIRM 270 inhibited arachidonate release from ionophore-stimulated neutrophils. The compound did not affect arachidonate reacylation. The blockage of arachidonate release coincided with inhibition of leukotriene B4 biosynthesis in these cells. BIRM 270 also inhibited ionophore-stimulated platelet-activating factor biosynthesis by human neutrophils. Although these results suggest that BIRM 270 inhibited phospholipase A2-mediated deacylation of membrane phospholipids, the compound did not directly inhibit the high molecular weight, cytosolic phospholipase A2 derived from human neutrophils or U937 cells. Thus, suppression of arachidonate mobilization by BIRM 270 may be due to indirect inhibition of intracellular phospholipase A2 or to inhibition of another acylhydrolase activity.

  14. 内皮中性粒细胞激活肽-78在哮喘大鼠中性粒细胞中的表达及意义%Expression of epithelial neutrophil activating protein-78 in neutrophil cell of asthma rats and its significance

    Institute of Scientific and Technical Information of China (English)

    周兴辉; 叶辉; 罗冬娇; 童夏生; 阮正英

    2011-01-01

    OBJECTIVE To observe the expression of neutrophil activating protein-78 (ENA-78) in asthma rats and the regulation by budesonide. And to investigate the role of neutrophil in the pathogenesis of asthma exacerbation. METHODS In this experiment, the rat model of asthma were randomly divided into three groups on average. ' including asthma group, control group and budesonide group. Levels of ENA-78 were detected by flow cytometry methods. RESULTS Levels of ENA-78 at blood neutrophil in asthma group (97. 65 ± 13. 99 mean fluorescence intensity) were significantly higher than those in control group (50. 79 ±8. 66 mean fluorescence intensity) (P< 0.01). And leveles of ENA-78 at blood neutrophil in budesonide group (75. 81 ±6. 10 mean fluorescence intensity) were significantly lower than those in asthma group, but higher than those in control group (all P< 0.01). There were significant positive correlation between ENA-78 expressions at blood neutrophil and numbers of total inflammatory cells in bronchoalveolar lavage fluid (BALF) (n = 29,r=0. 781, P<0. 01). CONCLUSION The level of ENA-78 increases at blood neutrophil of rat asthma. Neutrophil may be participated in inflammation of asthma exacerbation via ENA-78. The asthmatic airway inflammation may decreased through reducing NEU ENA-78 by Budesonide.%目的 观察大鼠哮喘内皮中性粒细胞激活肽-78(ENA-78),表达及布地奈德对其的影响,探讨中性粒细胞(NEU)参与哮喘发作的可能作用机制.方法采用大鼠哮喘模型,随机分成哮喘组、对照组、布地奈德组,流式细胞术测定血NEU ENA-78的表达.结果 哮喘组(97.65±13.99) MFI NEU ENA-78的平均荧光强度显著高于对照组(50.79±8.66)MFI(P<0.01);布地奈德组(75.81±6.10)MFI NEU ENA-78的平均荧光强度显著性低于哮喘组,且高于对照组(均P<0.01),NEU ENA-78的表达水平与支气管肺泡灌洗液(BALF)细胞总数呈显著正相关(n=29,r=0.781,P<0.01).结论 哮喘大鼠NEU ENA-78的

  15. Myocellular enzyme leakage, polymorphonuclear neutrophil activation and delayed onset muscle soreness induced by isokinetic eccentric exercise.

    Science.gov (United States)

    Croisier, J L; Camus, G; Deby-Dupont, G; Bertrand, F; Lhermerout, C; Crielaard, J M; Juchmès-Ferir, A; Deby, C; Albert, A; Lamy, M

    1996-01-01

    To address the question of whether delayed onset muscular soreness (DOMS) following intense eccentric muscle contraction could be due to increased production of the arachidonic acid derived product prostaglandin E2 (PGE2). 10 healthy male subjects were submitted to eccentric and concentric isokinetic exercises on a Kin Trex device at 60 degrees/s angular velocity. Exercise consisted of 8 stages of 5 maximal contractions of the knee extensor and flexor muscle groups of both legs separated by 1 min rest phases. There was an interval of at least 30 days between eccentric and concentric testing, and the order of the two exercise sessions was randomly assigned. The subjective presence and intensity of DOMS was evaluated using a visual analogue scale, immediately, following 24 h and 48 h after each test. Five blood samples were drawn from an antecubital vein: at rest before exercise, immediately after, after 30 min recovery, 24 h and 48 h after the tests. The magnitude of the acute inflammatory response to exercise was assessed by measuring plasma levels of polymorphonuclear elastase ([EL]), myeloperoxidase ([MPO]) and PGE2 ([PGE2]). Using two way analysis of variance, it appeared that only eccentric exercise significantly increased [EL] and DOMS, especially of the hamstring muscles. Furthermore, a significant decrease in eccentric peak torque of this muscle group only was observed on day 2 after eccentric work (- 21%; P < 0.002). Serum activity of creatine kinase and serum concentration of myoglobin increased significantly 24 and 48 h after both exercise tests. However, these variables reached significantly higher values following eccentric contractions 48 h after exercise. Mean [PGE2] in the two exercise modes remained unchanged over time and were practically equal at each time point. On the basis of these findings, we conclude that the magnitude of polymorphonuclear (PMN) activation, muscle damage, and DOMS are greater after eccentric than after concentric muscle

  16. A combretastatin-mediated decrease in neutrophil concentration in peripheral blood and the impact on the anti-tumor activity of this drug in two different murine tumor models.

    Directory of Open Access Journals (Sweden)

    Anja Bille Bohn

    Full Text Available The vascular disrupting agent combretastatin A-4 disodium phosphate (CA4P induces fluctuations in peripheral blood neutrophil concentration. Because neutrophils have the potential to induce both vascular damage and angiogenesis we analyzed neutrophil involvement in the anti-tumoral effects of CA4P in C3H mammary carcinomas in CDF1 mice and in SCCVII squamous cell carcinomas in C3H/HeN mice. Flow cytometry analyses of peripheral blood before and up to 144 h after CA4P administration (25 and 250 mg/kg revealed a decrease 1 h after treatment, followed by an early (3-6 h and a late (>72 h increase in the granulocyte concentration. We suggest that the early increase (3-6 h in granulocyte concentration was caused by the initial decrease at 1 h and found that the late increase was associated with tumor size, and hence independent of CA4P. No alterations in neutrophil infiltration into the C3H tumor after CA4P treatment (25 and 250 mg/kg were found. Correspondingly, neutrophil depletion in vivo, using an anti-neutrophil antibody, followed by CA4P treatment (25 mg/kg did not increase the necrotic fraction in C3H tumors significantly. However, by increasing the CA4P dose to 250 mg/kg we found a significant increase of 359% in necrotic fraction when compared to neutrophil-depleted mice; in mice with no neutrophil depletion CA4P induced an 89% change indicating that the presence of neutrophils reduced the effect of CA4P. In contrast, neither CA4P nor 1A8 affected the necrotic fraction in the SCCVII tumors significantly. Hence, we suggest that the initial decrease in granulocyte concentration was caused by non-tumor-specific recruitment of neutrophils and that neutrophils may attenuate CA4P-mediated anti-tumor effect in some tumor models.

  17. Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

    DEFF Research Database (Denmark)

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

    2007-01-01

    BACKGROUND: Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr...... human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs) in viral neutralization assays as compared to multimeric SP-D. CONCLUSION: These studies......-D can be useful for dissecting out different functional properties of the protein....

  18. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia.

    Science.gov (United States)

    Abraham, E; Arcaroli, J; Shenkar, R

    2001-01-01

    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

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

    Science.gov (United States)

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

    2015-01-15

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

  20. 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.

  1. Yersinia enterocolitica YopH-Deficient Strain Activates Neutrophil Recruitment to Peyer's Patches and Promotes Clearance of the Virulent Strain.

    Science.gov (United States)

    Dave, Mabel N; Silva, Juan E; Eliçabe, Ricardo J; Jeréz, María B; Filippa, Verónica P; Gorlino, Carolina V; Autenrieth, Stella; Autenrieth, Ingo B; Di Genaro, María S

    2016-11-01

    Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

    Directory of Open Access Journals (Sweden)

    Nauder Faraday

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

  3. Single platelets seal neutrophil-induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice.

    Science.gov (United States)

    Gros, Angèle; Syvannarath, Varouna; Lamrani, Lamia; Ollivier, Véronique; Loyau, Stéphane; Goerge, Tobias; Nieswandt, Bernhard; Jandrot-Perrus, Martine; Ho-Tin-Noé, Benoît

    2015-08-20

    Platelets protect vascular integrity during inflammation. Recent evidence suggests that this action is independent of thrombus formation and requires the engagement of glycoprotein VI (GPVI), but it remains unclear how platelets prevent inflammatory bleeding. We investigated whether platelets and GPVI act primarily by preventing detrimental effects of neutrophils using models of immune complex (IC)-mediated inflammation in mice immunodepleted in platelets and/or neutrophils or deficient in GPVI. Depletion of neutrophils prevented bleeding in thrombocytopenic and GPVI(-/-) mice during IC-mediated dermatitis. GPVI deficiency did not modify neutrophil recruitment, which was reduced by thrombocytopenia. Neutrophil cytotoxic activities were reduced in thrombocytopenic and GPVI(-/-) mice during IC-mediated inflammation. Intravital microscopy revealed that in this setting, intravascular binding sites for platelets were exposed by neutrophils, and GPVI supported the recruitment of individual platelets to these spots. Furthermore, the platelet secretory response accompanying IC-mediated inflammation was partly mediated by GPVI, and blocking of GPVI signaling impaired the vasculoprotective action of platelets. Together, our results show that GPVI plays a dual role in inflammation by enhancing neutrophil-damaging activities while supporting the activation and hemostatic adhesion of single platelets to neutrophil-induced vascular breaches.

  4. Activation of the human neutrophil NADPH oxidase results in coupling of electron carrier function between ubiquinone-10 and cytochrome b559.

    Science.gov (United States)

    Gabig, T G; Lefker, B A

    1985-04-10

    The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome b559 in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH----E-FAD----ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded: NADPH----E-FAD----ubiquinone-10----cytochrome b559----oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (i.e. E-FAD, ubiquinone-10, and cytochrome b559). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome b559 and was without demonstrable effect on proximal electron flow. The p-chloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.

  5. Role of Bcr1-activated genes Hwp1 and Hyr1 in Candida albicans oral mucosal biofilms and neutrophil evasion.

    Science.gov (United States)

    Dwivedi, Prabhat; Thompson, Angela; Xie, Zhihong; Kashleva, Helena; Ganguly, Shantanu; Mitchell, Aaron P; Dongari-Bagtzoglou, Anna

    2011-01-25

    Candida albicans triggers recurrent infections of the oropharyngeal mucosa that result from biofilm growth. Prior studies have indicated that the transcription factor Bcr1 regulates biofilm formation in a catheter model, both in vitro and in vivo. We thus hypothesized that Bcr1 plays similar roles in the formation of oral mucosal biofilms and tested this hypothesis in a mouse model of oral infection. We found that a bcr1/bcr1 mutant did not form significant biofilm on the tongues of immunocompromised mice, in contrast to reference and reconstituted strains that formed pseudomembranes covering most of the tongue dorsal surface. Overexpression of HWP1, which specifies an epithelial adhesin that is under the transcriptional control of Bcr1, partly but significantly rescued the bcr1/bcr1 biofilm phenotype in vivo. Since HWP1 overexpression only partly reversed the biofilm phenotype, we investigated whether additional mechanisms, besides adhesin down-regulation, were responsible for the reduced virulence of this mutant. We discovered that the bcr1/bcr1 mutant was more susceptible to damage by human leukocytes when grown on plastic or on the surface of a human oral mucosa tissue analogue. Overexpression of HYR1, but not HWP1, significantly rescued this phenotype. Furthermore a hyr1/hyr1 mutant had significantly attenuated virulence in the mouse oral biofilm model of infection. These discoveries show that Bcr1 is critical for mucosal biofilm infection via regulation of epithelial cell adhesin and neutrophil function.

  6. Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease.

    Directory of Open Access Journals (Sweden)

    Ama-Tawiah Essilfie

    2011-10-01

    Full Text Available A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR. Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD. BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate and T lymphocytes (late, adaptive in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.

  7. [Ambiguity role of neutrophils in oncogenesis].

    Science.gov (United States)

    Mal'tseva, V N; Safronova, V G

    2009-01-01

    The review is focused on the participation of polymorphonuclear granulocytes (neutrophils) in development and spreading of a tumor. We consider both the well known functions of neutrophils (degranulation, production of reactive oxygen species (ROS)) and the recently shown one (presentation of an antigene). The special attention is focused on the ambiguity of the neutrophil role in oncogenesis. The dominant view is that neutrophils display exclusively antitumor properties. The update information testifies about protumoral activity of neutrophils: they migrate to a tumor and promote angiogenesis and metastasis at late stages of the tumor. It is interesting that certain components of neutrophil cytotoxic arsenal (ROS, cytokines, specific enzymes) participate both in antitumoral defenses of an organism and protumoral activity.

  8. Mechanisms of Degranulation in Neutrophils

    Directory of Open Access Journals (Sweden)

    Lacy Paige

    2006-09-01

    Full Text Available Abstract Neutrophils are critical inflammatory cells that cause tissue damage in a range of diseases and disorders. Being bone marrow-derived white blood cells, they migrate from the bloodstream to sites of tissue inflammation in response to chemotactic signals and induce inflammation by undergoing receptor-mediated respiratory burst and degranulation. Degranulation from neutrophils has been implicated as a major causative factor in pulmonary disorders, including severe asphyxic episodes of asthma. However, the mechanisms that control neutrophil degranulation are not well understood. Recent observations indicate that granule release from neutrophils depends on activation of intracellular signalling pathways, including β-arrestins, the Rho guanosine triphosphatase Rac2, soluble NSF attachment protein (SNAP receptors, the src family of tyrosine kinases, and the tyrosine phosphatase MEG2. Some of these observations suggest that degranulation from neutrophils is selective and depends on nonredundant signalling pathways. This review focuses on new findings from the literature on the mechanisms that control the release of granule-derived mediators from neutrophils.

  9. Reducing Skin Picking via Competing Activities

    Science.gov (United States)

    Lane, Kathleen Lynne; Thompson, Ada; Reske, Cara L.; Gable, Lauren M.; Barton-Arwood, Sally

    2006-01-01

    This study examined the outcomes of a competing activities intervention to decrease skin picking exhibited by a 9-year-old student with comorbid diagnoses. Results of an ABCBAB design revealed that the use of student-selected manipulatives resulted in reduced skin picking. (Contains 1 figure.)

  10. Circulating histamine and neutrophil chemotactic activity during allergen-induced asthma: the effect of inhaled antihistamines and anti-allergic compounds.

    Science.gov (United States)

    Morgan, D J; Moodley, I; Cundell, D R; Sheinman, B D; Smart, W; Davies, R J

    1985-07-01

    Plasma histamine and serum neutrophil chemotactic activity (S-NCA) were measured in ten atopic asthmatic patients on four separate occasions after allergen bronchial provocation testing (BPT). Single doses of inhaled sodium cromoglycate (SCG; 20 mg), clemastine (0.5 mg), ketotifen (0.5 mg) and isotonic saline (0.9% NaCl) placebo were administered 30 min before bronchial provocation testing in random order and double-blind. The airflow obstruction after BPT was monitored by measurement of forced expiratory volume in 1 s (FEV1). Plasma histamine was measured by the double-isotope radioenzymatic assay and S-NCA by a modified Boyden chamber technique. A highly significant decrease in FEV1 after BPT occurred on the placebo pre-treatment visit (P less than 0.001). Prior administration of inhaled SCG, clemastine and ketotifen significantly reduced the decrease in airflow obstruction seen after BPT when compared with placebo treatment (P less than 0.01, P less than 0.02, P less than 0.05 respectively). No significant alteration in plasma histamine was detected during allergen-induced airflow obstruction. Levels of S-NCA were significantly higher 5, 10 and 15 min after BPT when compared with the pre-challenge level (P less than 0.01, P less than 0.01, P less than 0.001 respectively). These levels were not significantly decreased when airflow obstruction was inhibited by the prior inhalation of SCG, clemastine or ketotifen.

  11. IMMUNOBIOLOGICAL ACTIVITY OF REGULATORY PEPTIDE FRACTIONS SYNTHESIZED BY NEUTROPHILS, AS TESTED IN A MACROPHAGE MODEL

    Directory of Open Access Journals (Sweden)

    G. I. Vasilieva

    2010-01-01

    Full Text Available The article presents experimental data on regulatory effect of neutrophilokine helper fractions on the macrophage (Mph functional activity in the course of antiplague immunity formation. It has revealed that these fractions content biologically active, low-molecular weight peptides. They stimulate Mph killing activity by increasing phagosome-lysosome fusion, thus boosting transformation of monocytes to Mph, and causing redistribution of macrophage subpopulations in the total cellular pool. The helper effect of neutrophilokine fractions upon functional activity of MPh is more pronounced during secondary immune response.

  12. Neutrophil volume, conductivity and scatter parameters with effective modeling of molecular activity statistical program gives better results in neonatal sepsis.

    Science.gov (United States)

    Celik, I H; Demirel, G; Sukhachev, D; Erdeve, O; Dilmen, U

    2013-02-01

    Neonatal sepsis remains an important clinical syndrome despite advances in neonatology. Current hematology analyzers can determine cell volume (V), conductivity for internal composition of cell (C) and light scatter for cytoplasmic granularity and nuclear structure (S), and standard deviations which are effective in the diagnosis of sepsis. Statistical models can be used to strengthen the diagnosis. Effective modeling of molecular activity (EMMA) uses combinatorial algorithm of the selection parameters for regression equation based on modified stepwise procedure. It allows obtaining different regression models with different combinations of parameters. We investigated these parameters in screening of neonatal sepsis. We used LH780 hematological analyzer (Beckman Coulter, Fullerton, CA, USA). We combined these parameters with interleukin-6 (IL-6) and C-reactive protein (CRP) and developed models by EMMA. A total of 304 newborns, 76 proven sepsis, 130 clinical sepsis and 98 controls, were enrolled in the study. Mean neutrophil volume (MNV) and volume distribution width (VDW) were higher in both proven and clinical sepsis groups. We developed three models using MNV, VDW, IL-6, and CRP. These models gave more sensitivity and specificity than the usage of each marker alone. We suggest to use the combination of MNV and VDW with markers such as CRP and IL-6, and use diagnostic models created by EMMA. © 2012 Blackwell Publishing Ltd.

  13. Mitochondrial N-formyl peptides cause airway contraction and lung neutrophil infiltration via formyl peptide receptor activation.

    Science.gov (United States)

    Wenceslau, Camilla Ferreira; Szasz, Theodora; McCarthy, Cameron G; Baban, Babak; NeSmith, Elizabeth; Webb, R Clinton

    2016-04-01

    Respiratory failure is a common characteristic of systemic inflammatory response syndrome (SIRS) and sepsis. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns (DAMPs). Mitochondrial N-formyl peptides (F-MITs) are DAMPs that share similarities with bacterial N-formylated peptides, and are potent immune system activators. Recently, we observed that hemorrhagic shock-induced increases in plasma levels of F-MITs associated with lung damage, and that antagonism of formyl peptide receptors (FPR) ameliorated hemorrhagic shock-induced lung injury in rats. Corroborating these data, in the present study, it was observed that F-MITs expression is higher in plasma samples from trauma patients with SIRS or sepsis when compared to control trauma group. Therefore, to better understand the role of F-MITs in the regulation of lung and airway function, we studied the hypothesis that F-MITs lead to airway contraction and lung inflammation. We observed that F-MITs induced concentration-dependent contraction in trachea, bronchi and bronchioles. However, pre-treatment with mast cells degranulator or FPR antagonist decreased this response. Finally, intratracheal challenge with F-MITs increased neutrophil elastase expression in lung and inducible nitric oxide synthase and cell division control protein 42 expression in all airway segments. These data suggest that F-MITs could be a putative target to treat respiratory failure in trauma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Development of an in vitro model system to study the interactions between Mycobacterium marinum and teleost neutrophils.

    Science.gov (United States)

    Hodgkinson, Jordan W; Ge, Jun-Qing; Katzenback, Barbara A; Havixbeck, Jeffrey J; Barreda, Daniel R; Stafford, James L; Belosevic, Miodrag

    2015-12-01

    The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates.

  15. Propensity of crocin to offset Vipera russelli venom induced oxidative stress mediated neutrophil apoptosis: a biochemical insight.

    Science.gov (United States)

    Santhosh, M Sebastin; Sundaram, M Shanmuga; Sunitha, K; Jnaneshwari, S; Devaraja, S; Kemparaju, K; Girish, K S

    2016-01-01

    Viper envenomation results in inflammation at the bitten site as well as target organs. Neutrophils and other polymorphonuclear leukocytes execute inflammation resolving mechanism and will undergo apoptosis after completing the task. However, the target specific toxins induce neutrophil apoptosis at the bitten site and in circulation prior to their function, thus reducing their number. Circulating activated neutrophils are major source of inflammatory cytokines and leakage of reactive oxygen species (ROS)/other toxic intermediates resulting in aggravation of inflammatory response at the bitten/target site. Therefore, neutralization of venom induced neutrophil apoptosis reduces inflammation besides increasing the functional neutrophil population. Therefore, the present study investigates the venom induced perturbances in isolated human neutrophils and its neutralization by crocin (Crocus sativus) a potent antioxidant carotenoid. Human neutrophils on treatment with venom resulted in altered ROS generation, intracellular Ca(2+) mobilization, mitochondrial membrane depolarization, cyt-c translocation, caspase activation, phosphatidylserine externalization and DNA damage. On the other hand significant protection against oxidative stress and apoptosis were evidenced in crocin pre-treated groups. In conclusion the viper venom induces neutrophil apoptosis and results in aggravation of inflammation and tissue damage. The present study demands the necessity of an auxiliary therapy in addition to antivenin therapy to treat secondary/overlooked complications of envenomation.

  16. Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

    Science.gov (United States)

    Gorgojo, Juan; Scharrig, Emilia; Gómez, Ricardo M.; Harvill, Eric T.; Rodríguez, Maria Eugenia

    2017-01-01

    B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts. PMID:28095485

  17. Neutrophils and neutrophil serine proteases are increased in the spleens of estrogen-treated C57BL/6 mice and several strains of spontaneous lupus-prone mice

    Science.gov (United States)

    Dai, Rujuan; Cowan, Catharine; Heid, Bettina; Khan, Deena; Liang, Zhihong; Pham, Christine T. N.; Ahmed, S. Ansar

    2017-01-01

    Estrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types. There is now renewed attention on neutrophils and neutrophil serine proteases (NSPs) such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) in inflammation and autoimmunity. In this study, we found that although estrogen treatment significantly reduced total splenocytes number, it markedly increased the splenic neutrophil absolute numbers in estrogen-treated C57BL/6 (B6) mice when compared to placebo controls. Concomitantly, the levels of NSPs and myeloperoxidase (MPO) were highly upregulated in the splenocytes from estrogen-treated mice. Despite the critical role of NSPs in the regulation of non-infectious inflammation, by employing NE-/-/PR3-/-/CG-/- triple knock out mice, we demonstrated that the absence of NSPs affected neither estrogen’s ability to increase splenic neutrophils nor the induction of inflammatory mediators (IFNγ, IL-1β, IL-6, TNFα, MCP-1, and NO) from ex vivo activated splenocytes. Depletion of neutrophils in vitro in splenocytes with anti-Ly6G antibody also had no obvious effect on NSP expression or LPS-induced IFNγ and MCP-1. These data suggest that estrogen augments NSPs, which appears to be independent of enhancing ex vivo inflammatory responses. Since estrogen has been implicated in regulating several experimental autoimmune diseases, we extended our observations in estrogen-treated B6 mice to spontaneous autoimmune-prone female MRL-lpr, B6-lpr and NZB/WF1 mice. There was a remarkable commonality with regards to the increase of neutrophils and concomitant increase of NSPs and MPO in the splenic cells of different strains of autoimmune-prone mice and estrogen-treated B6 mice. Collectively, since NSPs and neutrophils are involved in diverse pro-inflammatory activities, these data suggest a potential pathologic implication of increased neutrophils and NSPs that merits further investigation. PMID:28192517

  18. Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein.

    Science.gov (United States)

    Eklund, E A; Marshall, M; Gibbs, J B; Crean, C D; Gabig, T G

    1991-07-25

    Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G

  19. Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions.

    Science.gov (United States)

    Sawant, Kirti V; Poluri, Krishna Mohan; Dutta, Amit K; Sepuru, Krishna Mohan; Troshkina, Anna; Garofalo, Roberto P; Rajarathnam, Krishna

    2016-09-14

    The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury.

  20. Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI by lung epithelial cells: role of charge of the proteinase-inhibitor complex

    Directory of Open Access Journals (Sweden)

    Hiemstra Pieter S

    2008-08-01

    Full Text Available Abstract Background Secretory leukoproteinase inhibitor (SLPI is an important inhibitor of neutrophil elastase (NE, a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes in vitro. Methods Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling. Results We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration. Conclusion These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon in vitro, which may correlate with changes observed in vivo.

  1. Fungal and bacterial killing by neutrophils.

    Science.gov (United States)

    Ermert, David; Zychlinsky, Arturo; Urban, Constantin

    2009-01-01

    Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.

  2. Neutrophils in Cancer: Two Sides of the Same Coin

    Directory of Open Access Journals (Sweden)

    Eileen Uribe-Querol

    2015-01-01

    Full Text Available Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

  3. A newly synthesized molecule derived from ruthenium cation, with antitumour activity, activates NADPH oxidase in human neutrophils.

    Science.gov (United States)

    Carballo, M; Vilaplana, R; Márquez, G; Conde, M; Bedoya, F J; González-Vílchez, F; Sobrino, F

    1997-12-01

    To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-. production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1, 2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-. production. This increase was associated with the translocation of cytosolic factors p47(phox) and p67(phox) of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-. production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.

  4. ACTIVITY ATTACK ON REDUCED VARIANTS OF RIJNDAEL

    Institute of Scientific and Technical Information of China (English)

    Wei Baodian; Liu Dongsu; Wang Xinmei

    2004-01-01

    The famous Square attacks against the Rijndael algorithm have taken advantage of the change of the balance of some bytes. Further study shows that the change of activity always happens before the change of balance, which builds the foundation for a new activity attack presented in this paper. In the activity attack, the round in which the activity changes is executed in an equivalent form to avoid the obstructive restriction of the subkeys of that round.The existence of the birthday paradox guarantees much fewer plaintexts necessary for activity attacks comparing with that for corresponding Square attacks. But no benefit may result from the new attacks performed independently because the activity attacks guess four instead of one key byte once. Only when both the balance property and the activity property are exploited at the same time can much better performance be obtained. The better performance in the simulation shows that the consuming time and chosen plaintexts necessary are both reduced to one tenth of those of the corresponding Square attacks. So the activity attacks could be viewed as an efficient supplement to the Square attacks.

  5. Neutrophil myeloperoxidase activity and the influence of two single-nucleotide promoter polymorphisms

    NARCIS (Netherlands)

    Rutgers, Abraham; Heeringa, Peter; Giesen, Joyce E H M; Theunissen, Ruud T; Jacobs, Heinz; Tervaert, Jan Willem Cohen

    2003-01-01

    Myeloperoxidase (MPO) catalyses the formation of hypochlorous acid and is involved in many (patho)physiological processes. The present study was designed to determine the effect of two MPO promoter polymorphisms (463G/A and 129G/A) on enzyme activity. In 243 healthy controls, genotypes were determin

  6. Entamoeba histolytica induces human neutrophils to form NETs.

    Science.gov (United States)

    Ventura-Juarez, J; Campos-Esparza, Mr; Pacheco-Yepez, J; López-Blanco, J A; Adabache-Ortíz, A; Silva-Briano, M; Campos-Rodríguez, R

    2016-08-01

    Entamoeba histolytica invades the intestine and other organs during the pathogenesis of amoebiasis. In the early stages, the host organism responds with an inflammatory infiltrate composed mostly of neutrophils. It has been reported that these immune cells, activated by E. histolytica, exert a protective role by releasing proteolytic enzymes and generating reactive oxygen/nitrogen species (ROS/RNS) and antimicrobial peptides. It is now known that neutrophils also produce neutrophil extracellular traps (NETs), which are able to damage and kill pathogens. Studies have shown that intracellular protozoan pathogens, including Toxoplasma gondi, Plasmodium falciparum and Leishmania spp, induce neutrophils to release NETs and are damaged by them. However, the action of this mechanism has not been explored in relation to E. histolytica trophozoites. Through scanning electron, epifluorescence microscopy and viability assays, we show for first time that during in vitro interaction with E. histolytica trophozoites, human neutrophils released NETs that covered amoebas and reduced amoebic viability. These NETs presented histones, myeloperoxidase and decondensed chromatin. The results suggest that NETs participate in the elimination of the parasite.

  7. Activity of Reducing Steel Slag of EAF

    Institute of Scientific and Technical Information of China (English)

    GUO Chinhsiang; HWANG Chaolung; LIN Tingyi

    2011-01-01

    Reducing steel slag (RSS) was mainly acquired from five electric-arc furnace (EAF)steelmaking plants (among them, the products of two plants were carbon steel and those of other plants were stainless steel) for research tests. The chemical properties, compound compositions, activities and contents of main expansive compounds were tested. The results showed that the field sampled RSS had a very high crystallinity and hydraulicity with main chemical compositions close to those of Portland cement. It can be known from the study that in case of C/S ratio higher than 2.0, the main compound compositions are C2S, C3S, C2F and f-CaO. However, after the RSS was stored for six months, an obvious variation occurred with potential pre-hydration in RSS, where the SO3 content was slightly reduced and the compressive activity index was obviously higher than that at the 28th day.

  8. Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hideshi, E-mail: h-yokoya@u-shizuoka-ken.ac.jp [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Tsuruta, Osamu; Akao, Naoya; Fujii, Satoshi [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Structures of a metal-bound Helicobacter pylori neutrophil-activating protein were determined. Black-Right-Pointing-Pointer Two zinc ions were tetrahedrally coordinated by ferroxidase center (FOC) residues. Black-Right-Pointing-Pointer Two cadmium ions were coordinated in a trigonal-bipyramidal and octahedral manner. Black-Right-Pointing-Pointer The second metal ion was more weakly coordinated than the first at the FOC. Black-Right-Pointing-Pointer A zinc ion was found in one negatively-charged pore suitable as an ion path. -- Abstract: Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn{sup 2+}- or Cd{sup 2+}-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.

  9. The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.

    Science.gov (United States)

    Tole, Soumitra; Mukovozov, Ilya M; Huang, Yi-Wei; Magalhaes, Marco A O; Yan, Ming; Crow, Min Rui; Liu, Guang Ying; Sun, Chun Xiang; Durocher, Yves; Glogauer, Michael; Robinson, Lisa A

    2009-12-01

    In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP-2. In all instances, Slit2 reduced neutrophil recruitment effectively (PSlit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

  10. (-)-Epicatechin regenerates the chlorinating activity of myeloperoxidase in vitro and in neutrophil granulocytes.

    Science.gov (United States)

    Flemmig, Jörg; Remmler, Johannes; Röhring, Fiete; Arnhold, Jürgen

    2014-01-01

    The heme-containing enzyme myeloperoxidase (MPO) is mainly expressed in polymorphonuclear leukocytes (PMNs), the most abundant immune cell type in the blood. Accordingly, MPO is classically attributed to the innate immune response against pathogens. Yet, new results also show immune-regulatory functions of the halogenating MPO activity including the formation of anti-inflammatory mediators. In this work we tested the ability of the flavonoid (-)-epicatechin to regenerate this enzymatic activity both in vitro at the isolated MPO-H2O2-Cl(-) system and ex vivo in human PMNs. For all experiments the non-fluorescent dye aminophenyl fluorescein (APF) was used. Upon oxidation by the MPO, the halogenation product hypochlorous acid (HOCl) fluorescein is formed which can be detected e.g. by flow cytometry. The in vitro- and ex vivo-results concordantly show that (-)-epicatechin is a suitable substrate to overcome a compound II accumulation of MPO which was experimentally forced by applying excess hydrogen peroxide. Thereby concentration-dependent effects of the flavan-3-ol were found in both cases and confirmed the proposed mode of action of (-)-epicatechin. The results are in accordance with previous stopped-flow kinetic studies which showed a high reactivity of the polyphenol with MPO compound II. The obtained data may contribute to the explanation of the well-known health promoting effects of (-)-epicatechin. Moreover, the presented study provides new insights into the role of MPO during inflammation.

  11. Binding of human serum albumin to single-walled carbon nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

    Science.gov (United States)

    Lu, Naihao; Li, Jiayu; Tian, Rong; Peng, Yi-Yuan

    2014-06-16

    Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl-) and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase (MPO). However, the importance of protein-SWCNT interactions in the biodegradation of SWCNTs was not stressed. Here, we used both experimental and theoretical approaches to investigate the interactions of SWCNTs with human serum albumin (HSA, one of the most abundant proteins in blood circulation) and found that the binding was involved in the electrostatic interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π-π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Compared with SWCNTs, the binding of HSA could result in a reduced effect for OCl- (or the human MPO system)-induced SWCNTs degradation in vitro. However, the HSA-SWCNT interactions would enhance cellular uptake of nanotubes and stimulate MPO release and OCl- generation in neutrophils, thereby creating the conditions favorable for the degradation of the nanotubes. Upon zymosan stimulation, both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was more for HSA-SWCNTs under these relevant in vivo conditions. Our findings suggest that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in human inflammatory cells and therefore shed light on the biomedical and biotechnological applications of safe carbon nanotubes by comprehensive preconsideration of their interactions with human serum proteins.

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

    Directory of Open Access Journals (Sweden)

    Bahareh Abd Nikfarjam

    2017-03-01

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

  13. The fibrinogen cleavage product Aα-Val360, a specific marker of neutrophil elastase activity in vivo

    DEFF Research Database (Denmark)

    Carter, Richard I; Mumford, Richard A; Treonze, Kelly M

    2011-01-01

    Alpha-1-antitrypsin (A1AT) deficiency is the only recognised genetic risk factor for chronic obstructive pulmonary disease (COPD), a leading cause of morbidity and mortality worldwide. Since A1AT is the major inhibitor of neutrophil elastase (NE), this enzyme has become widely implicated in the p...

  14. Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study

    Science.gov (United States)

    Ranguelova, Kalina; Rice, Annette B.; Khajo, Abdelahad; Triquigneaux, Mathilde; Garantziotis, Stavros; Magliozzo, Richard S.; Mason, Ronald P.

    2012-01-01

    The objective of the present study is to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In the present study sulfite (•SO3−) and sulfate (SO4•−) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, PMA-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The latter adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO4•−) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation. PMID:22326772

  15. Neutrophils support lung colonization of metastasis-initiating breast cancer cells.

    Science.gov (United States)

    Wculek, Stefanie K; Malanchi, Ilaria

    2015-12-17

    Despite progress in the development of drugs that efficiently target cancer cells, treatments for metastatic tumours are often ineffective. The now well-established dependency of cancer cells on their microenvironment suggests that targeting the non-cancer-cell component of the tumour might form a basis for the development of novel therapeutic approaches. However, the as-yet poorly characterized contribution of host responses during tumour growth and metastatic progression represents a limitation to exploiting this approach. Here we identify neutrophils as the main component and driver of metastatic establishment within the (pre-)metastatic lung microenvironment in mouse breast cancer models. Neutrophils have a fundamental role in inflammatory responses and their contribution to tumorigenesis is still controversial. Using various strategies to block neutrophil recruitment to the pre-metastatic site, we demonstrate that neutrophils specifically support metastatic initiation. Importantly, we find that neutrophil-derived leukotrienes aid the colonization of distant tissues by selectively expanding the sub-pool of cancer cells that retain high tumorigenic potential. Genetic or pharmacological inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase (Alox5) abrogates neutrophil pro-metastatic activity and consequently reduces metastasis. Our results reveal the efficacy of using targeted therapy against a specific tumour microenvironment component and indicate that neutrophil Alox5 inhibition may limit metastatic progression.

  16. 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.

    Directory of Open Access Journals (Sweden)

    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.

  17. Activation of recombinant human neutrophil procollagenase in the presence of doxycycline results in fragmentation of the enzyme and loss of enzyme activity.

    Science.gov (United States)

    Smith, G N; Brandt, K D; Hasty, K A

    1996-02-01

    To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction. Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C-acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,10-phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested. Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28- and 26-kd fragments while trypsin cleavage yield 40- and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as 500 microM. Inhibition of collagenase activity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. These data suggest that doxy alters the conformation of procollagenase or collagenase by binding enzyme-associated Ca++, rendering the proteins more susceptible to proteolysis and resulting in irreversible loss of enzyme protein.

  18. Ly6Chi Monocytes and Their Macrophage Descendants Regulate Neutrophil Function and Clearance in Acetaminophen-Induced Liver Injury

    Science.gov (United States)

    Graubardt, Nadine; Vugman, Milena; Mouhadeb, Odelia; Caliari, Gabriele; Pasmanik-Chor, Metsada; Reuveni, Debby; Zigmond, Ehud; Brazowski, Eli; David, Eyal; Chappell-Maor, Lousie; Jung, Steffen; Varol, Chen

    2017-01-01

    Monocyte-derived macrophages (MoMF) play a pivotal role in the resolution of acetaminophen-induced liver injury (AILI). Timely termination of neutrophil activity and their clearance are essential for liver regeneration following injury. Here, we show that infiltrating Ly6Chi monocytes, their macrophage descendants, and neutrophils spatially and temporally overlap in the centrilobular necrotic areas during the necroinflammatory and resolution phases of AILI. At the necroinflammatory phase, inducible ablation of circulating Ly6Chi monocytes resulted in reduced numbers and fractions of reactive oxygen species (ROS)-producing neutrophils. In alignment with this, neutrophils sorted from monocyte-deficient livers exhibited reduced expression of NADPH oxidase 2. Moreover, human CD14+ monocytes stimulated with lipopolysaccharide or hepatocyte apoptotic bodies directly induced ROS production by cocultured neutrophils. RNA-seq-based transcriptome profiling of neutrophils from Ly6Chi monocyte-deficient versus normal livers revealed 449 genes that were differentially expressed with at least twofold change (p ≤ 0.05). In the absence of Ly6Chi monocytes, neutrophils displayed gene expression alterations associated with decreased innate immune activity and increased cell survival. At the early resolution phase, Ly6Chi monocytes differentiated into ephemeral Ly6Clo MoMF and their absence resulted in significant accumulation of late apoptotic neutrophils. Further gene expression analysis revealed the induced expression of a specific repertoire of bridging molecules and receptors involved with apoptotic cell clearance during the transition from Ly6Chi monocytes to MoMF. Collectively, our findings establish a phase-dependent task division between liver-infiltrating Ly6Chi monocytes and their MoMF descendants with the former regulating innate immune functions and cell survival of neutrophils and the later neutrophil clearance. PMID:28620383

  19. 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

  20. Modulation of polymorphonuclear neutrophil functions by astrocytes

    Directory of Open Access Journals (Sweden)

    Xie Luokun

    2010-09-01

    Full Text Available Abstract Background Neuroinflammation is a complex process involving cells from the immune system and the central nerve system (CNS. Polymorphonuclear neutrophils (PMN are the most abundant class of white blood cells, and typically the first type of leukocyte recruited to sites of inflammation. In the CNS, astrocytes are the most abundant glial cell population and participate in the local innate immune response triggered by a variety of insults. In the present study, we investigated the impacts of astrocytes on PMN function. Methods Primary astrocyte cultures were derived from postnatal C57BL/6 mice and primary neutrophils were isolated from 8 to 12 weeks old C57BL/6 mice. PMNs respiratory burst was analyzed by H2DCFDA assay. For phagocytosis assay, neutrophils were incubated with FITC-labeled E. coli and the phagocytosis of E coli was determined by flow cytometer. PMNs degranulation was determined by myeloperoxidase assay. Cytokine expression was determined by real-time PCR. To determine the involvement of different signaling pathway, protein lysates were prepared and western blots were conducted to assess the activation of Akt, Erk1/2, and p38. Results Using ex vivo neutrophils and primary astrocyte cultures, our study demonstrated that astrocytes differentially regulate neutrophil functions, depending upon whether the interactions between the two cell types are direct or indirect. Upon direct cell-cell contact, astrocytes attenuate neutrophil apoptosis, respiratory bust, and degranulation, while enhancing neutrophil phagocytic capability and pro-inflammatory cytokine expression. Through indirect interaction with neutrophils, astrocytes attenuate apoptosis and enhance necrosis in neutrophils, augment neutrophil phagocytosis and respiratory burst, and inhibit neutrophil degranulation. In addition, astrocytes could augment Akt, Erk1/2, and p38 activation in neutrophils. Conclusions Astrocytes differentially regulate neutrophil functions through

  1. Surface expression of HSP72 by LPS-stimulated neutrophils facilitates gammadeltaT cell-mediated killing.

    Science.gov (United States)

    Hirsh, Mark I; Hashiguchi, Naoyuki; Chen, Yu; Yip, Linda; Junger, Wolfgang G

    2006-03-01

    During inflammation and sepsis, accumulation of activated neutrophils causes lung tissue damage and organ failure. Effective clearance of neutrophils reduces the risk of organ failure; however, its mechanisms are poorly understood. Because lungs are rich in gammadeltaT cells, we investigated the physiological role of these cells in the protection of lung tissue from infiltrating neutrophils. In a mouse model of sepsis, we found that the lungs of survivors contained significantly higher numbers of gammadeltaT cells than those of mice that died from sepsis. The number of gammadeltaT cells correlated inversely with the number of neutrophils in the lungs and with the degree of lung tissue damage. LPS rapidly elicited the expression of heat shock protein (HSP) 72 on the surface of human neutrophils. Inhibitors of transcription, protein synthesis, and intracellular protein transport blocked HSP72 expression, indicating that de novo synthesis is required. gammadeltaT cells targeted and rapidly killed LPS-treated neutrophils through direct cell-to-cell contact. Pre-treatment with neutralizing antibodies to HSP72 diminished neutrophil killing. Our data indicate that HSP72 expression on the cell surface predisposes inflamed neutrophils to killing by gammadeltaT cells. This intercellular exchange may allow gammadeltaT cells to resolve inflammation and limit host tissue damage during sepsis.

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

    Science.gov (United States)

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

    2017-01-01

    In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis (M.tb) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site. PMID:28373877

  3. Neutrophils: important contributors to tumor progression and metastasis.

    Science.gov (United States)

    Swierczak, Agnieszka; Mouchemore, Kellie A; Hamilton, John A; Anderson, Robin L

    2015-12-01

    The presence of neutrophils in tumors has traditionally been considered to be indicative of a failed immune response against cancers. However, there is now evidence showing that neutrophils can promote tumor growth, and increasingly, the data support an active role for neutrophils in tumor progression to distant metastasis. Neutrophils have been implicated in promoting metastasis in cancer patients, where neutrophil numbers and neutrophil-related factors and functions have been associated with progressive disease. Nevertheless, the role of neutrophils in tumors, both at the primary and secondary sites, remains controversial, with some studies reporting their anti-tumor functions. This review will focus on the data demonstrating a role for neutrophils in both tumor growth and metastasis and will attempt to clarify the discrepancies in the literature.

  4. IL-17 attenuates the anti-apoptotic effects of GM-CSF in human neutrophils.

    Science.gov (United States)

    Dragon, Stéphane; Saffar, Arash Shoja; Shan, Lianyu; Gounni, Abdelilah Soussi

    2008-01-01

    Interleukin (IL)-17A is a pleiotropic, pro-inflammatory cytokine that is implicated in chronic inflammatory and degenerative disorders. IL-17 has been demonstrated to link activated T-lymphocyte with the recruitment of neutrophils at sites of inflammation, however whether IL-17 can mediate neutrophil survival and subsequently affect inflammatory responses has not fully been elucidated. In our study, we demonstrate that human peripheral blood and HL-60 differentiated neutrophils express mRNA and cell surface IL-17A receptor. IL-17A does not affect the rate of spontaneous neutrophil apoptosis, however significantly decreased granulocyte macrophage-colony stimulating factor (GM-CSF)-mediated survival by antagonizing the signal transduction pathways of p38, Erk1/2 and signal transducer and activator of transcription (STAT) 5B. These events were associated with reduced myeloid cell lymphoma-1 (Mcl-1) protein levels, increased translocation and aggregation of Bax to mitochondria, decreased mitochondrial transmembrane potential and in an increase in caspase-3/7 activity. These events were independent of increased Fas or soluble Fas ligand expression levels. Taken together, our findings suggest that IL-17 may regulate neutrophil homeostasis and favor the resolution of inflamed tissues by attenuating the delay in neutrophil apoptosis induced by inflammatory cytokines.

  5. Ebola Virus Disease Is Characterized by Poor Activation and Reduced Levels of Circulating CD16+ Monocytes.

    Science.gov (United States)

    Lüdtke, Anja; Ruibal, Paula; Becker-Ziaja, Beate; Rottstegge, Monika; Wozniak, David M; Cabeza-Cabrerizo, Mar; Thorenz, Anja; Weller, Romy; Kerber, Romy; Idoyaga, Juliana; Magassouba, N'Faly; Gabriel, Martin; Günther, Stephan; Oestereich, Lisa; Muñoz-Fontela, César

    2016-10-15

    A number of previous studies have identified antigen-presenting cells (APCs) as key targets of Ebola virus (EBOV), but the role of APCs in human Ebola virus disease (EVD) is not known. We have evaluated the phenotype and kinetics of monocytes, neutrophils, and dendritic cells (DCs) in peripheral blood of patients for whom EVD was diagnosed by the European Mobile Laboratory in Guinea. Acute EVD was characterized by reduced levels of circulating nonclassical CD16(+) monocytes with a poor activation profile. In survivors, CD16(+) monocytes were activated during recovery, coincident with viral clearance, suggesting an important role of this cell subset in EVD pathophysiology. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  6. Contribution of neutrophils to acute lung injury.

    Science.gov (United States)

    Grommes, Jochen; Soehnlein, Oliver

    2011-01-01

    Treatment of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), remain unsolved problems of intensive care medicine. ALI/ARDS are characterized by lung edema due to increased permeability of the alveolar-capillary barrier and subsequent impairment of arterial oxygenation. Lung edema, endothelial and epithelial injury are accompanied by an influx of neutrophils into the interstitium and broncheoalveolar space. Hence, activation and recruitment of neutrophils are regarded to play a key role in progression of ALI/ARDS. Neutrophils are the first cells to be recruited to the site of inflammation and have a potent antimicrobial armour that includes oxidants, proteinases and cationic peptides. Under pathological circumstances, however, unregulated release of these microbicidal compounds into the extracellular space paradoxically can damage host tissues. This review focuses on the mechanisms of neutrophil recruitment into the lung and on the contribution of neutrophils to tissue damage in ALI.

  7. Effector responses of bovine blood neutrophils against Escherichia coli: Role of NOD1/NF-κB signalling pathway.

    Science.gov (United States)

    Tan, Xun; Wei, Liang-Jun; Fan, Guo-Juan; Jiang, Ya-Nan; Yu, Xu-Ping

    2015-11-15

    Neutrophils use a broad array of pattern recognition receptors to sense and respond to invading pathogens and are important in the early control of acute bacterial infections. Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan. Reduced neutrophil NOD1 expression has been reported in periparturient dairy cows. The aim of this study was to investigate the role of NOD1 signalling in the early responses of bovine neutrophils to bacterial infections. Blood neutrophils from healthy heifers were preincubated for 2h with ML130, a selective inhibitor of NOD1-dependent nuclear factor-κB (NF-κB) activation. Thereafter, cells were cultured with live Escherichia coli for additional 30 min or subjected to Boyden chamber cell migration assay with E. coli in the lower chamber. Results showed that ML130 inhibited E. coli-induced NF-κB nuclear translocation. There was an indication, although not significant, that ML130 down-regulated gene expression of proinflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, chemokines IL-8 and C-X-C motif ligand 2 (CXCL2), and adhesion molecule CD62L, in E. coli-challenged neutrophils. Flow cytometry-based Annexin V staining revealed a considerable increase in neutrophil survival upon E. coli infection, an effect that was attenuated in the presence of ML130. Additionally, inhibition of NOD1/NF-κB signalling resulted in reduced migration of neutrophils to E. coli, and impaired phagocytosis, intracellular bacterial killing and reactive oxygen species production by neutrophils. These results indicate that NOD1/NF-κB pathway plays a crucial role in modulating neutrophil responses that are important for early control of infections. Approaches aiming at restoring neutrophil NOD1 function could be beneficial for prevention or treatment of coliform mastitis.

  8. IgA Complexes in Plasma and Synovial Fluid of Patients with Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via FcαRI.

    Science.gov (United States)

    Aleyd, Esil; Al, Marjon; Tuk, Cornelis W; van der Laken, Conny J; van Egmond, Marjolein

    2016-12-15

    Autoantibodies, including rheumatoid factor (RF), are an important characteristic of rheumatoid arthritis (RA). Interestingly, several studies reported a correlation between the presence of IgA autoantibodies and worse disease course. We demonstrated previously that triggering the IgA Fc receptor (FcαRI) on neutrophils results in neutrophil recruitment and the release of neutrophil extracellular traps (NETs). Because this can lead to tissue damage, we investigated whether IgA immune complexes in plasma and synovial fluid of RA patients activate neutrophils. RF isotypes were measured with ELISA, and immune complexes were precipitated using polyethylene glycol 6000. Isolated neutrophils were incubated with immune complexes, and activation and release of NETs were determined in the presence or absence of FcαRI-blocking Abs. Plasma and SF of RA patients contained IgM, IgG, and IgA RFs. Patient plasma IgA RF and IgM RF showed a strong correlation. No uptake of IgM and minimal endocytosis of IgG immune complexes by neutrophils was observed, in contrast to avid uptake of IgA complexes. Incubation of neutrophils with immune complexes resulted in the production of reactive oxygen species, as well as the release of NETs, lactoferrin, and chemotactic stimuli. Importantly, activation of neutrophils was reduced when FcαRI was blocked. Neutrophils were activated by IgA immune complexes, which suggests that neutrophils play a role in inducing joint damage in RA patients who have IgA autoantibody complexes, thereby increasing the severity of disease. Blocking FcαRI inhibited neutrophil activation and, as such, may represent an additional attractive novel therapeutic strategy for the treatment of RA.

  9. Proteases, neutrophils, and periodontitis: the NET effect.

    Science.gov (United States)

    Nauseef, William M

    2014-10-01

    Neutrophils exert potent antimicrobial activities in their role as first-line cellular defenders against infection. The synergistic and collective actions of oxidants and granule proteins, including serine proteases, support the microbial killing in phagosomes, where most neutrophil-mediated antimicrobial action occurs. In addition to phagocytosis, specific stimuli prompt neutrophils to extrude a matrix of DNA, histones, and granule proteins to produce neutrophil extracellular traps (NETs), which can trap microbes. Mice lacking the serine proteases necessary for NET production are more susceptible to infection, an observation suggesting that functional NETs are required for host protection. In this issue of the JCI, Sørensen and colleagues characterize neutrophils from a patient with Papillon-Lefèvre syndrome. The patient has an inactivating mutation in the gene encoding dipeptidyl peptidase I, resulting in neutrophils lacking elastase, a serine protease required for NET production. Despite the inability to form NETS, neutrophils from this patient killed pathogens in vitro, and the patient did not exhibit evidence of an increased propensity toward bacterial infections. Together, these results suggest that proteases in human neutrophils are dispensable for protection against bacterial infection and that the ability to generate NETs in vitro does not compromise host defense.

  10. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site...... RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.......Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...

  11. 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.

  12. Neutrophilic dermatoses in children.

    Science.gov (United States)

    Berk, David R; Bayliss, Susan J

    2008-01-01

    The neutrophilic dermatoses are rare disorders, especially in children, and are characterized by neutrophilic infiltrates in the skin and less commonly in extracutaneous tissue. The neutrophilic dermatoses share similar clinical appearances and associated conditions, including inflammatory bowel disease, malignancies, and medications. Overlap forms of disease demonstrating features of multiple neutrophilic dermatoses may be seen. The manuscript attempts to provide an up-to-date review of (i) classical neutrophilic dermatoses, focusing on distinctive features in children and (ii) neutrophilic dermatoses which may largely be pediatric or genodermatosis-associated (Majeed, SAPHO [synovitis, severe acne, sterile palmoplantar pustulosis, hyperostosis, and osteitis] syndrome, PAPA (pyogenic sterile arthritis, pyoderma gangrenosum, and acne), PFAPA (periodic fever with aphthous stomatitis, pharyngitis, and cervical adenopathy), and other periodic fever syndromes, and congenital erosive and vesicular dermatosis healing with reticulated supple scarring).

  13. Separating the roles of nitrogen and oxygen in high pressure-induced blood-borne microparticle elevations, neutrophil activation, and vascular injury in mice.

    Science.gov (United States)

    Yang, Ming; Bhopale, Veena M; Thom, Stephen R

    2015-08-01

    An elevation in levels of circulating microparticles (MPs) due to high air pressure exposure and the associated inflammatory changes and vascular injury that occur with it may be due to oxidative stress. We hypothesized that these responses arise due to elevated partial pressures of N2 and not because of high-pressure O2. A comparison was made among high-pressure air, normoxic high-pressure N2, and high-pressure O2 in causing an elevation in circulating annexin V-positive MPs, neutrophil activation, and vascular injury by assessing the leakage of high-molecular-weight dextran in a murine model. After mice were exposed for 2 h to 790 kPa air, there were over 3-fold elevations in total circulating MPs as well as subgroups bearing Ly6G, CD41, Ter119, CD31, and CD142 surface proteins-evidence of neutrophil activation; platelet-neutrophil interaction; and vascular injury to brain, omentum, psoas, and skeletal muscles. Similar changes were found in mice exposed to high-pressure N2 using a gas mixture so that O2 partial pressure was the same as that of ambient air, whereas none of these changes occurred after exposures to 166 kPa O2, the same partial pressure that occurs during high-pressure air exposures. We conclude that N2 plays a central role in intra- and perivascular changes associated with exposure to high air pressure and that these responses appear to be a novel form of oxidative stress.

  14. Vestitol Isolated from Brazilian Red Propolis Inhibits Neutrophils Migration in the Inflammatory Process: Elucidation of the Mechanism of Action.

    Science.gov (United States)

    Franchin, Marcelo; Cólon, David F; Castanheira, Fernanda V S; da Cunha, Marcos G; Bueno-Silva, Bruno; Alencar, Severino M; Cunha, Thiago M; Rosalen, Pedro L

    2016-04-22

    Vestitol is an isoflavonoid isolated from Brazilian red propolis with potential anti-inflammatory activity. This study investigated the mechanism of action of vestitol on the modulation of neutrophil migration in the inflammatory process. Pre-treatment with vestitol at 1, 3, or 10 mg/kg reduced LPS- or mBSA-induced neutrophil migration and the release of CXCL1/KC and CXCL2/MIP-2 in vivo. Likewise, pre-treatment with vestitol at 1, 3, or 10 μM reduced the levels of CXCL1/KC and CXCL2/MIP-2 in macrophage supernatants in vitro. Moreover, the administration of vestitol (10 mg/kg) reduced leukocyte rolling and adherence in the mesenteric microcirculation of mice. The pre-treatment with vestitol (10 mg/kg) in iNOS(-/-) mice did not block its activity concerning neutrophil migration. With regard to the activity of vestitol on neutrophils isolated from the bone marrow of mice, there was a reduction on the chemotaxis of CXCL2/MIP-2 or LTB4-induced neutrophils and on calcium influx after pre-treatment with the compound at 3 or 10 μM. There was no change in CXCR2 expression by neutrophils treated with vestitol at 10 μM. These findings demonstrate that vestitol is a promising novel anti-inflammatory agent.

  15. Cryptococcus neoformans modulates extracellular killing by neutrophils.

    Science.gov (United States)

    Qureshi, Asfia; Grey, Angus; Rose, Kristie L; Schey, Kevin L; Del Poeta, Maurizio

    2011-01-01

    We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike Candida albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. We monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the conditioned medium and found that pre-incubation with live but not "heat-killed" fungal cells significantly inhibits further killing activity of the medium. We then studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption-ionization tissue imaging in infected lung we found that similar to previous observations in the isogenic wild-type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells, but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells.

  16. Cryptococcus neoformans modulates extracellular killing by neutrophils

    Directory of Open Access Journals (Sweden)

    Asfia eQureshi

    2011-09-01

    Full Text Available We recently established a key role for host sphingomyelin synthase (SMS in the regulation of the killing activity of neutrophils against Cryptococcus neoformans. In this work, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and NK cells (Tgε26 mice. To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike C. albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. Next, we monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the medium and found that pre-incubation with live but not heat-killed fungal cells significantly inhibits further killing activity of the medium. We next studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption-ionization (MALDI tissue imaging in infected lung we found that similarly to previous observations in the isogenic wild type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells.

  17. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.

    Science.gov (United States)

    Corleis, Björn; Korbel, Daniel; Wilson, Robert; Bylund, Johan; Chee, Ronnie; Schaible, Ulrich E

    2012-07-01

    Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

  18. [Ultrastructural location of enzymes in peripheral blood neutrophils and in cerebrospinal fluid neutrophils in neuroinfections].

    Science.gov (United States)

    Skotarczak, B

    1993-01-01

    Using cytochemical methods the location and activity were determined of alkaline phosphatase, ATP-ase and succinate dehydrogenase as representative enzymes for the metabolic processes in neutrophils isolated from blood and cerebrospinal fluid (CSF) of patients with meningococcal meningoencephalitis as compared with peripheral blood neutrophils in a control group. The study showed presence of phosphatase on the membranes of many intracellular structures. The activity of the enzymes was higher than in the control group in the membranes of neutrophils in blood and CSF. This is explained as an effect of action of the chemotactic factor on the cell membrane and activation of the cell to movements and phagocytosis. ATP-ase activity in peripheral blood neutrophils in controls was found in all membranous structures in the cell. However, in peripheral blood neutrophils and CSF neutrophils in the acute stage of the disease the active enzyme was noted, in the first place, in cell membranes and digesting vacuoles, which reflected probably the direction of metabolic processes for phagocytosis and destroying of bacteria. The activity of succinate dehydrogenase was found in mitochondrial membranes. Peripheral blood and CSF neutrophils showed a high activity of the enzyme. In the CSF cells in acute phase atypical sites of succinate dehydrogenase activity were noted, which was explained as a sign of cell destruction.

  19. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  20. THE PHENOTYPE AND METABOLISM RELATIONSHIP OF BLOOD NEUTROPHILS IN PATIENTS WITH WIDESPREAD PURULENT PERITONITIS IN THE POSTOPERATIVE PERIOD DYNAMICS

    Directory of Open Access Journals (Sweden)

    A. A. Savchenko

    2017-01-01

    Full Text Available The aim of the study was to examine the relationship of the phenotype and metabolism of neutrophils in patients with widespread purulent peritonitis (WPP in the dynamics of the postoperative period. The study involved 27 patients with acute surgical diseases and injuries of abdominal organs complicated by WPP. Blood sampling was performed prior to surgery (pre-operative period and at 7, 14 and 24 day post-operative period. As controls 67 respect healthy people were examined. Research blood neutrophilic granulocytes phenotype was performed by f low cytometry using a direct immunof luorescence whole peripheral blood. The levels of surface receptor expression was assessed by the mean f luorescence intensity. The NADand NADP-dependent dehydrogenases activity in the blood neutrophils studied using bioluminescence method. It was found that in patients with WPP in the preoperative period in the peripheral blood increased content of CD62L+-, HLA-DR+and CD64+-neutrophils. High levels of CD62L+-cells stored within 24 postoperative days, whereas the amount of HLA-DR+and CD64+-neutrophils on 24 postoperative day is reduced to the level of controls. The dynamics of changes in the content of CD64+-cells in the peripheral blood of patients with WPP corresponds to the expression level of CD64-receptor on the membrane of neutrophilic granulocytes. The metabolism of blood neutrophils in patients with WPP in the preand postoperative period is characterized by high intensity of the substrate stream on the citric acid cycle, low activity of NADP-dependent glutamate dehydrogenase and aerobic reaction of lactate dehydrogenase. In the preoperative period and within 14 days of the postoperative period in neutrophil granulocytes of the patients revealed high activity of anaerobic lactate dehydrogenase reaction characterized by increased activity of anaerobic glycolysis. In the late postoperative period the intensity of anaerobic energy in the neutrophils of patients with

  1. Clinical relevance of P-glycoprotein activity on peripheral blood mononuclear cells and polymorphonuclear neutrophils to methotrexate in systemic lupus erythematosus patients.

    Science.gov (United States)

    García-Carrasco, Mario; Mendoza-Pinto, Claudia; Macías-Díaz, Salvador; Etchegaray-Morales, Ivet; Méndez-Martínez, Socorro; Soto-Santillán, Pamela; Pérez-Romano, Beatriz; Jiménez-Herrera, Erick A; Guzmán-Ruiz, Omar; Ruiz-Argüelles, Alejandro

    2017-06-14

    To elucidate the relationship between P-glycoprotein activity on peripheral blood leukocytes of systemic lupus erythematosus (SLE) patients with lupus arthritis and the clinical response to methotrexate. An observational study was made in patients with SLE according to ACR criteria 1997 who had arthralgia and arthritis and received methotrexate for ≥3 months. Methotrexate responders and non-responders were compared according to the Clinical Disease Activity Index. Mononuclear cells and polymorphonuclear neutrophils were isolated from SLE patients and P-glycoprotein expression was measured using the relative fluorescence index and percentage of positive cells. The chi-square test was used to compare P-glycoprotein activity between responders and non-responders. Thirty-two patients with a mean age of 45.4 ± 10.7 years were included: 34.4% had a response to methotrexate and 65.6% did not. Mean relative fluorescence units of both mononuclear cells and polymorphonuclear neutrophils were significantly lower in patients with a good response (7.0 ± 4.3 vs. 9.6 ± 3.8; p = 0.041 and 4.2 ± 3.5 vs. 7.6 ± 4.0; p = 0.004). The prevalence of low fluorescence levels (P-glycoprotein activity of both mononuclear cells and polymorphonuclear neutrophils, was higher in methotrexate responders than in non-responders (27.3 vs. 4.8%; p = 0.10 and 81.8 vs. 23.8%; p = 0.003, respectively). In SLE patients with joint involvement treated with methotrexate, P-glycoprotein activity was higher in responders to methotrexate than in non-responders. Further studies are required to determine the mechanisms behind this finding and whether P-glycoprotein activity mediates alterations in methotrexate efficacy.

  2. Invariant NKT cells promote skin wound healing by preventing a prolonged neutrophilic inflammatory response.

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    Tanno, Hiromasa; Kawakami, Kazuyoshi; Kanno, Emi; Suzuki, Aiko; Takagi, Naoyuki; Yamamoto, Hideki; Ishii, Keiko; Imai, Yoshimichi; Maruyama, Ryoko; Tachi, Masahiro

    2017-09-23

    The wound-healing process consists of the inflammation, proliferation, and remodeling phases. In chronic wounds, the inflammation phase is prolonged with persistent neutrophil infiltration. The inflammatory response is critically regulated by cytokines and chemokines that are secreted from various immune cells. Recently, we showed that skin wound healing was delayed and the healing process was impaired under conditions lacking invariant natural killer T (iNKT) cells, an innate immune lymphocyte with potent immuno-regulatory activity. In the present study, we investigated the effect of iNKT cell deficiency on the neutrophilic inflammatory response during the wound healing process. Neutrophil infiltration was prolonged in wound tissue in mice genetically lacking iNKT cells (Jα18KO mice) compared with wild-type (WT) control mice on days 1 and 3 after wounding. MIP-2, KC, and IL-17A were produced at a significantly higher level in Jα18KO mice than in WT mice. In addition, neutrophil apoptosis was significantly reduced in the wound tissue in Jα18KO mice compared with WT mice. Treatment with either anti-IL-17A mAb, anti-Gr-1 mAb, or neutrophil elastase inhibitor reversed the impaired wound healing in Jα18KO mice. These results suggest that iNKT cells may promote the wound healing process through preventing the prolonged inflammatory response mediated by neutrophils. This article is protected by copyright. All rights reserved. © 2017 by the Wound Healing Society.

  3. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

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    Cindy Franklin

    Full Text Available Chronic graft-versus-host disease (cGVHD is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

  4. Ensemble models of neutrophil trafficking in severe sepsis.

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    Sang Ok Song

    Full Text Available A hallmark of severe sepsis is systemic inflammation which activates leukocytes and can result in their misdirection. This leads to both impaired migration to the locus of infection and increased infiltration into healthy tissues. In order to better understand the pathophysiologic mechanisms involved, we developed a coarse-grained phenomenological model of the acute inflammatory response in CLP (cecal ligation and puncture-induced sepsis in rats. This model incorporates distinct neutrophil kinetic responses to the inflammatory stimulus and the dynamic interactions between components of a compartmentalized inflammatory response. Ensembles of model parameter sets consistent with experimental observations were statistically generated using a Markov-Chain Monte Carlo sampling. Prediction uncertainty in the model states was quantified over the resulting ensemble parameter sets. Forward simulation of the parameter ensembles successfully captured experimental features and predicted that systemically activated circulating neutrophils display impaired migration to the tissue and neutrophil sequestration in the lung, consequently contributing to tissue damage and mortality. Principal component and multiple regression analyses of the parameter ensembles estimated from survivor and non-survivor cohorts provide insight into pathologic mechanisms dictating outcome in sepsis. Furthermore, the model was extended to incorporate hypothetical mechanisms by which immune modulation using extracorporeal blood purification results in improved outcome in septic rats. Simulations identified a sub-population (about 18% of the treated population that benefited from blood purification. Survivors displayed enhanced neutrophil migration to tissue and reduced sequestration of lung neutrophils, contributing to improved outcome. The model ensemble presented herein provides a platform for generating and testing hypotheses in silico, as well as motivating further experimental

  5. Candida albicans escapes from mouse neutrophils.

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    Ermert, David; Niemiec, Maria J; Röhm, Marc; Glenthøj, Andreas; Borregaard, Niels; Urban, Constantin F

    2013-08-01

    Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse 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 that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.

  6. Neutrophil Cathepsin G, but Not Elastase, Induces Aggregation of MCF-7 Mammary Carcinoma Cells by a Protease Activity-Dependent Cell-Oriented Mechanism

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    Satoru Yui

    2014-01-01

    Full Text Available We previously found that a neutrophil serine protease, cathepsin G, weakens adherence to culture substrates and induces E-cadherin-dependent aggregation of MCF-7 human breast cancer cells through its protease activity. In this study, we examined whether aggregation is caused by degradation of adhesion molecules on the culture substrates or through an unidentified mechanism. We compared the effect of treatment with cathepsin G and other proteases, including neutrophil elastase against fibronectin- (FN- coated substrates. Cathepsin G and elastase potently degraded FN on the substrates and induced aggregation of MCF-7 cells that had been subsequently seeded onto the substrate. However, substrate-bound cathepsin G and elastase may have caused cell aggregation. After inhibiting the proteases on the culture substrates using the irreversible inhibitor phenylmethylsulfonyl fluoride (PMSF, we examined whether aggregation of MCF-7 cells was suppressed. PMSF attenuated cell aggregation on cathepsin G-treated substrates, but the effect was weak in cells pretreated with high concentrations of cathepsin G. In contrast, PMSF did not suppress cell aggregation on elastase-treated FN. Moreover, cathepsin G, but not elastase, induced aggregation on poly-L-lysine substrates which are not decomposed by these enzymes, and the action of cathepsin G was nearly completely attenuated by PMSF. These results suggest that cathepsin G induces MCF-7 aggregation through a cell-oriented mechanism.

  7. The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

    LENUS (Irish Health Repository)

    Hayes, Elaine

    2011-04-01

    Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

  8. The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

    LENUS (Irish Health Repository)

    Hayes, Elaine

    2012-02-01

    Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

  9. Neutrophil-mediated phagocytosis of Staphylococcus aureus

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    Jos A.G. Van Strijp

    2014-09-01

    Full Text Available For invading staphylococci, phagocytosis an killing bij human neutrophils is the biggest threat. Neutrophils are the only cells that can effectively kill staphylococci by engulfment and subsequent bombardment with proteases, amidases, antimicrobial peptides and proteins in concert with reactive oxygen species that are generated during the metabolic burst.Both complement and antibodies are crucial for effective uptake and neutrophil activation. S. aureus is not an innocent bystander in this process. It actively secretes several proteins to impair every single step in this process from receptor modulation, to complement inhibition to neutrophil lysis to protease, antimicrobial peptide inhibition and resistance to reactive oxygen species. For the design of future novel antimicrobial strategies: therapeutic antibodies, vaccines, novel antibiotics, all this should be taken into account. Still the best way to treat diseases is to help to enhance the natural defence mechanism that are already in place.

  10. Non-steroidal anti-inflammatory drug, nabumetone, prevents indometacin-induced gastric damage via inhibition of neutrophil functions.

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    Ishiwata, Yoshiro; Okamoto, Masayuki; Yokochi, Shoji; Hashimoto, Hiroyuki; Nakamura, Takashi; Miyachi, Atsushi; Naito, Yuji; Yoshikawa, Toshikazu

    2003-02-01

    Nabumetone is a non-steroidal anti-inflammatory drug (NSAID). It works as a prodrug and is extensively metabolized to an active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA). It is well known that neutrophil infiltration and activation are critical in the pathogenesis of NSAID-induced gastric injury, and nabumetone shows less incidence of gastrointestinal irritancy. We examined the effects of nabumetone on neutrophil activation and on indometacin-induced gastric damage. In the indometacin-induced gastric mucosal injury, rats were treated with indometacin and then nabumetone or 6MNA was orally administered. Nabumetone prevented gastric damage accompanied by the reduction of neutrophil infiltration into gastric mucosa, but such an effect was not observed with 6MNA. Nabumetone reduced the formyl methionyl leucyl phenylalanine (fMLP)-induced respiratory burst of human neutrophils to 30% of the control level in-vitro, but 6MNA did not. In addition, nabumetone prevented the fMLP-induced migration of neutrophils. Nabumetone did not inhibit O2- generation in the xanthine-xanthine oxidase system. These results suggest that nabumetone prevents gastric damage induced by the active metabolite, 6MNA, via the suppression of neutrophil activation in gastric mucosa.

  11. Simvastatin antagonizes CD40L secretion, CXC chemokine formation, and pulmonary infiltration of neutrophils in abdominal sepsis.

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    Zhang, Su; Rahman, Milladur; Zhang, Songen; Qi, Zhongquan; Thorlacius, Henrik

    2011-05-01

    Statins have been reported to exert anti-inflammatory actions and protect against septic organ dysfunction. Herein, we hypothesized that simvastatin may attenuate neutrophil activation and lung damage in abdominal sepsis. Male C57BL/6 mice were pretreated with simvastatin (0.5 or 10 mg/kg) before CLP. In separate groups, mice received an anti-CD40L antibody or a CXCR2 antagonist (SB225002) prior to CLP. BALF and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 and CD40L expression on neutrophils and platelets, as well as soluble CD40L in plasma. Simvastatin decreased CLP-induced neutrophil infiltration and edema formation in the lung. Moreover, Mac-1 expression increased on septic neutrophils, which was significantly attenuated by simvastatin. Inhibition of CD40L reduced CLP-induced up-regulation of Mac-1 on neutrophils. Simvastatin prevented CD40L shedding from the surface of platelets and reduced circulating levels of CD40L in septic mice. CXC chemokine-induced migration of neutrophils in vitro was decreased greatly by simvastatin. Moreover, simvastatin abolished CLP-evoked formation of CXC chemokines in the lung, and a CXCR2 antagonist attenuated pulmonary accumulation of neutrophils. Our data suggest that the inhibitory effect of simvastatin on pulmonary accumulation of neutrophils may be related to a reduction of CD40L secretion into the circulation, as well as a decrease in CXC chemokine formation in the lung. Thus, these protective mechanisms help to explain the beneficial actions exerted by statins, such as simvastatin, in sepsis.

  12. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

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    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.

  13. Transcriptome kinetics of circulating neutrophils during human experimental endotoxemia.

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    Stan de Kleijn

    Full Text Available Polymorphonuclear cells (neutrophils play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4 administered a single dose of endotoxin (LPS, 2 ng/kg iv. In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.

  14. Heparins with reduced anti-coagulant activity reduce myocardial reperfusion injury.

    Science.gov (United States)

    Barry, William H; Kennedy, Thomas P

    2011-05-01

    Heparin which is desulfated at the 2-O and 3-O positions (ODSH) has reduced anti-coagulant properties, and reduced interaction with heparin antibodies. Because of the reduced anti-coagulant effect, ODSH can be safely administered to animals and humans intravenously at doses up to 20 mg/kg, resulting in a serum concentration of up to 250µg/ml. Administration of ODSH causes a 35% reduction in infarct size in dogs and pigs subjected to coronary artery occlusion and reperfusion when given 5 min before reperfusion. ODSH has anti-inflamatory effects, manifest as a decrease in neutrophil infiltration into ischemic tissue at high doses, but this effect does not entirely account for the reduction in infarct size. ODSH decreases Na(+) and Ca(2+) loading in isolated cardiac myocytes subjected to simulated ischemia. This effect appears due to an ODSH-induced reduction in an enhanced Na(+) influx via the Na channel in the membrane of cardiac myocyes caused by oxygen radicals generated during ischemia and reperfusion. Reduction in Na(+) influx decreases Ca(2+) loading by reducing Ca2(+) influx via Na/Ca exchange, thus reducing Ca(2+) - dependent reperfusion injury. ODSH does not appear to interact with antibodies to the heparin/platelet factor 4 complex, and does not cause heparin-induced thrombocytopenia. Because of these therapeutic and safety considerations, ODSH would appear to be a promising heparin derivative for prevention of reperfusion injury in humans undergoing thrombolytic or catheter-based reperfusion for acute myocardial infarction. The review article discussed the use of heparin and the discussion of some of the important patents, including: US6489311; US7478358; PCTUS2008070836 and PCTUS2009037836.

  15. Neutrophils in asthma--a review.

    Science.gov (United States)

    Ciepiela, Olga; Ostafin, Magdalena; Demkow, Urszula

    2015-04-01

    Asthma is a chronic inflammatory disease, with an array of cells involved in the pathogenesis of the disease. The role of neutrophils in the development of bronchial asthma is found to be complex, as they may trigger activation of immunocompetent cells and are a potent source of free oxygen radicals and enzymes participating in airway remodeling. The review highlights the role of neutrophils in bronchial asthma.

  16. TAK1 contributes to the enhanced responsiveness of LTB(4)-treated neutrophils to Toll-like receptor ligands.

    Science.gov (United States)

    Gaudreault, Éric; Paquet-Bouchard, Carine; Fiola, Stéphanie; Le Bel, Manon; Lacerte, Patricia; Shio, Marina Tiemi; Olivier, Martin; Gosselin, Jean

    2012-11-01

    Pattern-recognition receptors such as Toll-like receptors (TLRs) are essential sensors implicated in the early and efficient innate immune response against pathogens. We have previously demonstrated that leukotriene B(4)(LTB(4)) has the capacity to enhance leukocyte responses to TLR9 ligands and to control viral infection. In this report, we provide evidence that LTB(4) treatment of human neutrophils leads to a potentiation in proinflammatory cytokine secretion induced by various myeloid differentiation factor 88-dependent TLR agonists. LTB(4) failed to enhance TLR mRNA levels as well as expression of TLR2 and TLR4 receptors, suggesting that LTB(4) acts through intracellular mechanism(s) to potentiate neutrophil responses to TLR ligands. We found that while IRAK can be activated by LTB(4), this process is dispensable to LTB(4) to potentiate neutrophil responses to TLR ligands since pretreatment of neutrophils with IRAK1/4 inhibitor did not affect its potentiating effects. However, our data clearly show that LTB(4) treatment of neutrophils led to the phosphorylation of downstream signaling molecules, TAK1 and p38, a process found essential to observe an increased secretion of cytokines by neutrophils activated with TLR ligands. Pretreatment of neutrophils with TAK1 or p38 kinase inhibitors strongly repressed the effect of LTB(4) on cytokine synthesis by neutrophils stimulated with LTA, LPS or CpG. The same pattern was observed in agonist-treated human embryonic kidney 293 cells transfected with TAK1-targeting siRNA where secretion of IL-8 was significantly reduced to basal levels. These results indicate that TAK1 and p38 kinases appear to be central in the 'priming effect' of LTB(4) on neutrophils to enhance response to TLR ligands.

  17. Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein.

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    Gabig, T G; English, D; Akard, L P; Schell, M J

    1987-02-05

    Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

  18. Astragaloside IV alleviates E. coli-caused peritonitis via upregulation of neutrophil influx to the site of infection.

    Science.gov (United States)

    Huang, Ping; Lu, Xiaoyan; Yuan, Baohong; Liu, Tao; Dai, Liangcheng; Liu, Yunjun; Yin, Hui

    2016-10-01

    Astragaloside IV (AS-IV), an active saponin purified from Astragali Radix, has been identified with broad biological and pharmacological activities. In the present study, we continue to explore the potential effect of AS-IV on antibacterial response using an acute E. coli peritoneal infection model. Our findings implied that administration of AS-IV decreases mortality in mice challenged by lethal E. coli infection. The protection of AS-IV was related to promotion of neutrophil extravasation into the peritoneum and bacterial clearance. Toll-like receptor (TLR) activation in neutrophils has been reported to reduce CXCR2 expression and subsequent neutrophil migration. Our data indicated that AS-IV prevented the reduction of CXCR2 expression and neutrophil migration induced by LPS, the activator for TLR4. Moreover, we found that AS-IV blocks LPS-induced suppression of CXCR2 on neutrophils by inhibiting the expression of G protein-coupled receptor kinase-2 (GRK2), an agonist that regulates desensitization and internalization of chemokine receptors. Taken together, these data propose that AS-IV, through modulating GRK2-CXCR2 signal in neutrophils, offers an essential efficacy on host antibacterial immunity.

  19. Store-operated calcium signaling in neutrophils.

    Science.gov (United States)

    Clemens, Regina A; Lowell, Clifford A

    2015-10-01

    Calcium signals in neutrophils are initiated by a variety of cell-surface receptors, including formyl peptide and other GPCRs, FcRs, and integrins. The predominant pathway by which calcium enters immune cells is termed SOCE, whereby plasma membrane CRAC channels allow influx of extracellular calcium into the cytoplasm when intracellular ER stores are depleted. The identification of 2 key families of SOCE regulators, STIM calcium "sensors" and ORAI calcium channels, has allowed for genetic manipulation of SOCE pathways and provided valuable insight into the molecular mechanism of calcium signaling in immune cells, including neutrophils. This review focuses on our current knowledge of the molecules involved in neutrophil SOCE and how study of these molecules has further informed our understanding of the role of calcium signaling in neutrophil activation.

  20. Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis patients

    NARCIS (Netherlands)

    Al Laham, F.; Kaelsch, A. -I.; Heinrich, L.; Birck, R.; Kallenberg, C. G. M.; Heeringa, P.; Yard, B.

    2010-01-01

    P>Leucocyte transendothelial migration is strictly regulated to prevent undesired inflammation and collateral damage of endothelial cells by activated neutrophils/monocytes. We hypothesized that in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV) patients' dysregulation

  1. Ménage-à-trois: The ratio of bicarbonate to CO2 and the pH regulate the capacity of neutrophils to form NETs

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    Christian Maueröder

    2016-12-01

    Full Text Available In this study we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (PMA, ionomycin, monosodium urate and LPS. In contrast, an acidic environment impaired neutrophil extracellular trap formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to neutrophil extracellular trap formation. Inflammatory foci are characterized by an acidic environment. Our data indicates that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover our findings highlight the necessity for strict pH control during assays of neutrophil extracellular trap formation.

  2. Enhancement of neutrophil-mediated killing of Plasmodium falciparum asexual blood forms by fatty acids: importance of fatty acid structure.

    Science.gov (United States)

    Kumaratilake, L M; Ferrante, A; Robinson, B S; Jaeger, T; Poulos, A

    1997-10-01

    Effects of fatty acids on human neutrophil-mediated killing of Plasmodium falciparum asexual blood forms were investigated by using a quantitative radiometric assay. The results showed that the antiparasitic activity of neutrophils can be greatly increased (>threefold) by short-term treatment with fatty acids with 20 to 24 carbon atoms and at least three double bonds. In particular, the n-3 polyenoic fatty acids, eicosapentaenoic and docosahexaenoic acids, and the n-6 fatty acid, arachidonic acid, significantly enhanced neutrophil antiparasitic activity. This effect was >1.5-fold higher than that induced by an optical concentration of the known agonist cytokine tumor necrosis factor alpha (TNF-alpha). At suboptimal concentrations, the combination of arachidonic acid and TNF-alpha caused a synergistic increase in neutrophil-mediated parasite killing. The fatty acid-induced effect was independent of the availability of serum opsonins but dependent on the structure of the fatty acids. The length of the carbon chain, degree of unsaturation, and availability of a free carboxyl group were important determinants of fatty acid activity. The fatty acids which increased neutrophil-mediated killing primed the enhanced superoxide radical generation of neutrophils in response to P. falciparum as detected by chemiluminescence. Scavengers of oxygen radicals significantly reduced the fatty acid-enhanced parasite killing, but cyclooxygenase and lipoxygenase inhibitors had no effect. These findings have identified a new class of immunoenhancers that could be exploited to increase resistance against Plasmodium species.

  3. Neutrophil biology: an update

    OpenAIRE

    Kobayashi, Yoshiro

    2015-01-01

    Neutrophil extracellular traps (NETs) are involved in bacterial killing as well as autoimmunity, because NETs contain proteases, bactericidal peptides, DNA and ribonucleoprotein. NETs are formed via a novel type of cell death called NETosis. NETosis is distinct from apoptosis, but it resembles necrosis in that both membranes are not intact so that they allow intracellular proteins to leak outside of the cells. Removal of NETs and neutrophils undergoing NETosis by phagocytes and its subsequent...

  4. Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

    Energy Technology Data Exchange (ETDEWEB)

    Lee, T. (Oak Ridge Associated Univ., TN); Malone, B.; Wasserman, S.I.; Fitzgerald, V.; Snyder, F.

    1982-04-29

    Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (l-alkyl-2-acetyl-sn-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the O-alkyl linkage in lipids), l-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-sn-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-sn-glycero-3-phospho-choline synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.

  5. Deranged Bioenergetics and Defective Redox Capacity in T Lymphocytes and Neutrophils Are Related to Cellular Dysfunction and Increased Oxidative Stress in Patients with Active Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Ko-Jen Li

    2012-01-01

    Full Text Available Urinary excretion of N-benzoyl-glycyl-Nε-(hexanonyllysine, a biomarker of oxidative stress, was higher in 26 patients with active systemic lupus erythematosus (SLE than in 11 non-SLE patients with connective tissue diseases and in 14 healthy volunteers. We hypothesized that increased oxidative stress in active SLE might be attributable to deranged bioenergetics, defective reduction-oxidation (redox capacity, or other factors. We demonstrated that, compared to normal cells, T lymphocytes (T and polymorphonuclear neutrophils (PMN of active SLE showed defective expression of facilitative glucose transporters GLUT-3 and GLUT-6, which led to increased intracellular basal lactate and decreased ATP production. In addition, the redox capacity, including intracellular GSH levels and the enzyme activity of glutathione peroxidase (GSH-Px and γ-glutamyl-transpeptidase (GGT, was decreased in SLE-T. Compared to normal cells, SLE-PMN showed decreased intracellular GSH levels, and GGT enzyme activity was found in SLE-PMN and enhanced expression of CD53, a coprecipitating molecule for GGT. We conclude that deranged cellular bioenergetics and defective redox capacity in T and PMN are responsible for cellular immune dysfunction and are related to increased oxidative stress in active SLE patients.

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

    Directory of Open Access Journals (Sweden)

    Yuandani

    2013-01-01

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

  7. Paracoccidioides brasiliensis killing by IFN-gamma, TNF-alpha and GM-CSF activated human neutrophils: role for oxygen metabolites.

    Science.gov (United States)

    Rodrigues, D R; Dias-Melicio, L A; Calvi, S A; Peraçoli, M T S; Soares, A M V C

    2007-02-01

    Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against the fungus and several papers show the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, the studies focusing on polymorphonuclear neutrophils (PMNs) antifungal functions are scarcer. Thus, the objective of the present paper was to assess the capacity of human PMNs to kill virulent P. brasiliensis strain in vitro, before and after priming with different cytokines. Moreover, the involvement of oxygen metabolites in this activity was evaluated. Nonactivated cells failed to exhibit antifungal activity. However, when these cells were IFN-gamma, TNF-alpha or GM-CSF activated, a significative fungicidal activity was detected. This process was significantly inhibited when P. brasiliensis challenge occurred in presence of catalase (CAT - a scavenger of H2O2) and superoxide dismutase (SOD - a scavenger of superoxide anion). From these results it is concluded that cytokines activation is required for P. brasiliensis killing by human PMNs, and that H2O2 and superoxide anion participate as effectors molecules in this process.

  8. Antioxidant activity of Calendula officinalis extract: inhibitory effects on chemiluminescence of human neutrophil bursts and electron paramagnetic resonance spectroscopy.

    Science.gov (United States)

    Braga, Pier Carlo; Dal Sasso, Monica; Culici, Maria; Spallino, Alessandra; Falchi, Mario; Bertelli, Aldo; Morelli, Roberto; Lo Scalzo, Roberto

    2009-01-01

    There is growing interest in natural chemical compounds from aromatic, spicy, medicinal and other plants with antioxidant properties in order to find new sources of compounds inactivating free radicals generated by metabolic pathways within body tissue and cells, mainly polymorphonuclear leukocytes (PMNs) whose overregulated recruitment and activation generate a large amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to an imbalance of redox homeostasis and oxidative stress. The aim of this study was to examine whether a propylene glycol extract of Calendula officinalis interferes with ROS and RNS during the PMN respiratory bursts, and to establish the lowest concentration at which it still exerts antioxidant activity by means of luminol-amplified chemiluminescence. Electron paramagnetic resonance (EPR) spectroscopy was also used in order to confirm the activity of the C. officinalis extract. The C. officinalis extract exerted its anti-ROS and anti-RNS activity in a concentration-dependent manner, with significant effects being observed at even very low concentrations: 0.20 microg/ml without L-arginine, 0.10 microg/ml when L-arginine was added to the test with phorbol 12-myristate 13-acetate and 0.05 microg/ml when it was added to the test with N-formyl-methionyl-leucyl-phenylalanine. The EPR study confirmed these findings, 0.20 microg/ml being the lowest concentration of C. officinalis extract that significantly reduced 2,2-diphenyl-1-picrylhydrazyl. These findings are interesting for improving the antioxidant network and restoring the redox balance in human cells with plant-derived molecules as well as extending the possibility of antagonizing the oxidative stress generated in living organisms when the balance is in favor of free radicals as a result of the depletion of cell antioxidants.

  9. The Proteolytically Stable Peptidomimetic Pam-(Lys-ßNSpe)6-NH2 Selectively Inhibits Human Neutrophil Activation via Formyl Peptide Receptor 2

    DEFF Research Database (Denmark)

    Skovbakke, Sarah Line; Heegaard, Peter M. H.; Larsen, Camilla J.

    2015-01-01

    of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release...

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

    Science.gov (United States)

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

    2017-09-01

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

  11. CYTOKINE REGULATION OF RESPIRATORY BURST IN BLOOD NEUTROPHILS FOR PREDICTION OF ABDOMINAL SEPSIS IN PATIENTS WITH EXTENDED PURULENT PERITONITIS

    Directory of Open Access Journals (Sweden)

    A. A. Savchenko

    2016-01-01

    Full Text Available The aim of this study was to evaluate correlations between cytokine concentrations and parameters of respiratory burst in blood neutrophils, in order to predict potential development of abdominal sepsis in the patients with extended purulent peritonitis (EPP. The study involved fifteen patients with EPP. Peripheral blood samples were taken on the first day post-admissiona. Abdominal sepsis was diagnosed in thirty-five patients (70% within 5…10 days after surgical intervention. Clinical complications were absent in fifteen patients (30%. Sixty-seven healthy individuals were examined as a control group. Evaluation of the cytokine concentrations was performed by ELISA technique. The degree of respiratory burst in blood neutrophils was measured by means of chemiluminescence assay.It is revealed that the EPP patients exhibited increased levels of serum proinflammatory cytokines and IFNγ, along with higher intensity of respiratory burst in blood neutrophils, due to activated synthesis of of both primary and secondary reactive oxygen species (ROS. The EPP patients who later developed abdominal sepsis showed reduced spontaneous synthesis of primary ROS and increased spontaneous synthesis of secondary ROS. Upon zymosan induction of neutrophils, both primary and secondary ROS levels proved to be similar in the EPP subgroups with or without subsequent sepsis. EPP patients with uncomplicated post-surgical period still exhibited a predominant regulation of respiratory burst of neutrophils by IFNγ activity. Meanwhile, the neutrophil respiratory burst was correlated with TNFα and IL-6 in those patients who further developed abdominal sepsis. A stimulatory effect of IFNγ and a presumably inhibitory action of TNFα and IL-6 upon respiratory burst of blood neutrophils in EPP patients are associated with a release of large cytokine amounts during acute immune inflammatory events, and migration of activated cells to the inflammatory focus. In particular, the

  12. Quercetin inhibits degranulation and superoxide generation in PMA stimulated neutrophils

    OpenAIRE

    2012-01-01

    Activated neutrophils represent the main source of myeloperoxidase (MPO), superoxide (SO) and subsequently derived oxygen metabolites. They have important microbicidal activities, however in inflammatory conditions they may secondarily attack surrounding tissues. Overproduction of reactive oxygen species, prolonged or excessive liberation of MPO and other effective yet also toxic substances from neutrophils may participate in disturbed apoptosis, intensify the inflammatory processes and resul...

  13. Effects of Acer okamotoanum sap on the function of polymorphonuclear neutrophilic leukocytes in vitro and in vivo.

    Science.gov (United States)

    An, Beum-Soo; Kang, Ji-Houn; Yang, Hyun; Yang, Mhan-Pyo; Jeung, Eui-Bae

    2013-02-01

    Sap is a plant fluid that primarily consists of water and small amounts of mineral elements, sugars, hormones and other nutrients. Acer mono (A. mono) is an endemic Korean mono maple which was recently suggested to have health benefits due to its abundant calcium and magnesium ion content. In the present study, we examined the effects of sap from Acer okamotoanum (A. okamotoanum) on the phagocytic response of mouse neutrophils in vivo and rat and canine neutrophils in vitro. We tested the regulation of phagocytic activity, oxidative burst activity (OBA) and the levels of filamentous polymeric actin (F-actin) in the absence and presence of dexamethasone (DEX) in vitro and in vivo. Our results showed that DEX primarily reduced OBA in the mouse neutrophils, and that this was reversed in the presence of the sap. By contrast, the phagocytic activity of the mouse cells was not regulated by either DEX or the sap. Rat and canine polymorphonuclear neutrophilic leukocytes (PMNs) responded in vitro to the sap in a similar manner by increasing OBA. However, regulation of phagocytic activity by the sap was different between the species. In canine PMNs, phagocytic activity was enhanced by the sap at a high dose, while it did not significantly modulate this activity in rat PMNs. These findings suggest that the sap of A. okamotoanum stimulates neutrophil activity in the mouse, rat and canine by increasing OBA in vivo and in vitro, and thus may have a potential antimicrobial effect in the PMNs of patients with infections.

  14. Mechanism of neutrophil recruitment to the lung after pulmonary contusion.

    Science.gov (United States)

    Hoth, J Jason; Wells, Jonathan D; Hiltbold, Elizabeth M; McCall, Charles E; Yoza, Barbara K

    2011-06-01

    Blunt chest trauma resulting in pulmonary contusion is a common but poorly understood injury. We previously demonstrated that lung contusion activates localized and systemic innate immune mechanisms and recruits neutrophils to the injured lung. We hypothesized that the innate immune and inflammatory activation of neutrophils may figure prominently in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function and pulmonary neutrophil recruitment. Comparisons were made between injured mice with and without neutrophil depletion. We further examined the role of chemokines and adhesion receptors in neutrophil recruitment to the injured lung. We found that lung injury and resultant physiological dysfunction after contusion were dependent on the presence of neutrophils in the alveolar space. We show that CXCL1, CXCL2/3, and CXCR2 are involved in neutrophil recruitment to the lung after injury and that intercellular adhesion molecule 1 is locally expressed and actively participates in this process. Injured gp91-deficient mice showed improved lung function, indicating that oxidant production by neutrophil NADPH oxidase mediates lung dysfunction after contusion. These data suggest that both neutrophil presence and function are required for lung injury after lung contusion.

  15. Inhibitory effect of Chinese green tea on cigarette smoke-induced up-regulation of airway neutrophil elastase and matrix metalloproteinase-12 via antioxidant activity.

    Science.gov (United States)

    Chan, Ka Ho; Chan, Stanley Chi Hang; Yeung, Sze Chun; Man, Ricky Ying Keung; Ip, Mary Sau Man; Mak, Judith Choi Wo

    2012-09-01

    Our recent study has indicated that Chinese green tea (Lung Chen), in which epigallocatechin-3-gallate (EGCG) accounts for 60% of catechins, protected cigarette smoke-induced lung injury. We now hypothesized that Lung Chen tea may also have potential effect on lung oxidative stress and proteases/anti-proteases in a smoking rat model. Sprague-Dawley rats were exposed to either sham air (SA) or 4% cigarette smoke (CS) plus 2% Lung Chen tea or water by oral gavage. Serine proteases, matrix metalloproteinases (MMPs) and their respective endogenous inhibitors were determined in bronchoalveolar lavage (BAL) and lung tissues by gelatin/casein zymography and biochemical assays. Green tea consumption significantly decreased CS-induced elevation of lung lipid peroxidation marker, malondialdehyde (MDA), and CS-induced up-regulation of neutrophil elastase (NE) concentration and activity along with that of α(1)-antitrypsin (α(1)-AT) and secretory leukoproteinase inhibitor (SLPI) in BAL and lung. In parallel, significant elevation of MMP-12 activity was found in BAL and lung of the CS-exposed group, which returned to the levels of SA-exposed group after green tea consumption but not CS-induced reduction of tissue inhibitor of metalloproteinase (TIMP)-1 activity, which was not reversed by green tea consumption. Taken together, our data supported the presence of local oxidative stress and protease/anti-protease imbalance in the airways after CS exposure, which might be alleviated by green tea consumption through its biological antioxidant activity.

  16. Ascorbate recycling in human neutrophils: Induction by bacteria

    OpenAIRE

    Wang, Yaohui; Russo, Thomas A.; Kwon, Oran; Chanock, Stephen; Rumsey, Steven C.; Levine, Mark

    1997-01-01

    Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen C...

  17. INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS

    NARCIS (Netherlands)

    Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef

    2010-01-01

    INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron tr

  18. INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS

    NARCIS (Netherlands)

    Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef

    2010-01-01

    INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron tr

  19. INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS

    NARCIS (Netherlands)

    Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef

    2010-01-01

    INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron

  20. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

    Science.gov (United States)

    Ricci-Azevedo, Rafael; Oliveira, Aline Ferreira; Conrado, Marina C. A. V.; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina

    2016-01-01

    ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an

  1. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

    Science.gov (United States)

    Ricci-Azevedo, Rafael; Oliveira, Aline Ferreira; Conrado, Marina C A V; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina

    2016-04-01

    ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an

  2. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

    Directory of Open Access Journals (Sweden)

    Rafael Ricci-Azevedo

    2016-04-01

    Full Text Available ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1 immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS, form neutrophil extracellular traps (NETs and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF and interleukin-1beta (IL-1β release; otherwise, transforming growth factor-beta (TGF-β production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be

  3. Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

    Directory of Open Access Journals (Sweden)

    Sorensen Grith L

    2007-02-01

    Full Text Available Abstract Background Surfactant protein D (SP-D plays important roles in innate host defense against influenza A virus (IAV infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr/Thr 11 form of SP-D is associated with low serum levels and assembles predominantly as trimers as opposed to the more common multimeric forms of SP-D. Methods Preliminary experiments were done to establish the effects of different monoclonal antibodies against SP-D on ability of SP-D to bind to or neutralize the virus. We then purified natural human trimeric and multimeric forms of SP-D from amniotic fluid and tested ability of these preparations to bind to IAV, to inhibit infectivity and hemagglutination activity of IAV in vitro. Results In initial experiments mAbs directed against different areas on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs in viral neutralization assays as compared to multimeric SP-D. Conclusion These studies indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP-D can be useful for dissecting out different functional properties of the protein.

  4. Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst

    Indian Academy of Sciences (India)

    David Alan Thompson; Bruce D Hammock

    2007-03-01

    The leukotoxins [9(10)- and 12(13)-EpOME] are produced by activated inflammatory leukocytes such as neutrophils. High EpOME levels are observed in disorders such as acute respiratory distress syndrome and in patients with extensive burns. Although the physiological significance of the EpOMEs remains poorly understood, in some systems, the EpOMEs act as a protoxin, with their corresponding epoxide hydrolase metabolites, 9,10- and 12,13-DiHOME, specifically exerting toxicity. Both the EpOMEs and the DiHOMEs were also recently shown to have neutrophil chemotactic activity. We evaluated whether the neutrophil respiratory burst, a surge of oxidant production thought to play an important role in limiting certain bacterial and fungal infections, is modulated by members of the EpOME metabolic pathway. We present evidence that the DiHOMEs suppress the neutrophil respiratory burst by a mechanism distinct from that of respiratory burst inhibitors such as cyclosporin H or lipoxin A4, which inhibit multiple aspects of neutrophil activation.

  5. Increased neutrophil priming and sensitization before commencing cardiopulmonary bypass in cardiac surgical patients

    NARCIS (Netherlands)

    Gu, YJ; Schoen, P; Tigchelaar, [No Value; Loef, BG; Ebels, T; Rankin, AJ; van Oeveren, W

    2002-01-01

    Background. Neutrophil activation is implicated in postoperative complications in patients having cardiac surgery with cardiopulmonary bypass (CPB). This study was designed to determine the temporal fluctuations in the primability of neutrophils in the preoperative, intraoperative, and postoperative

  6. Oral neutrophils are an independent marker of the systemic inflammatory response after cardiac bypass

    National Research Council Canada - National Science Library

    Wilcox, Mary Elizabeth; Charbonney, Emmanuel; d'Empaire, Pablo Perez; Duggal, Abhijit; Pinto, Ruxandra; Javid, Ashkan; Dos Santos, Claudia; Rubenfeld, Gordon David; Sutherland, Susan; Liles, Wayne Conrad; Glogauer, Michael

    2014-01-01

    .... No method exists to noninvasively assess in vivo neutrophil activity. The objective of this study was to determine if neutrophil recruitment to the oral cavity would correlate with specific biomarkers after coronary bypass surgery (CPB...

  7. Taurine modulates neutrophil function but potentiates uropathogenic E. coli infection in the murine bladder.

    LENUS (Irish Health Repository)

    Condron, Claire

    2010-08-01

    Eradication of a urinary tract infection (UTI) appears to be related to a number of innate host defence mechanisms and their interactions with invading bacteria. Recurrent UTIs (rUTIs) pose a difficult problem in that these bacteria use both host and bacterial factors to evade elimination. Neutrophil bactericidal function is depressed, both systemically and in urine, in patients with a history of recurrent UTI. Taurine is a semi-essential amino acid and is successful in preserving neutrophil bactericidal function in urine. Taurine may preserve neutrophil function at the urothelium and thus aid UTI resolution. Adult female (6 weeks old) C57Bl\\/6 mice were randomised into three groups: a saline gavage only control group, a saline gavage + E. coli group, and a taurine gavage + E. coli group [21 g\\/70 kg taurine in 0.9% normal saline (N\\/S) for 5 days]. Whilst taurine gavage pre-treatment resulted in increased serum neutrophils respiratory burst activity, at the urothelial-endothelial interface it caused higher colony forming units in the urine and a higher incidence of E. coli invasion in the bladder wall with no evidence of increased bladder wall neutrophils infiltration on MPO assay of histological assessment. Histologically there was also evidence of reduced bladder inflammation and urothelial cell apoptosis. In conclusion, taurine effectively increases neutrophils activity but given its anti-inflammatory properties, at the expense of decreased urothelial-endothelial activation thus preventing clearance of active E. coli infection in the bladder. Despite the negative results, this study demonstrates the importance of modulating interactions at the urothelial interface.

  8. Green tea catechins alone or in combination alter functional parameters of human neutrophils via suppressing the activation of TLR-4/NFκB p65 signal pathway.

    Science.gov (United States)

    Marinovic, M P; Morandi, A C; Otton, R

    2015-10-01

    The purpose of this study was to evaluate the potential of a mixture containing the four main catechins found in green tea, as well it separately, as modulators of the functional parameters of human neutrophils. The cells were obtained from peripheral blood of healthy individuals isolated and cultured with a mix: 30 μM of EGCG, 3 μM of EGC, 2 μM of ECG and 1.4 μM of EC, as well as each one alone. We evaluated the cytotoxicity of catechins, production of several reactive oxygen species (ROS), antioxidant enzymes (SOD, CAT, GPx and GR), Nrf2, TLR4/IKK/NFκB, CD11b mRNA levels, intracellular calcium release, chemotactic and phagocytic capacity, myeloperoxidase (MPO), and G6PDH activities, hypochlorous acid (HOCl) and pro-inflammatory cytokines release, protein levels of TLR4, p38 MAPK, iNOS and p-65 NFκB. The actions of the catechins were evidenced by the reduction in inflammatory parameters, including the suppression of TLR4, NFκB and iNOS protein expression, decreased release of TNF-α, IL-1β and IL-6, migration capacity, MPO activity and HOCl production and the suppression of ROS, nitric oxide and peroxynitrite production, while inducing antioxidant enzyme activities and Nrf2 mRNA levels, phagocytic capacity and calcium release. Our results demonstrate that catechins present marked immunomodulatory actions, either alone or in combination.

  9. The neutrophil-activating Dps protein of Helicobacter pylori, HP-NAP, adopts a mechanism different from Escherichia coli Dps to bind and condense DNA.

    Science.gov (United States)

    Ceci, Pierpaolo; Mangiarotti, Laura; Rivetti, Claudio; Chiancone, Emilia

    2007-01-01

    The Helicobacter pylori neutrophil-activating protein (HP-NAP), a member of the Dps family, is a fundamental virulence factor involved in H.pylori-associated disease. Dps proteins protect bacterial DNA from oxidizing radicals generated by the Fenton reaction and also from various other damaging agents. DNA protection has a chemical component based on the highly conserved ferroxidase activity of Dps proteins, and a physical one based on the capacity of those Dps proteins that contain a positively charged N-terminus to bind and condense DNA. HP-NAP does not possess a positively charged N-terminus but, unlike the other members of the family, is characterized by a positively charged protein surface. To establish whether this distinctive property could be exploited to bind DNA, gel shift, fluorescence quenching and atomic force microscopy (AFM) experiments were performed over the pH range 6.5-8.5. HP-NAP does not self-aggregate in contrast to Escherichia coli Dps, but is able to bind and even condense DNA at slightly acid pH values. The DNA condensation capacity acts in concert with the ferritin-like activity and could be used to advantage by H.pylori to survive during host-infection and other stress challenges. A model for DNA binding/condensation is proposed that accounts for all the experimental observations.

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

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    María A. Hidalgo

    2015-01-01

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

  11. Achyrocline satureioides (Lam. D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense

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    Eric Diego Barioni

    2013-01-01

    Full Text Available Achyrocline satureioides (Lam. D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4 expressions and phorbol-myristate-acetate- (PMA- stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity.

  12. Reduced-activation steels: Future development for improved creep strength

    Science.gov (United States)

    Klueh, R. L.

    2008-08-01

    Reduced-activation steels for fusion applications were developed in the 1980s to replace the elevated-temperature commercial steels first considered. The new steels were patterned after the commercial steels, with the objective that the new steels have yield stress and ultimate tensile strength and impact toughness in a Charpy test comparable to or better than the steels they replaced. That objective was achieved in reduced-activation steels developed in Japan, Europe, and the United States. Although tensile and impact toughness of the reduced-activation steels exceed those of the commercial steels they were patterned after, their creep-rupture properties are inferior to some commercial steels they replaced. They are even more inferior to commercial steels developed since the 1980s. In this paper, compositional differences between reduced-activation steels and new commercial steels are examined, and compositions are proposed for development of new-and-improved reduced-activation steels.

  13. 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

  14. False-positive myeloperoxidase binding activity due to DNA/anti-DNA antibody complexes: a source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic autoantibodies.

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    Jethwa, H S; Nachman, P H; Falk, R J; Jennette, J C

    2000-09-01

    Anti-myeloperoxidase antibodies (anti-MPO) are a major type of anti-neutrophil cytoplasmic antibody (ANCA). While evaluating anti-MPO monoclonal antibodies from SCG/Kj mice, we observed several hybridomas that appeared to react with both MPO and DNA. Sera from some patients with systemic lupus erythematosus (SLE) also react with MPO and DNA. We hypothesized that the MPO binding activity is a false-positive result due to the binding of DNA, contained within the antigen binding site of anti-DNA antibodies, to the cationic MPO. Antibodies from tissue culture supernatants from 'dual reactive' hybridomas were purified under high-salt conditions (3 M NaCl) to remove any antigen bound to antibody. The MPO and DNA binding activity were measured by ELISA. The MPO binding activity was completely abrogated while the DNA binding activity remained. The MPO binding activity was restored, in a dose-dependent manner, by the addition of increasing amount of calf-thymus DNA (CT-DNA) to the purified antibody. Sera from six patients with SLE that reacted with both MPO and DNA were treated with DNase and showed a decrease in MPO binding activity compared with untreated samples. MPO binding activity was observed when CT-DNA was added to sera from SLE patients that initially reacted with DNA but not with MPO. These results suggest that the DNA contained within the antigen binding site of anti-DNA antibodies could bind to the highly cationic MPO used as substrate antigen in immunoassays, resulting in a false-positive test.

  15. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma.

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    Kharazmi, A; Nielsen, H; Hovgaard, D; Borregaard, N; Nissen, N I

    1991-04-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma undergoing GM-CSF treatment. Patients with either Hodgkin's or non-Hodgkin's lymphoma were treated with various dosages (2-16 micrograms kg-1 body weight per day for 5 days) of rhGM-CSF by intravenous or subcutaneous route. Prior to and on day 5 of rhGM-CSF treatment, neutrophil and monocyte chemotaxis and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Me