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Sample records for neutrophil extracellular traps

  1. Neutrophil extracellular traps in physiology and pathology

    PubMed Central

    Manda, Aneta; Araźna, Magdalena; Demkow, Urszula A.

    2014-01-01

    Neutrophil extracellular traps (NETs) are developed by nature to protect the body from furious invaders. On the other hand NET s can play an important role in human pathology. Recent studies have shown that neutrophils are able to perform beneficial suicide to create an unique microbicidal net composed from cellular content attached to chromatic frame. It is a powerful tool that primary serve as protector from severe infections, but this weapon is also a double ended sword of the immunity. If overproduced NET s provoke certain autoimmune diseases, coagulation disorders and even cancer metastases. Moreover, due to the competition between host and pathogens, the microorganism have developed a width repertoire of sophisticated evading mechanisms, like creation of polysaccharide capsule or changes in cell wall charge. Therefore it is important to increase the knowledge about paths underlying NET s formation and degradation processes if we want to efficiently fight with bacterial infections and certain diseases. PMID:26155111

  2. Pneumolysin activates neutrophil extracellular trap formation.

    PubMed

    G Nel, J; Theron, A J; Durandt, C; Tintinger, G R; Pool, R; Mitchell, T J; Feldman, C; Anderson, R

    2016-06-01

    The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 μM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P < 0·05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection. PMID:26749379

  3. Metabolic requirements for neutrophil extracellular traps formation

    PubMed Central

    Rodríguez-Espinosa, Oscar; Rojas-Espinosa, Oscar; Moreno-Altamirano, María Maximina Bertha; López-Villegas, Edgar Oliver; Sánchez-García, Francisco Javier

    2015-01-01

    As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 1010 to 1011 cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis. PMID:25545227

  4. Neutrophil extracellular traps in sheep mastitis.

    PubMed

    Pisanu, Salvatore; Cubeddu, Tiziana; Pagnozzi, Daniela; Rocca, Stefano; Cacciotto, Carla; Alberti, Alberto; Marogna, Gavino; Uzzau, Sergio; Addis, Maria Filippa

    2015-01-01

    Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis. PMID:26088507

  5. Neutrophil extracellular traps - the dark side of neutrophils.

    PubMed

    Sørensen, Ole E; Borregaard, Niels

    2016-05-01

    Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those originally described in vitro. Citrullination of histones by peptidyl arginine deiminase 4 (PAD4) is central for NET formation in vivo. NETs may spur formation of autoantibodies and may also serve as scaffolds for thrombosis, thereby providing a link among infection, autoimmunity, and thrombosis. In this review, we present the mechanisms by which NETs are formed and discuss the physiological and pathophysiological consequences of NET formation. We conclude that NETs may be of more importance in autoimmunity and thrombosis than in innate immune defense. PMID:27135878

  6. How Neutrophil Extracellular Traps Become Visible

    PubMed Central

    2016-01-01

    Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one hand in vitro live-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital, in vivo, and in situ microscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages. PMID:27294157

  7. Neutrophil extracellular traps: Their role in periodontal disease

    PubMed Central

    Kolaparthy, Lakshmi Kanth; Sanivarapu, Sahitya; Swarna, Chakrapani; Devulapalli, Narasimha Swamy

    2014-01-01

    Neutrophils are the first line of innate immune defense against infectious diseases. Since their discovery, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during infections. Extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation along with other antimicrobial molecules. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophil extracellular trap production in the regulation of immune response and its role in periodontal disease. PMID:25624623

  8. Neutrophil extracellular traps: Their role in periodontal disease.

    PubMed

    Kolaparthy, Lakshmi Kanth; Sanivarapu, Sahitya; Swarna, Chakrapani; Devulapalli, Narasimha Swamy

    2014-01-01

    Neutrophils are the first line of innate immune defense against infectious diseases. Since their discovery, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during infections. Extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation along with other antimicrobial molecules. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophil extracellular trap production in the regulation of immune response and its role in periodontal disease. PMID:25624623

  9. Neutrophil extracellular traps: Is immunity the second function of chromatin?

    PubMed Central

    2012-01-01

    Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed “NETosis.” Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation. PMID:22945932

  10. P-selectin promotes neutrophil extracellular trap formation in mice.

    PubMed

    Etulain, Julia; Martinod, Kimberly; Wong, Siu Ling; Cifuni, Stephen M; Schattner, Mirta; Wagner, Denisa D

    2015-07-01

    Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin(-/-) mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin(ΔCT/ΔCT) mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis) were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin(ΔCT/ΔCT) neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases. PMID:25979951

  11. Neutrophil extracellular traps promote deep vein thrombosis in mice

    PubMed Central

    Brill, A.; Fuchs, T.A.; Savchenko, A.S.; Thomas, G.M.; Martinod, K.; De Meyer, S.F.; Bhandari, A.A.; Wagner, D.D.

    2011-01-01

    Summary Background Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be pro-thrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). Objective To explore the source and role of extracellular chromatin in DVT. Methods We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). Results We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared to sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs’ structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Conclusions Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development. PMID:22044575

  12. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.

    PubMed

    Najmeh, Sara; Cools-Lartigue, Jonathan; Giannias, Betty; Spicer, Jonathan; Ferri, Lorenzo E

    2015-01-01

    Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil's antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions. In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves. A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained. We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs. PMID:25938591

  13. At the Bedside: Neutrophil extracellular traps (NETs) as targets for biomarkers and therapies in autoimmune diseases.

    PubMed

    Barnado, April; Crofford, Leslie J; Oates, Jim C

    2016-02-01

    Neutrophil extracellular traps are associated with a unique form of cell death distinct from apoptosis or necrosis, whereby invading microbes are trapped and killed. Neutrophil extracellular traps can contribute to autoimmunity by exposing autoantigens, inducing IFN-α production, and activating the complement system. The association of neutrophil extracellular traps with autoimmune diseases, particularly systemic lupus erythematosus, will be reviewed. Increased neutrophil extracellular trap formation is seen in psoriasis, antineutrophil cytoplasmic antibody-associated vasculitis, antiphospholipid antibody syndrome rheumatoid arthritis, and systemic lupus erythematosus. Neutrophil extracellular traps may promote thrombus formation in antineutrophil cytoplasmic antibody-associated vasculitis and antiphospholipid antibody syndrome. In systemic lupus erythematosus, increased neutrophil extracellular trap formation is associated with increased disease activity and renal disease, suggesting that neutrophil extracellular traps could be a disease activity marker. Neutrophil extracellular traps can damage and kill endothelial cells and promote inflammation in atherosclerotic plaques, which may contribute to accelerated atherosclerosis in systemic lupus erythematosus. As neutrophil extracellular traps induce IFN-α production, measuring neutrophil extracellular traps may estimate IFN-α levels and identify which systemic lupus erythematosus patients have elevated levels and may be more likely to respond to emerging anti-IFN-α therapies. In addition to anti-IFN-α therapies, other novel agents, such as N-acetyl-cysteine, DNase I, and peptidylarginine deiminase inhibitor 4, target neutrophil extracellular traps. Neutrophil extracellular traps offer insight into the pathogenesis of autoimmune diseases and provide promise in developing disease markers and novel therapeutic agents in systemic lupus erythematosus. Priority areas for basic research based on clinical research

  14. Capillary plexuses are vulnerable to neutrophil extracellular traps.

    PubMed

    Boneschansker, Leo; Inoue, Yoshitaka; Oklu, Rahmi; Irimia, Daniel

    2016-02-01

    Capillary plexuses are commonly regarded as reliable networks for blood flow and robust oxygen delivery to hypoxia sensitive tissues. They have high levels of redundancy to assure adequate blood supply when one or more of the capillaries in the network are blocked by a clot. However, despite having extensive capillary plexuses, many vital organs are often subject to secondary organ injury in patients with severe inflammation. Recent studies have suggested that neutrophils play a role in this pathology, even though their precise contribution remains elusive. Here we investigate the effect of chromatin fibres released from overly-activated neutrophils (neutrophil extracellular traps, NETs) on the flow of blood through microfluidic networks of channels replicating geometrical features of capillary plexuses. In an in vitro setting, we show that NETs can decouple the traffic of red blood cells from that of plasma in microfluidic networks. The effect is astonishingly disproportionate, with NETs from less than 200 neutrophils resulting in more than half of a 0.6 mm(2) microfluidic network to become void of red blood cell traffic. Importantly, the NETs are able to perturb the blood flow in capillary networks despite the presence of anti-coagulants. If verified to occur in vivo, this finding could represent a novel mechanism for tissue hypoxia and secondary organ injury during severe inflammation in patients already receiving antithrombotic and anticoagulant therapies. PMID:26797289

  15. Platelets: New Bricks in the Building of Neutrophil Extracellular Traps

    PubMed Central

    Carestia, Agostina; Kaufman, Tomas; Schattner, Mirta

    2016-01-01

    In addition to being key elements in hemostasis and thrombosis, platelets have an important role in the inflammatory and innate immune response. This activity is associated with their capability to recognize pathogens through the expression of toll-like receptors, the secretion of various cytokines, chemokines, and growth factors stored within their granules, and the expression of cell adhesion molecules that allows interaction with other immune cells, mainly neutrophils and monocytes. As part of the first line of defense, neutrophils control invading pathogens by phagocytosis, the release of antimicrobial proteins during degranulation, or through the formation of web-like structures named neutrophil extracellular traps (NETs). NETs are formed by chromatin, proteases, and antimicrobial proteins, and their main function is to trap and kill bacteria, virus, and fungi, avoiding their dissemination. Besides microorganisms, NET formation is also triggered by proinflammatory molecules and platelets. The uncontrolled formation of NETs might exert tissue damage and has been involved in a pathogenic mechanism of autoimmune and prothrombotic clinical conditions. In this review, we discuss the role of platelets in NET generation highlighting the mediators, stimuli, and molecular mechanisms involved in this phenomenon, both in human and murine models. PMID:27458459

  16. NADPH oxidase promotes neutrophil extracellular trap formation in pulmonary aspergillosis.

    PubMed

    Röhm, Marc; Grimm, Melissa J; D'Auria, Anthony C; Almyroudis, Nikolaos G; Segal, Brahm H; Urban, Constantin F

    2014-05-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  17. Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation.

    PubMed

    Hahn, Jonas; Knopf, Jasmin; Maueröder, Christian; Kienhöfer, Deborah; Leppkes, Moritz; Herrmann, Martin

    2016-01-01

    Neutrophils, the most abundant leukocytes in the human body, are considered to be the first line of defense in the fight against microorganisms. In this fight neutrophils employ weaponry such as reactive oxygen species produced via the NADPH oxidase complex 2 together with the release of intracellular granules containing antimicrobial agents. The discovery that activated neutrophils release decondensed chromatin as DNase-sensitive neutrophil extracellular traps (NETs) lead to a renewed interest in these leukocytes and the function of NETs in vivo. In this review, we will focus on desirable as well as detrimental features of NETs by the example of gout and pancreatitis. In our models we observed that neutrophils drive the initiation of inflammation and are required for the resolution of inflammation. PMID:27586795

  18. Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

    PubMed

    Möllerherm, Helene; Neumann, Ariane; Schilcher, Katrin; Blodkamp, Stefanie; Zeitouni, Nathalie E; Dersch, Petra; Lüthje, Petra; Naim, Hassan Y; Zinkernagel, Annelies S; von Köckritz-Blickwede, Maren

    2015-12-01

    Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens. PMID:26459885

  19. Neutrophil Extracellular Traps in ANCA-Associated Vasculitis

    PubMed Central

    Söderberg, Daniel; Segelmark, Mårten

    2016-01-01

    A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3- and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3- and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced

  20. Neutrophil Extracellular Traps in ANCA-Associated Vasculitis.

    PubMed

    Söderberg, Daniel; Segelmark, Mårten

    2016-01-01

    A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3- and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3- and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced

  1. Neutrophil extracellular traps promote differentiation and function of fibroblasts.

    PubMed

    Chrysanthopoulou, Akrivi; Mitroulis, Ioannis; Apostolidou, Eirini; Arelaki, Stella; Mikroulis, Dimitrios; Konstantinidis, Theocharis; Sivridis, Efthimios; Koffa, Maria; Giatromanolaki, Alexandra; Boumpas, Dimitrios T; Ritis, Konstantinos; Kambas, Konstantinos

    2014-07-01

    Neutrophil activation by inflammatory stimuli and the release of extracellular chromatin structures (neutrophil extracellular traps - NETs) have been implicated in inflammatory disorders. Herein, we demonstrate that NETs released by neutrophils treated either with fibrosis-related agents, such as cigarette smoke, magnesium silicate, bleomycin, or with generic NET inducers, such as phorbol 12-myristate 13-acetate, induced activation of lung fibroblasts (LFs) and differentiation into myofibroblast (MF) phenotype. Interestingly, the aforementioned agents or IL-17 (a primary initiator of inflammation/fibrosis) had no direct effect on LF activation and differentiation. MFs treated with NETs demonstrated increased connective tissue growth factor expression, collagen production, and proliferation/migration. These fibrotic effects were significantly decreased after degradation of NETs with DNase1, heparin or myeloperoxidase inhibitor, indicating the key role of NET-derived components in LF differentiation and function. Furthermore, IL-17 was expressed in NETs and promoted the fibrotic activity of differentiated LFs but not their differentiation, suggesting that priming by DNA and histones is essential for IL-17-driven fibrosis. Additionally, autophagy was identified as the orchestrator of NET formation, as shown by inhibition studies using bafilomycin A1 or wortmannin. The above findings were further supported by the detection of NETs in close proximity to alpha-smooth muscle actin (α-SMA)-expressing fibroblasts in biopsies from patients with fibrotic interstitial lung disease or from skin scar tissue. Together, these data suggest that both autophagy and NETs are involved not only in inflammation but also in the ensuing fibrosis and thus may represent potential therapeutic targets in human fibrotic diseases. PMID:24740698

  2. Neutrophil extracellular traps (Nets) impact upon autoimmune disorders

    PubMed Central

    Kotuła, Iwona; Manda-Handzlik, Aneta

    2015-01-01

    Friend or foe? This is often asked question when it comes to neutrophil extracellular traps studies. There is no simple answer to that. At the time of their discovery they were considered to be protectors of our well-being. Excellent pathogen fighting skills were described as purely beneficial. But it was not long before those guardians of immunity reveal their dark side. What seemed to be profitable could also take its toll. They are perfectly constructed, made from nucleic deoxyribonucleic acid ornamented with cytoplasmic and granular proteins, to fight invaders. But this unique build is prone to become considered by our body as a threat. Since there is a thin line which when crossed turns a savior into enemy, it was postulated that Nets can play a significant role in autoimmune disorders pathogenesis and disease exacerbation. Recent years have brought a new insight into autoimmune disorders trying to connect the old knowledge and suspicions with modern discoveries. PMID:26557037

  3. Excessive Neutrophils and Neutrophil Extracellular Traps Contribute to Acute Lung Injury of Influenza Pneumonitis

    PubMed Central

    Narasaraju, Teluguakula; Yang, Edwin; Samy, Ramar Perumal; Ng, Huey Hian; Poh, Wee Peng; Liew, Audrey-Ann; Phoon, Meng Chee; van Rooijen, Nico; Chow, Vincent T.

    2011-01-01

    Complications of acute respiratory distress syndrome (ARDS) are common among critically ill patients infected with highly pathogenic influenza viruses. Macrophages and neutrophils constitute the majority of cells recruited into infected lungs, and are associated with immunopathology in influenza pneumonia. We examined pathological manifestations in models of macrophage- or neutrophil-depleted mice challenged with sublethal doses of influenza A virus H1N1 strain PR8. Infected mice depleted of macrophages displayed excessive neutrophilic infiltration, alveolar damage, and increased viral load, later progressing into ARDS-like pathological signs with diffuse alveolar damage, pulmonary edema, hemorrhage, and hypoxemia. In contrast, neutrophil-depleted animals showed mild pathology in lungs. The brochoalveolar lavage fluid of infected macrophage-depleted mice exhibited elevated protein content, T1-α, thrombomodulin, matrix metalloproteinase-9, and myeloperoxidase activities indicating augmented alveolar-capillary damage, compared to neutrophil-depleted animals. We provide evidence for the formation of neutrophil extracellular traps (NETs), entangled with alveoli in areas of tissue injury, suggesting their potential link with lung damage. When co-incubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induced NETs generation, and augmented endothelial damage. NETs induction was abrogated by anti-myeloperoxidase antibody and an inhibitor of superoxide dismutase, thus implying that NETs generation is induced by redox enzymes in influenza pneumonia. These findings support the pathogenic effects of excessive neutrophils in acute lung injury of influenza pneumonia by instigating alveolar-capillary damage. PMID:21703402

  4. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

    PubMed

    Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

    2016-09-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

  5. Neutrophil extracellular traps involvement in corneal fungal infection

    PubMed Central

    Zhao, Yingying; Zhang, Fan; Wan, Ting; Fan, Fangli; Xie, Xin; Lin, Zhenyun

    2016-01-01

    Purpose Neutrophils release neutrophil extracellular traps (NETs) when defending against invading microorganisms. We investigated the existence of NETs in fungal keratitis. Methods Fourteen patients with unilateral fungal keratitis were included. Detailed information about each patient was recorded, including (1) patient history (onset of symptoms and previous therapy), (2) ocular examination findings by slit-lamp biomicroscopy, (3) laboratory findings from direct smear examination and culture of corneal scrapings, (4) NET formation, and (5) treatment strategy and prognosis. Immunofluorescence staining was used to evaluate the existence of NETs on corneal scrapings. The relationship between the quantification of NETs and the clinical character of the fungal keratitis was identified. Results NETs were identified in all 14 patients. Patients with a higher grade of NET formation and fewer fungal hyphae always showed a good treatment response and a short course of infection. NETs were consistently found mixed with fungal hyphae in the corneal scrapings from infected patients. No statistical significance was found between the grade of NETs formed and the course of infection before presentation, and no relationship between the quantification of NETs and the size of the ulcer was found. Conclusions The results suggest that NETs are involved in fungal keratitis. The number of NETs in infected corneas may provide a tool for evaluating the prognosis for fungal keratitis. PMID:27559290

  6. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps.

    PubMed

    Ávila, Eva E; Salaiza, Norma; Pulido, Julieta; Rodríguez, Mayra C; Díaz-Godínez, César; Laclette, Juan P; Becker, Ingeborg; Carrero, Julio C

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  7. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

    PubMed Central

    Ávila, Eva E.; Rodríguez, Mayra C.; Díaz-Godínez, César; Laclette, Juan P.; Becker, Ingeborg; Carrero, Julio C.

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  8. Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

    PubMed

    Rocha, Juliana D B; Nascimento, Michelle T C; Decote-Ricardo, Debora; Côrte-Real, Suzana; Morrot, Alexandre; Heise, Norton; Nunes, Marise P; Previato, José Osvaldo; Mendonça-Previato, Lucia; DosReis, George A; Saraiva, Elvira M; Freire-de-Lima, Célio G

    2015-01-01

    In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen. PMID:25620354

  9. Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?

    PubMed

    Cooper, Paul R; Palmer, Lisa J; Chapple, Iain L C

    2013-10-01

    The discovery of neutrophil extracellular traps in 2004 opened a fascinating new chapter in immune-mediated microbial killing. Brinkman et al. demonstrated that neutrophils, when catastrophically stimulated, undergo a novel form of programmed cell death (neutrophil extracellular trap formation) whereby they decondense their entire nuclear chromatin/DNA and release the resulting structure into the cytoplasm to mix with granule-derived antimicrobial peptides before extruding these web-like structures into the extracellular environment. The process requires the activation of the granule enzyme peptidyl arginine deiminase-4, the formation of reactive oxygen species (in particular hypochlorous acid), the neutrophil microtubular system and the actin cytoskeleton. Recent work by Yousefi et al. demonstrated that exposure to different agents for shorter stimulation periods resulted in neutrophil extracellular trap release from viable granulocytes, and that such neutrophil extracellular traps comprised mitochondrial DNA rather than nuclear DNA and were also capable of microbial entrapment and destruction. Deficiency in NADPH-oxidase production (as found in patients with chronic granulomatous disease) results in an inability to produce neutrophil extracellular traps and, along with their failure to produce antimicrobial reactive oxygen species, these patients suffer from severe, and sometimes life-threatening, infections. However, conversely the release of nuclear chromatin into tissues is also potentially autoimmunogenic and is now associated with the generation of anti-citrullinated protein antibodies in seropositive rheumatoid arthritis. Other neutrophil-derived nuclear and cytoplasmic contents are also pathogenic, either through direct effects on tissues or via autoimmune processes (e.g. autoimmune vasculitis). In this review, we discuss the plant origins of a highly conserved innate immune method of microbial killing, the history and biology of neutrophil extracellular

  10. Mitochondrial DNA Released by Trauma Induces Neutrophil Extracellular Traps

    PubMed Central

    Itagaki, Kiyoshi; Kaczmarek, Elzbieta; Lee, Yen Ting; Tang, I. Tien; Isal, Burak; Adibnia, Yashar; Sandler, Nicola; Grimm, Melissa J.; Segal, Brahm H.; Otterbein, Leo E.; Hauser, Carl J.

    2015-01-01

    Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers’ PMN and in trauma patients’ PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to

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

    PubMed Central

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

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil. PMID:27034964

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

    PubMed

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

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil. PMID:27034964

  13. Cationic liposomes evoke proinflammatory mediator release and neutrophil extracellular traps (NETs) toward human neutrophils.

    PubMed

    Hwang, Tsong-Long; Hsu, Ching-Yun; Aljuffali, Ibrahim A; Chen, Chun-Han; Chang, Yuan-Ting; Fang, Jia-You

    2015-04-01

    Cationic liposomes are widely used as nanocarriers for therapeutic and diagnostic purposes. The cationic components of liposomes can induce inflammatory responses. This study examined the effect of cationic liposomes on human neutrophil activation. Cetyltrimethylammonium bromide (CTAB) or soyaethyl morpholinium ethosulfate (SME) was incorporated into liposomes as the cationic additive. The liposomes' cytotoxicity and their induction of proinflammatory mediators, intracellular calcium, and neutrophil extracellular traps (NETs) were investigated. The interaction of the liposomes with the plasma membrane triggered the stimulation of neutrophils. CTAB liposomes induced complete leakage of lactate dehydrogenase (LDH) at all concentrations tested, whereas SME liposomes released LDH in a concentration-dependent manner. CTAB liposomes proved to more effectively activate neutrophils compared with SME liposomes, as indicated by increased superoxide anion and elastase levels. Calcium influx increased 9-fold after treatment with CTAB liposomes. This influx was not changed by SME liposomes compared with the untreated control. Scanning electron microscopy (SEM) and immunofluorescence images indicated the presence of NETs after treatment with cationic liposomes. NETs could be quickly formed, within minutes, after CTAB liposomal treatment. In contrast to this result, NET formation was slowly and gradually increased by SME liposomes, within 4h. Based on the data presented here, it is important to consider the toxicity of cationic liposomes during administration in the body. This is the first report providing evidence of NET production induced by cationic liposomes. PMID:25731102

  14. Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

    PubMed

    Hollands, Andrew; Corriden, Ross; Gysler, Gabriela; Dahesh, Samira; Olson, Joshua; Raza Ali, Syed; Kunkel, Maya T; Lin, Ann E; Forli, Stefano; Newton, Alexandra C; Kumar, Geetha B; Nair, Bipin G; Perry, J Jefferson P; Nizet, Victor

    2016-07-01

    Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound. PMID:27226531

  15. Neutrophils of Scophthalmus maximus produce extracellular traps that capture bacteria and inhibit bacterial infection.

    PubMed

    Chi, Heng; Sun, Li

    2016-03-01

    Neutrophils constitute an essential part of the innate immune system. Recently, neutrophils have been found to produce a complex extracellular structure called neutrophil extracellular traps (NETs) that capture bacteria, fungi, and parasites. In fish, a few studies on NETs production have been reported, however, the function of fish NETs is unknown. In this study, we examined the ability of turbot (Scophthalmus maximus) neutrophils to produce NETs and investigated the effect of turbot NETs on bacterial infection. We found that upon lipopolysaccharides treatment, turbot head kidney neutrophils produced typical NETs structures that contained DNA and histones. Bacteria treatment also induced production of NETs, which in turn entrapped the bacterial cells and inhibited bacterial replication. Furthermore, when introduced into turbot, NETs-trapped bacteria exhibited significantly weakened ability of tissue dissemination and colonization. These results indicate for the first time that teleost NETs possess apparent antibacterial effect both in vitro and in vivo. PMID:26586641

  16. Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN.

    PubMed

    Kumar, Santhosh V R; Kulkarni, Onkar P; Mulay, Shrikant R; Darisipudi, Murthy N; Romoli, Simone; Thomasova, Dana; Scherbaum, Christina R; Hohenstein, Bernd; Hugo, Christian; Müller, Susanna; Liapis, Helen; Anders, Hans-Joachim

    2015-10-01

    Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN. PMID:25644111

  17. Neutrophil Extracellular Trap Formation Is Independent of De Novo Gene Expression.

    PubMed

    Sollberger, Gabriel; Amulic, Borko; Zychlinsky, Arturo

    2016-01-01

    Neutrophils are essential innate immune cells whose responses are crucial in the clearance of invading pathogens. Neutrophils can respond to infection by releasing neutrophil extracellular traps (NETs). NETs are formed of chromatin and specific granular proteins and are released after execution of a poorly characterized cell death pathway. Here, we show that NET formation induced by PMA or Candida albicans is independent of RNA polymerase II and III-mediated transcription as well as of protein synthesis. Thus, neutrophils contain all the factors required for NET formation when they emerge from the bone marrow as differentiated cells. PMID:27310721

  18. Neutrophil Extracellular Trap Formation Is Independent of De Novo Gene Expression

    PubMed Central

    Zychlinsky, Arturo

    2016-01-01

    Neutrophils are essential innate immune cells whose responses are crucial in the clearance of invading pathogens. Neutrophils can respond to infection by releasing neutrophil extracellular traps (NETs). NETs are formed of chromatin and specific granular proteins and are released after execution of a poorly characterized cell death pathway. Here, we show that NET formation induced by PMA or Candida albicans is independent of RNA polymerase II and III-mediated transcription as well as of protein synthesis. Thus, neutrophils contain all the factors required for NET formation when they emerge from the bone marrow as differentiated cells. PMID:27310721

  19. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps

    PubMed Central

    Guimarães-Costa, Anderson B.; Nascimento, Michelle T. C.; Froment, Giselle S.; Soares, Rodrigo P. P.; Morgado, Fernanda N.; Conceição-Silva, Fátima; Saraiva, Elvira M.

    2009-01-01

    Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania–neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania–neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site. PMID:19346483

  20. Human resistin promotes neutrophil proinflammatory activation and neutrophil extracellular trap formation and increases severity of acute lung injury.

    PubMed

    Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Gregoire, Murielle; Deshane, Jessy; Pittet, Jean Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2014-05-15

    Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies also suggested that resistin has proinflammatory properties. We examined whether the human-specific variant of resistin affects neutrophil activation and the severity of LPS-induced acute lung injury. Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using humanized resistin mice that exclusively express human resistin (hRTN(+/-)(/-)) but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn(+/-/-) neutrophils compared with control Rtn(-/-/-) neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase, a major sensor and regulator of cellular bioenergetics that also is implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin enhanced neutrophil extracellular trap formation. In LPS-induced acute lung injury, humanized resistin mice demonstrated enhanced production of proinflammatory cytokines, more severe pulmonary edema, increased neutrophil extracellular trap formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4-induced inflammatory responses, and it may be a target for future therapies aimed at reducing the severity of acute lung injury and other inflammatory situations in which neutrophils play a major role. PMID:24719460

  1. Ethylmercury and Hg2+ induce the formation of neutrophil extracellular traps (NETs) by human neutrophil granulocytes.

    PubMed

    Haase, Hajo; Hebel, Silke; Engelhardt, Gabriela; Rink, Lothar

    2016-03-01

    Humans are exposed to different mercurial compounds from various sources, most frequently from dental fillings, preservatives in vaccines, or consumption of fish. Among other toxic effects, these substances interact with the immune system. In high doses, mercurials are immunosuppressive. However, lower doses of some mercurials stimulate the immune system, inducing different forms of autoimmunity, autoantibodies, and glomerulonephritis in rodents. Furthermore, some studies suggest a connection between mercury exposure and the occurrence of autoantibodies against nuclear components and granulocyte cytoplasmic proteins in humans. Still, the underlying mechanisms need to be clarified. The present study investigates the formation of neutrophil extracellular traps (NETs) in response to thimerosal and its metabolites ethyl mercury (EtHg), thiosalicylic acid, and mercuric ions (Hg(2+)). Only EtHg and Hg(2+) triggered NETosis. It was independent of PKC, ERK1/2, p38, and zinc signals and not affected by the NADPH oxidase inhibitor DPI. Instead, EtHg and Hg(2+) triggered NADPH oxidase-independent production of ROS, which are likely to be involved in mercurial-induced NET formation. This finding might help understanding the autoimmune potential of mercurial compounds. Some diseases, to which a connection with mercurials has been shown, such as Wegener's granulomatosis and systemic lupus erythematosus, are characterized by high prevalence of autoantibodies against neutrophil-specific auto-antigens. Externalization in the form of NETs may be a source for exposure to these self-antigens. In genetically susceptible individuals, this could be one step in the series of events leading to autoimmunity. PMID:25701957

  2. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.

    PubMed

    Carestia, Agostina; Kaufman, Tomás; Rivadeneyra, Leonardo; Landoni, Verónica Inés; Pozner, Roberto Gabriel; Negrotto, Soledad; D'Atri, Lina Paola; Gómez, Ricardo Martín; Schattner, Mirta

    2016-01-01

    In addition to being key elements in hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram-negative bacteria, Pam3-cysteine-serine-lysine 4, a mimetic of lipopeptide from gram-positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase-associated DNA activity under static and flow conditions. Although P-selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A(2) production triggered by lipopolysaccharide or Pam3-cysteine-serine-lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet-mediated neutrophil extracellular trap generation. Signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet-triggered neutrophil extracellular trap release. Platelet-mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium-derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death. PMID:26320263

  3. High glucose modulates IL-6 mediated immune homeostasis through impeding neutrophil extracellular trap formation.

    PubMed

    Joshi, Manjunath B; Lad, Apurva; Bharath Prasad, Alevoor S; Balakrishnan, Aswath; Ramachandra, Lingadakai; Satyamoorthy, Kapaettu

    2013-07-11

    Neutrophils serve as an active constituent of innate immunity and are endowed with distinct ability for producing neutrophil extracellular traps (NETs) to eliminate pathogens. Earlier studies have demonstrated a dysfunction of the innate immune system in diabetic subjects leading to increased susceptibility to infections; however, the influence of hyperglycemic conditions on NETs is unknown. In the present study we demonstrate that (a) NETs are influenced by glucose homeostasis, (b) IL-6 is a potent inducer of energy dependent NET formation and (c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible to infections. PMID:23735697

  4. Complement factor H modulates the activation of human neutrophil granulocytes and the generation of neutrophil extracellular traps.

    PubMed

    Schneider, Andrea E; Sándor, Noémi; Kárpáti, Éva; Józsi, Mihály

    2016-04-01

    Factor H (FH) is a major inhibitor of the alternative pathway of complement activation in plasma and on certain host surfaces. In addition to being a complement regulator, FH can bind to various cells via specific receptors, including binding to neutrophil granulocytes through complement receptor type 3 (CR3; CD11b/CD18), and modulate their function. The cellular roles of FH are, however, poorly understood. Because neutrophils are important innate immune cells in inflammatory processes and the host defense against pathogens, we aimed at studying the effects of FH on various neutrophil functions, including the generation of extracellular traps. FH co-localized with CD11b on the surface of neutrophils isolated from peripheral blood of healthy individuals, and cell-bound FH retained its cofactor activity and enhanced C3b degradation. Soluble FH supported neutrophil migration and immobilized FH induced cell spreading. In addition, immobilized but not soluble FH enhanced IL-8 release from neutrophils. FH alone did not trigger the cells to produce neutrophil extracellular traps (NETs), but NET formation induced by PMA and by fibronectin plus fungal β-glucan were inhibited by immobilized, but not by soluble, FH. Moreover, in parallel with NET formation, immobilized FH also inhibited the production of reactive oxygen species induced by PMA and by fibronectin plus β-glucan. Altogether, these data indicate that FH has multiple regulatory roles on neutrophil functions. While it can support the recruitment of neutrophils, FH may also exert anti-inflammatory effects and influence local inflammatory and antimicrobial reactions, and reduce tissue damage by modulating NET formation. PMID:26938503

  5. Role of PTEN in neutrophil extracellular trap formation.

    PubMed

    Teimourian, Shahram; Moghanloo, Ehsan

    2015-08-01

    NETosis has been associated with a particular mode of cell death although it is still controversial as to what extent autophagy is involved in NETosis. Class I/AKT/mTOR pathway is a key regulator of autophagy. PTEN tumor suppressor gene encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase in class the I/AKT/mTOR pathway. In this study, we investigated the effects of PTEN down-regulation as well as overexpression on NETosis. Our results show that 35% of HL-60 differentiated neutrophil-like cells generated NETs by PMA. The portion of the population that produced NETs in PTEN knockdown HL-60 differentiated neutrophils was 9% and in PTEN overexpressed HL-60 differentiated neutrophils, it was 56%. Our results show that increasing PTEN expression increases NETs formation in neutrophils, and its suppression reduces NETs. PMID:25913476

  6. Possible implication of disordered neutrophil extracellular traps in the pathogenesis of MPO-ANCA-associated vasculitis.

    PubMed

    Nakazawa, Daigo; Tomaru, Utano; Ishizu, Akihiro

    2013-10-01

    Neutrophil extracellular traps (NETs) are characterized by the presence of extracellular DNA fibers studded with antimicrobial proteins, including myeloperoxidase (MPO). Although NETs play an important role in the innate immune system, the scattered extracellular enzymes, such as MPO, pose risks to the host. Therefore, NETs are strictly regulated by DNase I in the serum, which prevents them from persisting. Recent studies have demonstrated that dysregulation of NETs could be involved in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. In this review, we interpret the association of disordered NETs with autoimmune diseases, especially propylthiouracil-induced MPO-ANCA-associated vasculitis. PMID:23224024

  7. How neutrophil extracellular traps orchestrate the local immune response in gout.

    PubMed

    Maueröder, Christian; Kienhöfer, Deborah; Hahn, Jonas; Schauer, Christine; Manger, Bernhard; Schett, Georg; Herrmann, Martin; Hoffmann, Markus H

    2015-07-01

    Neutrophil granulocytes possess a large arsenal of pro-inflammatory substances and mechanisms that empower them to drive local acute immune reactions to invading microorganisms or endogenous inflammatory triggers. The use of this armory needs to be tightly controlled to avoid chronic inflammation and collateral tissue damage. In gout, inflammation arises from precipitation of uric acid in the form of needle-shaped monosodium urate crystals. Inflammasome activation by these crystals in local immune cells results in a rapid and dramatic recruitment of neutrophils. This neutrophil influx is accompanied by the infamously intense clinical symptoms of inflammation during an acute gout attack. Neutrophilic inflammation however is equipped with a built-in safeguard; activated neutrophils form neutrophil extracellular traps (NETs). At the very high neutrophil densities that occur at the site of inflammation, NETs build aggregates that densely pack the monosodium urate (MSU) crystals and trap and degrade pro-inflammatory mediators by inherent proteases. Local removal of cytokines and chemokines by aggregated NETs explains how acute inflammation can stop in the consistent presence of the inflammatory trigger. Aggregated NETs resemble early stages of the typical large MSU deposits that constitute the pathognomonic structures of gout, tophi. Although tophi contribute to muscosceletal damage and mortality in patients with chronic gout, they can therefore be considered as a payoff that is necessary to silence the intense inflammatory response during acute gout. PMID:26002146

  8. PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling.

    PubMed

    Desai, Jyaysi; Kumar, Santhosh V; Mulay, Shrikant R; Konrad, Lukas; Romoli, Simone; Schauer, Christine; Herrmann, Martin; Bilyy, Rostyslav; Müller, Susanna; Popper, Bastian; Nakazawa, Daigo; Weidenbusch, Marc; Thomasova, Dana; Krautwald, Stefan; Linkermann, Andreas; Anders, Hans-Joachim

    2016-01-01

    Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside-in signaling pathway triggering NET formation is unknown. Here, we show that the receptor-interacting protein kinase (RIPK)-1-stabilizers necrostatin-1 or necrostatin-1s and the mixed lineage kinase domain-like (MLKL)-inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal- or PMA-induced NET formation in human and mouse neutrophils. These compounds do not affect PMA- or urate crystal-induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA-induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal-induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies. PMID:26531064

  9. Do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases?

    PubMed Central

    Rao, Ashish N; Kazzaz, Nayef M; Knight, Jason S

    2015-01-01

    Thrombotic events, both arterial and venous, are a major health concern worldwide. Further, autoimmune diseases, such as systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and antiphospholipid syndrome, predispose to thrombosis, and thereby push the risk for these morbid events even higher. In recent years, neutrophils have been identified as important players in both arterial and venous thrombosis. Specifically, chromatin-based structures called neutrophil extracellular traps (NETs) play a key role in activating the coagulation cascade, recruiting platelets, and serving as scaffolding upon which the thrombus can be assembled. At the same time, neutrophils and NETs are emerging as important mediators of pathogenic inflammation in the aforementioned autoimmune diseases. Here, we first review the general role of NETs in thrombosis. We then posit that exaggerated NET release contributes to the prothrombotic diatheses of systemic lupus erythematosus, ANCA-associated vasculitis, and antiphospholipid syndrome. PMID:26730289

  10. At the Bench: Neutrophil extracellular traps (NETs) highlight novel aspects of innate immune system involvement in autoimmune diseases.

    PubMed

    Grayson, Peter C; Kaplan, Mariana J

    2016-02-01

    The putative role of neutrophils in host defense against pathogens is a well-recognized aspect of neutrophil function. The discovery of neutrophil extracellular traps has expanded the known range of neutrophil defense mechanisms and catalyzed a discipline of research focused upon ways in which neutrophils can shape the immunologic landscape of certain autoimmune diseases, including systemic lupus erythematosus. Enhanced neutrophil extracellular trap formation and impaired neutrophil extracellular trap clearance may contribute to immunogenicity in systemic lupus erythematosus and other autoimmune diseases by promoting the externalization of modified autoantigens, inducing synthesis of type I IFNs, stimulating the inflammasome, and activating both the classic and alternative pathways of the complement system. Vasculopathy is a central feature of many autoimmune diseases, and neutrophil extracellular traps may contribute directly to endothelial cell dysfunction, atherosclerotic plaque burden, and thrombosis. The elucidation of the subcellular events of neutrophil extracellular trap formation may generate novel, therapeutic strategies that target the innate immune system in autoimmune and vascular diseases. PMID:26432901

  11. A Lipid Mediator Hepoxilin A3 Is a Natural Inducer of Neutrophil Extracellular Traps in Human Neutrophils

    PubMed Central

    Douda, David N.; Grasemann, Hartmut; Pace-Asciak, Cecil

    2015-01-01

    Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases. PMID:25784781

  12. Iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils.

    PubMed

    Völlger, Lena; Akong-Moore, Kathryn; Cox, Linda; Goldmann, Oliver; Wang, Yanming; Schäfer, Simon T; Naim, Hassan Y; Nizet, Victor; von Köckritz-Blickwede, Maren

    2016-07-01

    Neutrophil extracellular trap (NET) formation is a significant innate immune defense mechanism against microbial infection that complements other neutrophil functions including phagocytosis and degranulation of antimicrobial peptides. NETs are decondensed chromatin structures in which antimicrobial components (histones, antimicrobial peptides and proteases) are deployed and mediate immobilization of microbes. Here we describe an effect of iron chelation on the phenotype of NET formation. Iron-chelating agent desferrioxamine (DFO) showed a modest but significant induction of NETs by freshly isolated human neutrophils as visualized and quantified by immunocytochemistry against histone-DNA complexes. Further analyses revealed that NET induction by iron chelation required NADPH-dependent production of reactive oxygen species (ROS) as well as protease and peptidyl-arginine-deiminase 4 (PAD4) activities, three key mechanistic pathways previously linked to NET formation. Our results demonstrate that iron chelation by DFO contributes to the formation of NETs and suggest a target for pharmacological manipulation of NET activity. PMID:27129288

  13. The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release.

    PubMed

    Bystrzycka, Weronika; Moskalik, Aneta; Sieczkowska, Sandra; Manda-Handzlik, Aneta; Demkow, Urszula; Ciepiela, Olga

    2016-01-01

    Neutrophil extracellular traps (NETs) are threads of nuclear DNA complexed with antimicrobial proteins released by neutrophils to extracellular matrix to bind, immobilise, and kill different pathogens. NET formation is triggered by different physiological and non-physiological stimulants. It is also suggested that antibiotics could be non-physiological compounds that influence NET release. The aim of the study was to investigate the effect of clindamycin and amoxicillin on NET release and the phagocyte function of neutrophils. Neutrophils isolated from healthy donors by density centrifugation method were incubated with amoxicillin or clindamycin for two hours, and then NET release was stimulated with phorbol 12-myristate 13-acetate (PMA). After three hours of incubation with PMA NETs were quantified as amount of extracellular DNA by fluorometry and visualised by immunofluorescent microscopy. The percent of phagocyting cells was measured by flow cytometry. We showed that amoxicillin induces NET formation (increase of extracellular DNA fluorescence, p = 0.03), while clindamycin had no influence on NET release (p > 0.05), as confirmed by quantitative measurement and fluorescent microscopy. Regarding phagocyte function, both antibiotics increased bacterial uptake (43.3% and 61.6% median increase for amoxicillin and clindamycin, respectively). We concluded that the ability of antibiotics to modulate NET release depends on the antibiotic used and is not associated with their ability to influence phagocytosis. PMID:27095915

  14. Hyperosmolar Stress Induces Neutrophil Extracellular Trap Formation: Implications for Dry Eye Disease

    PubMed Central

    Tibrewal, Sapna; Ivanir, Yair; Sarkar, Joy; Nayeb-Hashemi, Neema; Bouchard, Charles S.; Kim, Eunjae; Jain, Sandeep

    2014-01-01

    Purpose. To determine if hyperosmolar stress can stimulate human neutrophils to form neutrophil extracellular traps (NETs) and to investigate potential strategies to reduce formation of NETs (NETosis) in a hyperosmolar environment. Methods. Neutrophils were isolated from peripheral venous blood of healthy subjects and incubated in iso-osmolar (280 mOsM) or hyperosmolar (420 mOsM) media for 4 hours. Neutrophil extracellular traps were quantified using a PicoGreen dye assay to measure extracellular DNA. Two known inhibitors of NETosis, staurosporine and anti-β2 integrin blocking antibody, and two proresolution formyl peptide receptor 2 (FPR2) agonists, annexin/lipocortin-1 mimetic peptide and 15-epi-lipoxin A4, were evaluated as possible strategies to reduce hyperosmolarity-induced NETosis. Results. The amount of NETs induced by hyperosmolar medium (420 mOsM) increased linearly over time to 3.2 ± 0.3 times that induced by iso-osmolar medium at 4 hours (P < 0.05). NETosis increased exponentially with increasing osmolarity and was independent of the stimulus used to increase osmolarity. Upon neutrophil exposure to hyperosmolar stress, restoration of iso-osmolar conditions decreased NET formation by 52.7% ± 5% (P < 0.05) but did not completely abrogate it. Among the strategies tested to reduce NETosis in a hyperosmolar environment, annexin-1 peptide was the most efficacious. Conclusions. Hyperosmolarity induces formation of NETs by neutrophils. This NETosis mechanism may explain the presence of excessive NETs on the ocular surface of patients with dry eye disease. Because they reduce hyperosmolarity-induced NETosis, FPR2 agonists may have therapeutic potential in these patients. PMID:25406284

  15. Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways.

    PubMed

    Pijanowski, L; Golbach, L; Kolaczkowska, E; Scheer, M; Verburg-van Kemenade, B M L; Chadzinska, M

    2013-05-01

    Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish. PMID:23422817

  16. Flavonoids and 5-Aminosalicylic Acid Inhibit the Formation of Neutrophil Extracellular Traps

    PubMed Central

    Möller, Sonja; Klinger, Matthias; Solbach, Werner; Laskay, Tamás

    2013-01-01

    Neutrophil extracellular traps (NETs) have been suggested to play a pathophysiological role in several autoimmune diseases. Since NET-formation in response to several biological and chemical stimuli is mostly ROS dependent, in theory any substance that inhibits or scavenges ROS could prevent ROS-dependent NET release. Therefore, in the present comprehensive study, several antioxidative substances were assessed for their capacity to inhibit NET formation of primary human neutrophils in vitro. We could show that the flavonoids (−)-epicatechin, (+)-catechin hydrate, and rutin trihydrate as well as vitamin C and the pharmacological substances N-acetyl-L-cysteine and 5-aminosalicylic acid inhibited PMA induced ROS production and NET formation. Therefore, a broad spectrum of antioxidative substances that reduce ROS production of primary human neutrophils also inhibits ROS-dependent NET formation. It is tempting to speculate that such antioxidants can have beneficial therapeutic effects in diseases associated with ROS-dependent NET formation. PMID:24381411

  17. Deficient Neutrophil Extracellular Trap Formation in Patients Undergoing Bone Marrow Transplantation

    PubMed Central

    Glenn, Jared W.; Cody, Mark J.; McManus, Meghann P.; Pulsipher, Michael A.; Schiffman, Joshua D.; Yost, Christian Con

    2016-01-01

    Overwhelming infection causes significant morbidity and mortality among patients treated with bone marrow transplantation (BMT) for primary immune deficiencies, syndromes of bone marrow failure, or cancer. The polymorphonuclear leukocyte (PMN; neutrophil) is the first responder to microbial invasion and acts within the innate immune system to contain and clear infections. PMNs contain, and possibly clear, infections in part by forming neutrophil extracellular traps (NETs). NETs are extensive lattices of extracellular DNA and decondensed chromatin decorated with antimicrobial proteins and degradative enzymes, such as histones, myeloperoxidase, and neutrophil elastase. They trap and contain microbes, including bacteria and fungi, and may directly affect extracellular microbial killing. Whether or not deficient NET formation contributes to the increased risk for overwhelming infection in patients undergoing BMT remains incompletely characterized, especially in the pediatric population. We examined NET formation in vitro in PMNs isolated from 24 patients who had undergone BMT for 13 different clinical indications. For these 24 study participants, the median age was 7 years. For 6 of the 24 patients, we examined NET formation by PMNs isolated from serial, peripheral blood samples drawn at three different clinical time points: pre-BMT, pre-engraftment, and post-engraftment. We found decreased NET formation by PMNs isolated from patients prior to BMT and during the pre-engraftment and post-engraftment phases, with decreased NET formation compared with healthy control PMNs detected even out to 199 days after their BMT. This decrease in NET formation after BMT did not result from neutrophil developmental immaturity as we demonstrated that >80% of the PMNs tested using flow cytometry expressed both CD10 and CD16 as markers of terminal differentiation along the neutrophilic lineage. These pilot study results mandate further exploration regarding the mechanisms or factors

  18. Deficient Neutrophil Extracellular Trap Formation in Patients Undergoing Bone Marrow Transplantation.

    PubMed

    Glenn, Jared W; Cody, Mark J; McManus, Meghann P; Pulsipher, Michael A; Schiffman, Joshua D; Yost, Christian Con

    2016-01-01

    Overwhelming infection causes significant morbidity and mortality among patients treated with bone marrow transplantation (BMT) for primary immune deficiencies, syndromes of bone marrow failure, or cancer. The polymorphonuclear leukocyte (PMN; neutrophil) is the first responder to microbial invasion and acts within the innate immune system to contain and clear infections. PMNs contain, and possibly clear, infections in part by forming neutrophil extracellular traps (NETs). NETs are extensive lattices of extracellular DNA and decondensed chromatin decorated with antimicrobial proteins and degradative enzymes, such as histones, myeloperoxidase, and neutrophil elastase. They trap and contain microbes, including bacteria and fungi, and may directly affect extracellular microbial killing. Whether or not deficient NET formation contributes to the increased risk for overwhelming infection in patients undergoing BMT remains incompletely characterized, especially in the pediatric population. We examined NET formation in vitro in PMNs isolated from 24 patients who had undergone BMT for 13 different clinical indications. For these 24 study participants, the median age was 7 years. For 6 of the 24 patients, we examined NET formation by PMNs isolated from serial, peripheral blood samples drawn at three different clinical time points: pre-BMT, pre-engraftment, and post-engraftment. We found decreased NET formation by PMNs isolated from patients prior to BMT and during the pre-engraftment and post-engraftment phases, with decreased NET formation compared with healthy control PMNs detected even out to 199 days after their BMT. This decrease in NET formation after BMT did not result from neutrophil developmental immaturity as we demonstrated that >80% of the PMNs tested using flow cytometry expressed both CD10 and CD16 as markers of terminal differentiation along the neutrophilic lineage. These pilot study results mandate further exploration regarding the mechanisms or factors

  19. Interaction of Bacterial Exotoxins with Neutrophil Extracellular Traps: Impact for the Infected Host.

    PubMed

    von Köckritz-Blickwede, Maren; Blodkamp, Stefanie; Nizet, Victor

    2016-01-01

    Since their discovery in 2004, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense against various pathogens. Released in response to infectious and pro-inflammatory stimuli, NETs can immobilize invading pathogens within a fibrous matrix consisting of DNA, histones, and antimicrobial peptides. Conversely, excessive or dysregulated NET release may hold a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during infectious challenge. In recent years, a number of microbial virulence factors have been shown to modulate formation of NETs, thereby facilitating colonization or spread within the host. In this mini-review we summarize the contemporary research on the interaction of bacterial exotoxins with neutrophils that modulate NET production, focusing particular attention on consequences for the host. Understanding host-pathogen dynamics in this extracellular battlefield of innate immunity may provide novel therapeutic approaches for infectious and inflammatory disorders. PMID:27064864

  20. Interaction of Bacterial Exotoxins with Neutrophil Extracellular Traps: Impact for the Infected Host

    PubMed Central

    von Köckritz-Blickwede, Maren; Blodkamp, Stefanie; Nizet, Victor

    2016-01-01

    Since their discovery in 2004, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense against various pathogens. Released in response to infectious and pro-inflammatory stimuli, NETs can immobilize invading pathogens within a fibrous matrix consisting of DNA, histones, and antimicrobial peptides. Conversely, excessive or dysregulated NET release may hold a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during infectious challenge. In recent years, a number of microbial virulence factors have been shown to modulate formation of NETs, thereby facilitating colonization or spread within the host. In this mini-review we summarize the contemporary research on the interaction of bacterial exotoxins with neutrophils that modulate NET production, focusing particular attention on consequences for the host. Understanding host–pathogen dynamics in this extracellular battlefield of innate immunity may provide novel therapeutic approaches for infectious and inflammatory disorders. PMID:27064864

  1. Neutrophil extracellular traps (NETs): Double-edged swords of innate immunity1

    PubMed Central

    Kaplan, Mariana J.; Radic, Marko

    2012-01-01

    Spectacular images of neutrophils ejecting nuclear chromatin and bactericidal proteins, in response to microbes, were first reported in 2004. As externalized chromatin could entangle bacteria, these structures were named neutrophil extracellular traps (NETs). Subsequent studies identified microorganisms and sterile conditions that stimulate NETs, and additional cell types that release extracellular chromatin. NETs’ release is the most dramatic stage in a cell death process called NETosis. Experimental evidence suggests that NETs participate in pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis and vascular disorders. Exaggerated NETosis or diminished NET clearance likely increases risk of autoreactivity to NET components. The biological significance of NETs is just beginning to be explored. A more complete integration of NETosis within immunology and pathophysiology will require better understanding of NET properties associated with specific disease states and microbial infections. This may lead to the identification of important therapeutic targets. PMID:22956760

  2. [Discovery of the neutrophil extracellular traps begins a new stage in the study of neutrophil morphogenesis and function].

    PubMed

    Perova, M D; Shubich, M G

    2011-01-01

    The purpose of the present review was to analyze the accumulating evidence regarding recently discovered novel defense mechanism of neutrophils - capacity to form neutrophil extracellular traps (NETs). Contact with pathogenic microbes and/or exposure to proinflammatory cytokines trigger the respiratory burst in the neutrophils with a subsequent initiation of a cell death (NETosis) which differs from apoptosis and necrosis. NETs are formed by the fibrils of decondensed chromatin (DNA/ histones), released from the neutrophil, which is closely associated with the antimicrobial proteins of cytoplasmic granules. Due to its three-dimensional structure, NETs are capable of retaining the microorganisms (bacteria, fungi and protozoa), while high local concentration of the antimicrobial substances provides their killing. The review presents the evidence of a potential defensive role of NETs in infectious diseases, traumas and surgical operations, as well as during the early stage of a repair process. Considering the role played by neutrophils in the immune response orientation via pentraxin-3 (PTX3), including the switching to adaptive immunity, it is necessary to study the subsequent interaction of DNA/histone exrtacellular structures with the tissue microenvironment. PMID:21954717

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

    PubMed

    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. PMID:27333499

  4. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    PubMed

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri. PMID:27189133

  5. Inhibitors of Serine Proteases in Regulating the Production and Function of Neutrophil Extracellular Traps

    PubMed Central

    Majewski, Pawel; Majchrzak-Gorecka, Monika; Grygier, Beata; Skrzeczynska-Moncznik, Joanna; Osiecka, Oktawia; Cichy, Joanna

    2016-01-01

    Neutrophil extracellular traps (NETs), DNA webs released into the extracellular environment by activated neutrophils, are thought to play a key role in the entrapment and eradication of microbes. However, NETs are highly cytotoxic and a likely source of autoantigens, suggesting that NET release is tightly regulated. NET formation involves the activity of neutrophil elastase (NE), which cleaves histones, leading to chromatin decondensation. We and others have recently demonstrated that inhibitors of NE, such as secretory leukocyte protease inhibitor (SLPI) and SerpinB1, restrict NET production in vitro and in vivo. SLPI was also identified as a NET component in the lesional skin of patients suffering from the autoinflammatory skin disease psoriasis. SLPI-competent NET-like structures (a mixture of SLPI with neutrophil DNA and NE) stimulated the synthesis of interferon type I (IFNI) in plasmacytoid dendritic cells (pDCs) in vitro. pDCs uniquely respond to viral or microbial DNA/RNA but also to nucleic acids of “self” origin with the production of IFNI. Although IFNIs are critical in activating the antiviral/antimicrobial functions of many cells, IFNIs also play a role in inducing autoimmunity. Thus, NETs decorated by SLPI may regulate skin immunity through enhancing IFNI production in pDCs. Here, we review key aspects of how SLPI and SerpinB1 can control NET production and immunogenic function. PMID:27446090

  6. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis

    PubMed Central

    Demers, Mélanie; Krause, Daniela S.; Schatzberg, Daphne; Martinod, Kimberly; Voorhees, Jaymie R.; Fuchs, Tobias A.; Scadden, David T.; Wagner, Denisa D.

    2012-01-01

    Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients. PMID:22826226

  7. In Vivo Characterization of Neutrophil Extracellular Traps in Various Organs of a Murine Sepsis Model

    PubMed Central

    Tanaka, Koji; Koike, Yuhki; Shimura, Tadanobu; Okigami, Masato; Ide, Shozo; Toiyama, Yuji; Okugawa, Yoshinaga; Inoue, Yasuhiro; Araki, Toshimitsu; Uchida, Keiichi; Mohri, Yasuhiko; Mizoguchi, Akira; Kusunoki, Masato

    2014-01-01

    Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis. PMID:25372699

  8. Regulation of neutrophil extracellular trap formation by anti-inflammatory drugs.

    PubMed

    Lapponi, María José; Carestia, Agostina; Landoni, Verónica Inés; Rivadeneyra, Leonardo; Etulain, Julia; Negrotto, Soledad; Pozner, Roberto Gabriel; Schattner, Mirta

    2013-06-01

    The formation of neutrophil extracellular traps (NETs) is a newly described phenomenon that increases the bacteria-killing ability and the inflammatory response of neutrophils. Because NET generation occurs in an inflammatory microenvironment, we examined its regulation by anti-inflammatory drugs. Treatment of neutrophils with dexamethasone had no effect, but acetylsalicylic acid (ASA) treatment prevented NET formation. NETosis was also abrogated by the presence of BAY 11-7082 [(E)-3-[4-methylphenylsulfonyl]-2-propenenitrile] and Ro 106-9920 [6-(phenylsulfinyl)tetrazolo[1,5-b]pyridazine], two structurally unrelated nuclear factor-κB (NF-κB) inhibitors. The decrease in NET formation mediated by ASA, BAY-11-7082, and Ro 106-9920 was correlated with a significant reduction in the phosphorylation of NF-κB p65 subunit, indicating that the activation of this transcription factor is a relevant signaling pathway involved in the generation of DNA traps. The inhibitory effect of these drugs was also observed when NET generation was induced under acidic or hyperthermic conditions, two stress signals of the inflammatory microenvironment. In a mouse peritonitis model, while pretreatment of animals with ASA or BAY 11-7082 resulted in a marked suppression of NET formation along with increased bacteremia, dexamethasone had no effect. Our results show that NETs have an important role in the local control of infection and that ASA and NF-κB blockade could be useful therapies to avoid undesired effect of persistent neutrophil activation. PMID:23536315

  9. Neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease.

    PubMed

    Cortjens, Bart; de Boer, Onno J; de Jong, Rineke; Antonis, Adriaan Fg; Sabogal Piñeros, Yanaika S; Lutter, René; van Woensel, Job Bm; Bem, Reinout A

    2016-02-01

    Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA-rich mucus plugs are characteristic features of severe RSV-LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first-line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti-viral effect in vitro. Second, we studied NET formation in vivo during severe RSV-LRTD in infants and bovine RSV-LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV-LRTD. Detailed histopathological examination of calves with RSV-LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV-LRTD contributes to airway obstruction. PMID:26468056

  10. Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus

    PubMed Central

    Pieterse, E; Hofstra, J; Berden, J; Herrmann, M; Dieker, J; van der Vlag, J

    2015-01-01

    In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE. PMID:24758196

  11. Immobilized immune complexes induce neutrophil extracellular trap release by human neutrophil granulocytes via FcγRIIIB and Mac-1.

    PubMed

    Behnen, Martina; Leschczyk, Christoph; Möller, Sonja; Batel, Tobit; Klinger, Matthias; Solbach, Werner; Laskay, Tamás

    2014-08-15

    Canonical neutrophil antimicrobial effector mechanisms, such as degranulation, production of reactive oxygen species, and release of neutrophil extracellular traps (NETs), can result in severe pathology. Activation of neutrophils through immune complexes (ICs) plays a central role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report that immobilized ICs (iICs), which are hallmarks of several autoimmune diseases, induce the release of NETs from primary human neutrophils. The iIC-induced NET formation was found to require production of reactive oxygen species by NADPH oxidase and myeloperoxidase and to be mediated by FcγRIIIb. Blocking of the β2 integrin macrophage-1 Ag but not lymphocyte function-associated Ag-1 abolished iIC-induced NET formation. This suggests that FcγRIIIb signals in association with macrophage-1 Ag. As intracellular signaling pathways involved in iIC-induced NET formation we identified the tyrosine kinase Src/Syk pathway, which downstream regulates the PI3K/Akt, p38 MAPK, and ERK1/2 pathways. To our knowledge, the present study shows for the first time that iICs induce NET formation. Thus, we conclude that NETs contribute to pathology in autoimmune inflammatory disorders associated with surface-bound ICs. PMID:25024378

  12. Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira

    PubMed Central

    Scharrig, Emilia; Carestia, Agostina; Ferrer, María F.; Cédola, Maia; Pretre, Gabriela; Drut, Ricardo; Picardeau, Mathieu; Schattner, Mirta; Gómez, Ricardo M.

    2015-01-01

    NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden. PMID:26161745

  13. Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis1

    PubMed Central

    Lin, Andrew M.; Rubin, Cory J.; Khandpur, Ritika; Wang, Jennifer Y.; Riblett, MaryBeth; Yalavarthi, Srilakshmi; Villanueva, Eneida C.; Shah, Parth; Kaplan, Mariana J.; Bruce, Allen T.

    2011-01-01

    IL-17 and IL-23 are absolutely central to psoriasis pathogenesis as drugs targeting either cytokine are highly effective treatments for this disease. The efficacy of these drugs has been attributed to blocking the function of IL-17-producing T cells and their IL-23-induced expansion. However, we demonstrate that mast cells and neutrophils, not T cells, are the predominant cell types that contain IL-17 in human skin. IL-17+ mast cells and neutrophils are found at higher densities than IL-17+ T cells in psoriasis lesions and frequently release IL-17 in the process of forming specialized structures called extracellular traps (MCETs and NETs, respectively). Furthermore, we find that IL-23 and IL-1β can induce MCET formation and degranulation of human mast cells. Release of IL-17 from innate immune cells may be central to the pathogenesis of psoriasis, representing a fundamental mechanism by which the IL-23-IL-17 axis mediates host defense and autoimmunity. PMID:21606249

  14. Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

    PubMed

    Arai, Yasuyuki; Nishinaka, Yoko; Arai, Toshiyuki; Morita, Makiko; Mizugishi, Kiyomi; Adachi, Souichi; Takaori-Kondo, Akifumi; Watanabe, Tomohiro; Yamashita, Kouhei

    2014-01-10

    Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia. PMID:24326071

  15. Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development

    PubMed Central

    Savchenko, A. S.; Martinod, K.; Seidman, M. A.; Wong, S. L.; Borissoff, J. I.; Piazza, G.; Libby, P.; Goldhaber, S. Z.; Mitchell, R. N.; Wagner, D. D.

    2014-01-01

    Background A growing health problem, venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), requires refined diagnostic and therapeutic approaches. Neutrophils contribute to thrombus initiation and development in experimental DVT. Recent animal studies recognized neutrophil extracellular traps (NETs) as an important scaffold supporting thrombus stability. However, the hypothesis that human venous thrombi involve NETs has not undergone rigorous testing. Objective To explore the cellular composition and the presence of NETs within human venous thrombi at different stages of development. Patients and Methods We examined sixteen thrombi obtained from 11 patients during surgery or at autopsy using histomorphological, immunohistochemical and immunofluorescence analyses. Results We classified thrombus regions as unorganized, organizing, and organized according to their morphological characteristics. We then evaluated them focusing on neutrophil and platelet deposition as well as micro-vascularization of the thrombus body. We observed evidence of NET accumulation, including the presence of citrullinated histone H3 (H3Cit)-positive cells. NETs, defined as extracellular diffuse H3Cit areas associated with myeloperoxidase and DNA, localized predominantly during the phase of organization in human venous thrombi. Conclusions NETs are present in organizing thrombi in patients with VTE. They are associated with thrombus maturation in humans. Dissolution of NETs might thus facilitate thrombolysis. This finding provides new insights into the clinical development and pathology of thrombosis and provides new perspectives for therapeutic advances. PMID:24674135

  16. Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

    PubMed Central

    Saffarzadeh, Mona; Juenemann, Christiane; Queisser, Markus A.; Lochnit, Guenter; Barreto, Guillermo; Galuska, Sebastian P.; Lohmeyer, Juergen; Preissner, Klaus T.

    2012-01-01

    Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction. PMID:22389696

  17. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype.

    PubMed

    Akk, Antonina; Springer, Luke E; Pham, Christine T N

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  18. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade.

  19. Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition

    PubMed Central

    Hopke, Alex; Nicke, Nadine; Hidu, Erica E.; Degani, Genny; Popolo, Laura

    2016-01-01

    Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. The opportunistic human fungal pathogen Candida albicans, which can cause fatal disease in immunocompromised patient populations, offers a good example as it masks the inflammatory epitope β-glucan in its cell wall from host recognition. It has been demonstrated previously that β-glucan becomes exposed during infection in vivo but the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance immune recognition by the Dectin-1 β-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog. PMID:27223610

  20. Effects of the antioxidants Trolox, Tiron and Tempol on neutrophil extracellular trap formation.

    PubMed

    Vorobjeva, Nina V; Pinegin, Boris V

    2016-02-01

    Neutrophils can entrap and kill pathogens by releasing of neutrophil extracellular traps (NETs), in addition to their routine functions such as phagocytosis and degranulation. NETs consist of a DNA backbone supplemented by multiple bactericidal proteins from the nucleus, the cytoplasm and the granules. Neutrophils release NETs after their activation by a number of physiological and pharmacological stimuli. In addition to the antimicrobial function, NETs are involved in the pathogenesis of various autoimmune and inflammatory diseases. Since NET formation predominantly depends on the generation of reactive oxygen species (ROS), all substances that are capable of scavenging ROS or inhibiting the enzymes responsible for their synthesis should prevent ROS-associated NET release. The aim of this study was to test substances with an antioxidant activity, such as Trolox, Tiron, and Tempol, for their capacity to inhibit NET formation by primary human neutrophils in vitro. We revealed for the first time an inhibitory effect of Trolox on ROS-dependent NET release. We also established a suppressive effect of Tempol on NET formation that manifested itself in a wide range of concentrations. In this study, no inhibitory influence of Tiron on NET release was revealed. All tested substances exerted a significant dose-dependent antioxidative effect on ROS generation induced by phorbol 12-myristate 13-acetate (PMA). We suggest that the antioxidants Trolox and Tempol should be recommended for treating autoimmune and inflammatory diseases that implicate ROS-dependent NET release. PMID:26371849

  1. Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition.

    PubMed

    Hopke, Alex; Nicke, Nadine; Hidu, Erica E; Degani, Genny; Popolo, Laura; Wheeler, Robert T

    2016-05-01

    Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. The opportunistic human fungal pathogen Candida albicans, which can cause fatal disease in immunocompromised patient populations, offers a good example as it masks the inflammatory epitope β-glucan in its cell wall from host recognition. It has been demonstrated previously that β-glucan becomes exposed during infection in vivo but the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance immune recognition by the Dectin-1 β-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog. PMID:27223610

  2. Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.

    PubMed

    Tohme, Samer; Yazdani, Hamza O; Al-Khafaji, Ahmed B; Chidi, Alexis P; Loughran, Patricia; Mowen, Kerri; Wang, Yanming; Simmons, Richard L; Huang, Hai; Tsung, Allan

    2016-03-15

    Risks of tumor recurrence after surgical resection have been known for decades, but the mechanisms underlying treatment failures remain poorly understood. Neutrophils, first-line responders after surgical stress, may play an important role in linking inflammation to cancer progression. In response to stress, neutrophils can expel their protein-studded chromatin to form local snares known as neutrophil extracellular traps (NET). In this study, we asked whether, as a result of its ability to ensnare moving cells, NET formation might promote metastasis after surgical stress. Consistent with this hypothesis, in a cohort of patients undergoing attempted curative liver resection for metastatic colorectal cancer, we observed that increased postoperative NET formation was associated with a >4-fold reduction in disease-free survival. In like manner, in a murine model of surgical stress employing liver ischemia-reperfusion, we observed an increase in NET formation that correlated with an accelerated development and progression of metastatic disease. These effects were abrogated by inhibiting NET formation in mice through either local treatment with DNAse or inhibition of the enzyme peptidylarginine deaminase, which is essential for NET formation. In growing metastatic tumors, we found that intratumoral hypoxia accentuated NET formation. Mechanistic investigations in vitro indicated that mouse neutrophil-derived NET triggered HMGB1 release and activated TLR9-dependent pathways in cancer cells to promote their adhesion, proliferation, migration, and invasion. Taken together, our findings implicate NET in the development of liver metastases after surgical stress, suggesting that their elimination may reduce risks of tumor relapse. PMID:26759232

  3. Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps.

    PubMed

    Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

    2016-01-01

    Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system. PMID:27512953

  4. Reactive oxidants and myeloperoxidase and their involvement in neutrophil extracellular traps.

    PubMed

    Parker, Heather; Winterbourn, Christine C

    2012-01-01

    Neutrophils release extracellular traps (NETs) in response to a variety of inflammatory stimuli. These structures are composed of a network of chromatin strands associated with a variety of neutrophil-derived proteins including the enzyme myeloperoxidase (MPO). Studies into the mechanisms leading to the formation of NETs indicate a complex process that differs according to the stimulus. With some stimuli an active nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is required. However, assigning specific reactive oxygen species involved downstream of the oxidase is a difficult task and definitive proof for any single oxidant is still lacking. Pharmacological inhibition of MPO and the use of MPO-deficient neutrophils indicate active MPO is required with phorbol myristate acetate as a stimulus but not necessarily with bacteria. Reactive oxidants and MPO may also play a role in NET-mediated microbial killing. MPO is present on NETs and maintains activity at this site. Therefore, MPO has the potential to generate reactive oxidants in close proximity to trapped microorganisms and thus effect microbial killing. This brief review discusses current evidence for the involvement of reactive oxidants and MPO in NET formation and their potential contribution to NET antimicrobial activity. PMID:23346086

  5. The impact of cationic solid lipid nanoparticles on human neutrophil activation and formation of neutrophil extracellular traps (NETs).

    PubMed

    Hwang, Tsong-Long; Aljuffali, Ibrahim A; Hung, Chi-Feng; Chen, Chun-Han; Fang, Jia-You

    2015-06-25

    Cationic solid lipid nanoparticles (cSLNs) are extensively employed as the nanocarriers for drug/gene targeting to tumors and the brain. Investigation into the possible immune response of cSLNs is still lacking. The aim of this study was to evaluate the impact of cSLNs upon the activation of human polymorphonuclear neutrophil cells (PMNs). The cytotoxicity, pro-inflammatory mediators, Ca(2+) mobilization, mitogen-activated protein kinases (MAPKs), and neutrophil extracellular traps (NETs) as the indicators of PMN stimulation were examined in this work. The cSLNs presented a diameter of 195 nm with a zeta potential of 44 mV. The cSLNs could interact with the cell membrane to produce a direct membrane lysis and the subsequent cytotoxicity according to lactate dehydrogenase (LDH) elevation. The interaction of cSLNs with the membrane also triggered a Ca(2+) influx, followed by the induction of oxidative stress and degranulation. The cationic nanoparticles elevated the levels of superoxide anion and elastase by 24- and 9-fold, respectively. The PMN activation by cSLNs promoted the phosphorylation of p38 and Jun-N-terminal kinases (JNK) but not extracellular signal-regulated kinases (ERK). The imaging of scanning electron microscopy (SEM) and immunofluorescence demonstrated the production of NETs by cSLNs. This phenomenon was not significant for the neutral SLNs (nSLNs), although histones in NETs also increased after treatment of nSLNs. Our results suggest an important role of cSLNs in governing the activation of human neutrophils. PMID:25920576

  6. Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease.

    PubMed

    He, Zhangxiu; Si, Yu; Jiang, Tao; Ma, Ruishuang; Zhang, Yan; Cao, Muhua; Li, Tao; Yao, Zhipeng; Zhao, Lu; Fang, Shaohong; Yu, Bo; Dong, Zengxiang; Thatte, Hemant S; Bi, Yayan; Kou, Junjie; Yang, Shufen; Piao, Daxun; Hao, Lirong; Zhou, Jin; Shi, Jialan

    2016-04-01

    Inflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients. PMID:26660948

  7. Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCA-associated vasculitis

    PubMed Central

    Tang, S; Zhang, Y; Yin, S-W; Gao, X-J; Shi, W-W; Wang, Y; Huang, X; Wang, L; Zou, L-Y; Zhao, J-H; Huang, Y-J; Shan, L-Y; Gounni, A S; Wu, Y-Z; Zhang, J-B

    2015-01-01

    Increasing evidence indicates that aberrant neutrophil extracellular trap (NET) formation could contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Recent research has provided evidence that a novel type of ANCA autoantibody, anti-lysosomal membrane protein-2 (LAMP-2) antibody, may have a pathogenic role in AAV. We have shown previously that anti-LAMP-2 antibody-stimulated NET formation contains autoantigens and anti-microbial peptides. The current study sought to determine whether LAMP-2, as a novel antigen of ANCA, was present on NETs in AAV patients, the influence of the anti-LAMP-2 antibody on the neutrophil apoptosis rate and the role of autophagy in anti-LAMP-2 antibody-induced NET formation. NET formation was assessed using immunofluorescence microscopy, scanning electron microscopy or live cell imaging. The neutrophil apoptosis rate was analysed using fluorescence activated cell sorting (FACS). Autophagy was detected using LC3B accumulation and transmission electron microscopy. The results showed that enhanced NET formation, which contains LAMP-2, was observed in kidney biopsies and neutrophils from AAV patients. The apoptosis rate decreased significantly in human neutrophils stimulated with anti-LAMP-2 antibody, and this effect was attenuated by the inhibitors of autophagy 3-methyladenine (3MA) and 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002). The anti-LAMP-2 antibody-stimulated NET formation was unaffected by benzyloxycarbonyl-Val- Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) and necrostatin-1 (Nec-1), which are inhibitors of apoptosis and necrosis, respectively, but was inhibited by 3MA and LY294002. Moreover, the proportion of LC3BI that was converted to LC3BII increased significantly (P = 0·0057), and massive vacuolizations that exhibited characteristics typical of autophagy were detected in neutrophils stimulated with anti-LAMP-2 antibody. Our results provide further evidence that

  8. New Insights into Neutrophil Extracellular Traps: Mechanisms of Formation and Role in Inflammation.

    PubMed

    Yang, Hang; Biermann, Mona Helena; Brauner, Jan Markus; Liu, Yi; Zhao, Yi; Herrmann, Martin

    2016-01-01

    Recent data suggest that NETosis plays a crucial role in the innate immune response and disturbs the homeostasis of the immune system. NETosis is a form of neutrophil-specific cell death characterized by the release of large web-like structures referred to as neutrophil extracellular traps (NETs). NETs are composed of DNA strands associated with histones and decorated with about 20 different proteins, including neutrophil elastase, myeloperoxidase, cathepsin G, proteinase 3, high mobility group protein B1, and LL37. Reportedly, NETosis can be induced by several microbes, and particulate matter including sterile stimuli, via distinct cellular mechanisms. Meanwhile, suicidal NETosis and vital NETosis are controversial. As we enter the second decade of research on NETosis, we have partly understood NETs as double-edged swords of innate immunity. In this review, we will discuss the mechanisms of NETosis, its antimicrobial action, and role in autoimmune diseases, as well as the relatively new field of NET-associated mitochondrial DNA. PMID:27570525

  9. New Insights into Neutrophil Extracellular Traps: Mechanisms of Formation and Role in Inflammation

    PubMed Central

    Yang, Hang; Biermann, Mona Helena; Brauner, Jan Markus; Liu, Yi; Zhao, Yi; Herrmann, Martin

    2016-01-01

    Recent data suggest that NETosis plays a crucial role in the innate immune response and disturbs the homeostasis of the immune system. NETosis is a form of neutrophil-specific cell death characterized by the release of large web-like structures referred to as neutrophil extracellular traps (NETs). NETs are composed of DNA strands associated with histones and decorated with about 20 different proteins, including neutrophil elastase, myeloperoxidase, cathepsin G, proteinase 3, high mobility group protein B1, and LL37. Reportedly, NETosis can be induced by several microbes, and particulate matter including sterile stimuli, via distinct cellular mechanisms. Meanwhile, suicidal NETosis and vital NETosis are controversial. As we enter the second decade of research on NETosis, we have partly understood NETs as double-edged swords of innate immunity. In this review, we will discuss the mechanisms of NETosis, its antimicrobial action, and role in autoimmune diseases, as well as the relatively new field of NET-associated mitochondrial DNA. PMID:27570525

  10. Placental histology and neutrophil extracellular traps in lupus and pre-eclampsia pregnancies

    PubMed Central

    Marder, Wendy; Knight, Jason S; Kaplan, Mariana J; Somers, Emily C; Zhang, Xu; O'Dell, Alexander A; Padmanabhan, Vasantha; Lieberman, Richard W

    2016-01-01

    Objective Systemic lupus erythematosus (SLE) is associated with increased risk of adverse pregnancy outcomes, including pre-eclampsia, particularly in association with antiphospholipid antibody syndrome (APS). While significant placental abnormalities are expected in pre-eclampsia, less is known about how lupus activity and APS in pregnancy affect the placenta. We describe placental pathology from a population of lupus pregnancies, several of which were complicated by APS-related thromboses, in which pre-eclampsia and other complications developed. We performed standard histopathological placental review and quantified neutrophils and neutrophil extracellular traps (NETs) in the intervillous space, given the recognised association of NETs with lupus, APS and pre-eclampsia. Methods Pre-eclampsia, SLE and control placentas were scored for histological features, and neutrophils were quantified on H&E and immunohistochemical staining for the granular protein myeloperoxidase. NETs were identified by extracellular myeloperoxidase staining in the setting of decondensed nuclei. Non-parametric analysis was used to evaluate differences in netting and intact neutrophils between groups, with Kruskal–Wallis testing for associations between histological findings and neutrophils. Results Placentas were evaluated from 35 pregnancies: 10 controls, 11 pre-eclampsia, 4 SLE+pre-eclampsia and 10 SLE, including one complicated by catastrophic APS and one complicated by hepatic and splenic vein thromboses during pregnancy. Intrauterine growth restriction and oligohydramnios were observed in lupus cases but not controls. Significantly more NETs were found infiltrating placental intervillous spaces in pre-eclampsia, SLE+pre-eclampsia and all 10 SLE non-pre-eclampsia cases. The ratio of NETs to total neutrophils was significantly increased in all case groups compared with controls. When present, NETs were associated with maternal vasculitis, laminar decidual necrosis, maternal

  11. Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.

    PubMed

    Ostafin, Magdalena; Pruchniak, Michal Przemyslaw; Ciepiela, Olga; Reznick, Abraham Zeev; Demkow, Urszula

    2016-09-15

    A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample. PMID:27179553

  12. Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.

    PubMed

    Jeffery, Unity; Kimura, Kayoko; Gray, Robert; Lueth, Paul; Bellaire, Bryan; LeVine, Dana

    2015-12-15

    Neutrophil extracellular traps (NETs) are webs of DNA and protein with both anti-microbial and pro-thrombotic properties which have not been previously reported in dogs. To confirm dog neutrophils can form NETs, neutrophils were isolated from healthy dogs, and stimulated in vitro with 2μM, 8μM, 31μM, and 125μM platelet activating factor (PAF) or 0.03μM, 0.1μM, 0.4μM, 1.6μM and 6.4μM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4h. At 4h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31μM and ≥0.1μM for PAF and PMA, respectively. Cells stimulated with 31.25μM PAF reached maximal fluorescence by 1h, whereas maximal fluorescence was not achieved until 2h for cells stimulated with 0.1μM PMA. Immunofluorescent imaging using DAPI and anti-elastase antibody confirmed that extracellular DNA is released as NETs. As NETs have been implicated in thrombosis, nucleosomes, a marker correlated with NET formation, were measured in the serum of dogs with the thrombotic disorder primary immune-mediated hemolytic anemia (IMHA) (n=7) and healthy controls (n=20) using a commercially available ELISA. NETs were significantly higher in IMHA cases than controls (median 0.12 and 0.90, respectively, p=0.01), but there were large positive interferences associated with hemolysis and icterus. In summary, the study is the first to describe NET generation by canine neutrophils and provides preliminary evidence that a marker associated with NETs is elevated in IMHA. However, this apparent elevation must be interpreted with caution due to the effect of interference, emphasizing the need for a more specific and robust assay for NETs in clinical samples. PMID:26574161

  13. Neutrophil Extracellular Traps in Pulmonary Diseases: Too Much of a Good Thing?

    PubMed Central

    Porto, Bárbara Nery; Stein, Renato Tetelbom

    2016-01-01

    Neutrophil extracellular traps (NETs) arise from the release of granular and nuclear contents of neutrophils in the extracellular space in response to different classes of microorganisms, soluble factors, and host molecules. NETs are composed by decondensed chromatin fibers coated with antimicrobial granular and cytoplasmic proteins, such as myeloperoxidase, neutrophil elastase (NE), and α-defensins. Besides being expressed on NET fibers, NE and MPO also regulate NET formation. Furthermore, histone deimination by peptidylarginine deiminase 4 (PAD4) is a central step to NET formation. NET formation has been widely demonstrated to be an effective mechanism to fight against invading microorganisms, as deficiency in NET release or dismantling NET backbone by bacterial DNases renders the host susceptible to infections. Therefore, the primary role of NETs is to prevent microbial dissemination, avoiding overwhelming infections. However, an excess of NET formation has a dark side. The pathogenic role of NETs has been described for many human diseases, infectious and non-infectious. The detrimental effect of excessive NET release is particularly important to lung diseases, because NETs can expand more easily in the pulmonary alveoli, causing lung injury. Moreover, NETs and its associated molecules are able to directly induce epithelial and endothelial cell death. In this regard, massive NET formation has been reported in several pulmonary diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory syncytial virus bronchiolitis, influenza, bacterial pneumonia, and tuberculosis, among others. Thus, NET formation must be tightly regulated in order to avoid NET-mediated tissue damage. Recent development of therapies targeting NETs in pulmonary diseases includes DNA disintegration with recombinant human DNase, neutralization of NET proteins, with anti-histone antibodies and protease inhibitors. In this review, we summarize the recent

  14. DAMPs-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury

    PubMed Central

    Huang, Hai; Tohme, Samer; Al-Khafaji, Ahmed B; Tai, Sheng; Loughran, Patricia; Chen, Li; Wang, Shu; Kim, Jiyun; Billiar, Timothy; Wang, Yanming; Tsung, Allan

    2015-01-01

    Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage associated molecular patterns (DAMPs), which trigger innate immune and inflammatory cascade via pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to the organ damage, innate immune and inflammatory responses. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. DAMPs, such as HMGB1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. In addition, we found inhibition of NET formation by PAD4 inhibitor or DNase I reduces HMGB1 and histone-mediated liver I/R injury. Conclusion DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R. PMID:25855125

  15. A PPARγ AGONIST ENHANCES BACTERIAL CLEARANCE THROUGH NEUTROPHIL EXTRACELLULAR TRAP FORMATION AND IMPROVES SURVIVAL IN SEPSIS.

    PubMed

    Araújo, Cláudia V; Campbell, Clarissa; Gonçalves-de-Albuquerque, Cassiano F; Molinaro, Raphael; Cody, Mark J; Yost, Christian C; Bozza, Patricia T; Zimmerman, Guy A; Weyrich, Andrew S; Castro-Faria-Neto, Hugo C; Silva, Adriana R

    2016-04-01

    Dysregulation of the inflammatory response against infection contributes to mortality in sepsis. Inflammation provides critical host defense, but it can cause tissue damage, multiple organ failure, and death. Because the nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) exhibits therapeutic potential, we characterized the role of PPARγ in sepsis. We analyzed severity of clinical signs, survival rates, cytokine production, leukocyte influx, and bacterial clearance in a cecal ligation and puncture (CLP) model of sepsis in Swiss mice. The PPARγ agonist rosiglitazone treatment improved clinical status and mortality, while increasing IL-10 production and decreasing TNF-α and IL-6 levels, and peritoneal neutrophil accumulation 24 h after CLP. We noted increased bacterial killing in rosiglitazone treated mice, correlated with increased generation of reactive oxygen species. Polymorphonuclear leukocytes (PMN) incubated with LPS or Escherichia coli and rosiglitazone increased peritoneal neutrophil extracellular trap (NET)-mediated bacterial killing, an effect reversed by the PPARγ antagonist (GW 9662) treatment. Rosiglitazone also enhanced the release of histones by PMN, a surrogate marker of NET formation, effect abolished by GW 9662. Rosiglitazone modulated the inflammatory response and increased bacterial clearance through PPARγ activation and NET formation, combining immunomodulatory and host-dependent anti-bacterial effects and, therefore, warrants further study as a potential therapeutic agent in sepsis. PMID:26618986

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

    PubMed

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

    2014-12-01

    Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare. PMID:25218891

  17. DOCK2 and DOCK5 Act Additively in Neutrophils To Regulate Chemotaxis, Superoxide Production, and Extracellular Trap Formation

    PubMed Central

    Watanabe, Mayuki; Terasawa, Masao; Miyano, Kei; Yanagihara, Toyoshi; Uruno, Takehito; Sanematsu, Fumiyuki; Nishikimi, Akihiko; Côté, Jean-François; Sumimoto, Hideki; Fukui, Yoshinori

    2015-01-01

    Neutrophils are highly motile leukocytes that play important roles in the innate immune response to invading pathogens. Neutrophils rapidly migrate to the site of infections and kill pathogens by producing reactive oxygen species (ROS). Neutrophil chemotaxis and ROS production require activation of Rac small GTPase. DOCK2, an atypical guanine nucleotide exchange factor (GEF), is one of the major regulators of Rac in neutrophils. However, because DOCK2 deficiency does not completely abolish fMLF-induced Rac activation, other Rac GEFs may also participate in this process. In this study, we show that DOCK5 acts with DOCK2 in neutrophils to regulate multiple cellular functions. We found that fMLF- and PMA-induced Rac activation were almost completely lost in mouse neutrophils lacking both DOCK2 and DOCK5. Although β2 integrin–mediated adhesion occurred normally even in the absence of DOCK2 and DOCK5, mouse neutrophils lacking DOCK2 and DOCK5 exhibited a severe defect in chemotaxis and ROS production. Similar results were obtained when human neutrophils were treated with CPYPP, a small-molecule inhibitor of these DOCK GEFs. Additionally, we found that DOCK2 and DOCK5 regulate formation of neutrophil extracellular traps (NETs). Because NETs are involved in vascular inflammation and autoimmune responses, DOCK2 and DOCK5 would be a therapeutic target for controlling NET-mediated inflammatory disorders. PMID:25339677

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

    PubMed Central

    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.; F. Pimentel, Maria Ines; 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

  19. Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production

    PubMed Central

    Kusunoki, Yoshihiro; Nakazawa, Daigo; Shida, Haruki; Hattanda, Fumihiko; Miyoshi, Arina; Masuda, Sakiko; Nishio, Saori; Tomaru, Utano; Atsumi, Tatsuya; Ishizu, Akihiro

    2016-01-01

    Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 106/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared

  20. Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production.

    PubMed

    Kusunoki, Yoshihiro; Nakazawa, Daigo; Shida, Haruki; Hattanda, Fumihiko; Miyoshi, Arina; Masuda, Sakiko; Nishio, Saori; Tomaru, Utano; Atsumi, Tatsuya; Ishizu, Akihiro

    2016-01-01

    Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 10(6)/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared

  1. Neutrophil Extracellular Traps Are Pathogenic in Primary Graft Dysfunction after Lung Transplantation

    PubMed Central

    Mallavia, Beñat; Liu, Fengchun; Ortiz-Muñoz, Guadalupe; Caudrillier, Axelle; DerHovanessian, Ariss; Ross, David J.; Lynch III, Joseph P.; Saggar, Rajan; Ardehali, Abbas; Ware, Lorraine B.; Christie, Jason D.; Belperio, John A.; Looney, Mark R.

    2015-01-01

    Rationale: Primary graft dysfunction (PGD) causes early mortality after lung transplantation and may contribute to late graft failure. No effective treatments exist. The pathogenesis of PGD is unclear, although both neutrophils and activated platelets have been implicated. We hypothesized that neutrophil extracellular traps (NETs) contribute to lung injury in PGD in a platelet-dependent manner. Objectives: To study NETs in experimental models of PGD and in lung transplant patients. Methods: Two experimental murine PGD models were studied: hilar clamp and orthotopic lung transplantation after prolonged cold ischemia (OLT-PCI). NETs were assessed by immunofluorescence microscopy and ELISA. Platelet activation was inhibited with aspirin, and NETs were disrupted with DNaseI. NETs were also measured in bronchoalveolar lavage fluid and plasma from lung transplant patients with and without PGD. Measurements and Main Results: NETs were increased after either hilar clamp or OLT-PCI compared with surgical control subjects. Activation and intrapulmonary accumulation of platelets were increased in OLT-PCI, and platelet inhibition reduced NETs and lung injury, and improved oxygenation. Disruption of NETs by intrabronchial administration of DNaseI also reduced lung injury and improved oxygenation. In bronchoalveolar lavage fluid from human lung transplant recipients, NETs were more abundant in patients with PGD. Conclusions: NETs accumulate in the lung in both experimental and clinical PGD. In experimental PGD, NET formation is platelet-dependent, and disruption of NETs with DNaseI reduces lung injury. These data are the first description of a pathogenic role for NETs in solid organ transplantation and suggest that NETs are a promising therapeutic target in PGD. PMID:25485813

  2. The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

    PubMed Central

    Lee, Mark J.; Liu, Hong; Barker, Bridget M.; Snarr, Brendan D.; Gravelat, Fabrice N.; Al Abdallah, Qusai; Gavino, Christina; Baistrocchi, Shane R.; Ostapska, Hanna; Xiao, Tianli; Ralph, Benjamin; Solis, Norma V.; Lehoux, Mélanie; Baptista, Stefanie D.; Thammahong, Arsa; Cerone, Robert P.; Kaminskyj, Susan G. W.; Guiot, Marie-Christine; Latgé, Jean-Paul; Fontaine, Thierry; Vinh, Donald C.; Filler, Scott G.; Sheppard, Donald C.

    2015-01-01

    Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs. PMID:26492565

  3. Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing.

    PubMed

    Van Avondt, Kristof; van der Linden, Maarten; Naccache, Paul H; Egan, David A; Meyaard, Linde

    2016-05-01

    In response to microbial invasion, neutrophils release neutrophil extracellular traps (NETs) to trap and kill extracellular microbes. Alternatively, NET formation can result in tissue damage in inflammatory conditions and may perpetuate autoimmune disease. Intervention strategies that are aimed at modifying pathogenic NET formation should ideally preserve other neutrophil antimicrobial functions. We now show that signal inhibitory receptor on leukocytes-1 (SIRL-1) attenuates NET release by human neutrophils in response to distinct triggers, including opsonized Staphylococcus aureus and inflammatory danger signals. NET release has different kinetics depending on the stimulus, and rapid NET formation is independent of NADPH oxidase activity. In line with this, we show that NET release and reactive oxygen species production upon challenge with opsonized S. aureus require different signaling events. Importantly, engagement of SIRL-1 does not affect bacterially induced production of reactive oxygen species, and intracellular bacterial killing by neutrophils remains intact. Thus, our studies define SIRL-1 as an intervention point of benefit to suppress NET formation in disease while preserving intracellular antimicrobial defense. PMID:27016607

  4. Beta-hydroxybutyrate abrogates formation of bovine neutrophil extracellular traps and bactericidal activity against mammary pathogenic Escherichia coli.

    PubMed

    Grinberg, Navit; Elazar, Sharon; Rosenshine, Ilan; Shpigel, Nahum Y

    2008-06-01

    Escherichia coli is an important bacterial species isolated from bovine mastitis. The rate of neutrophil recruitment into the mammary gland and their bactericidal activity largely affect the severity and outcome of the disease. Ketosis is a common metabolic disease, and affected dairy cows are known to have increased risk for mastitis and other infectious conditions. The disease is associated with high blood and milk levels of beta-hydroxybutyrate (BHBA), previously shown to negatively affect neutrophil function by unknown mechanisms. We show here that the mammary pathogenic E. coli strain P4 activates normal bovine neutrophils to form neutrophil extracellular traps (NETs), which are highly bactericidal against this organism. Preincubation of these neutrophils with increasing concentrations (0.1 to 8 mmol/liter) of BHBA caused a fivefold decrease of E. coli P4 phagocytosis, though intracellular killing was unaffected. Furthermore, BHBA caused a 10-fold decrease in the NETs formed by E. coli P4-activated neutrophils and a similar decrease in NET bactericidal activity against this organism. These negative effects of BHBA on bovine neutrophils might explain the increased susceptibility of ketotic cows to mastitis and other infectious conditions. PMID:18411287

  5. Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase

    PubMed Central

    Knudsen, Giselle M.; Perera, Natascha C.; Jenne, Dieter E.; Murphy, John E.; Craik, Charles S.; Hermiston, Terry W.

    2013-01-01

    Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases. PMID:24073241

  6. The architecture of neutrophil extracellular traps investigated by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Pires, Ricardo H.; Felix, Stephan B.; Delcea, Mihaela

    2016-07-01

    Neutrophils are immune cells that engage in a suicidal pathway leading to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). NETs behave as a double edged sword; they can bind to pathogens thereby ensnaring them and limiting their spread during infection; however, they may bind to host circulating materials and trigger thrombotic events, and are associated with autoimmune disorders. Despite the fundamental role of NETs as part of an immune system response, there is currently a very poor understanding of how their nanoscale properties are reflected in their macroscopic impact. In this work, using a combination of fluorescence and atomic force microscopy, we show that NETs appear as a branching filament network that results in a substantially organized porous structure with openings with 0.03 +/- 0.04 μm2 on average and thus in the size range of small pathogens. Topological profiles typically up to 3 +/- 1 nm in height are compatible with a ``beads on a string'' model of nucleosome chromatin. Typical branch lengths of 153 +/- 103 nm appearing as rigid rods and height profiles of naked DNA in NETs of 1.2 +/- 0.5 nm are indicative of extensive DNA supercoiling throughout NETs. The presence of DNA duplexes could also be inferred from force spectroscopy and the occurrence of force plateaus that ranged from ~65 pN to 300 pN. Proteolytic digestion of NETs resulted in widespread disassembly of the network structure and considerable loss of mechanical properties. Our results suggest that the underlying structure of NETs is considerably organized and that part of its protein content plays an important role in maintaining its mesh architecture. We anticipate that NETs may work as microscopic mechanical sieves with elastic properties that stem from their DNA-protein composition, which is able to segregate particles also as a result of their size. Such a behavior may explain their participation in capturing pathogens and their association

  7. The architecture of neutrophil extracellular traps investigated by atomic force microscopy.

    PubMed

    Pires, Ricardo H; Felix, Stephan B; Delcea, Mihaela

    2016-08-01

    Neutrophils are immune cells that engage in a suicidal pathway leading to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). NETs behave as a double edged sword; they can bind to pathogens thereby ensnaring them and limiting their spread during infection; however, they may bind to host circulating materials and trigger thrombotic events, and are associated with autoimmune disorders. Despite the fundamental role of NETs as part of an immune system response, there is currently a very poor understanding of how their nanoscale properties are reflected in their macroscopic impact. In this work, using a combination of fluorescence and atomic force microscopy, we show that NETs appear as a branching filament network that results in a substantially organized porous structure with openings with 0.03 ± 0.04 μm(2) on average and thus in the size range of small pathogens. Topological profiles typically up to 3 ± 1 nm in height are compatible with a "beads on a string" model of nucleosome chromatin. Typical branch lengths of 153 ± 103 nm appearing as rigid rods and height profiles of naked DNA in NETs of 1.2 ± 0.5 nm are indicative of extensive DNA supercoiling throughout NETs. The presence of DNA duplexes could also be inferred from force spectroscopy and the occurrence of force plateaus that ranged from ∼65 pN to 300 pN. Proteolytic digestion of NETs resulted in widespread disassembly of the network structure and considerable loss of mechanical properties. Our results suggest that the underlying structure of NETs is considerably organized and that part of its protein content plays an important role in maintaining its mesh architecture. We anticipate that NETs may work as microscopic mechanical sieves with elastic properties that stem from their DNA-protein composition, which is able to segregate particles also as a result of their size. Such a behavior may explain their participation in capturing pathogens and their association

  8. Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis

    PubMed Central

    Nascimento, Daniele Carvalho; Sônego, Fabiane; Castanheira, Fernanda Vargas e Silva; Melo, Paulo Henrique; Scortegagna, Gabriela Trentin; Silva, Rangel Leal; Barroso-Sousa, Romualdo; Souto, Fabrício Oliveira; Pazin-Filho, Antonio; Figueiredo, Florencio; Alves-Filho, José Carlos; Cunha, Fernando Queiróz

    2016-01-01

    Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are

  9. Gallic acid reduces the effect of LPS on apoptosis and inhibits the formation of neutrophil extracellular traps.

    PubMed

    Haute, Gabriela Viegas; Caberlon, Eduardo; Squizani, Eamim; de Mesquita, Fernanda Cristina; Pedrazza, Leonardo; Martha, Bianca Andrade; da Silva Melo, Denizar Alberto; Cassel, Eduardo; Czepielewski, Rafael Sanguinetti; Bitencourt, Shanna; Goettert, Márcia Inês; de Oliveira, Jarbas Rodrigues

    2015-12-25

    Apoptosis and NETosis of neutrophils are two major mechanisms of programmed cell death that differ in their morphological characteristics and effects on the immune system. Apoptosis can be delayed by the presence of pathogens or chemical components such as lipopolysaccharide (LPS). Neutrophils have other antimicrobial strategy, called neutrophil extracellular traps (NETs), which contributes to the elimination and control of the pathogen. NETosis is induced by infection, inflammation or trauma and represents an innate immune activation mechanism. The objective of this study was to evaluate the effect of gallic acid (GA) in the modulation of apoptosis and NETs release. The results show that GA decreased the anti-apoptotic effect of LPS, blocked the induction of NETs and prevented the formation of free radicals induced by LPS. These findings demonstrate that the GA is a novel therapeutic agent for decreasing the exacerbated response of the body against an infectious agent. PMID:26475966

  10. Proteins derived from neutrophil extracellular traps may serve as self-antigens and mediate organ damage in autoimmune diseases

    PubMed Central

    Knight, Jason S.; Carmona-Rivera, Carmelo; Kaplan, Mariana J.

    2012-01-01

    Neutrophils are the most abundant leukocytes in circulation and represent one of the first lines of defense against invading pathogens. Neutrophils possess a vast arsenal of antimicrobial proteins, which can be released from the cell by a death program termed NETosis. Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin decorated with granular and cytosolic proteins. Both exuberant NETosis and impaired clearance of NETs have been implicated in the organ damage of autoimmune diseases, such as systemic lupus erythematosus (SLE), small vessel vasculitis (SVV), and psoriasis. NETs may also represent an important source of modified autoantigens in SLE and SVV. Here, we review the autoimmune diseases linked to NETosis, with a focus on how modified proteins externalized on NETs may trigger loss of immune tolerance and promote organ damage. PMID:23248629

  11. Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx, necrosis and neutrophil extracellular trap formation

    PubMed Central

    Francis, R J; Butler, R E; Stewart, G R

    2014-01-01

    Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca2+ overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca2+ activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca2+, stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission. PMID:25321481

  12. Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2.

    PubMed

    Lee, Sung Kyun; Kim, Sang Doo; Kook, Minsoo; Lee, Ha Young; Ghim, Jaewang; Choi, Youngwoo; Zabel, Brian A; Ryu, Sung Ho; Bae, Yoe-Sik

    2015-08-24

    We determined the function of phospholipase D2 (PLD2) in host defense in highly lethal mouse models of sepsis using PLD2(-/-) mice and a PLD2-specific inhibitor. PLD2 deficiency not only increases survival but also decreases vital organ damage during experimental sepsis. Production of several inflammatory cytokines (TNF, IL-1β, IL-17, and IL-23) and the chemokine CXCL1, as well as cellular apoptosis in immune tissues, kidney, and liver, are markedly decreased in PLD2(-/-) mice. Bactericidal activity is significantly increased in PLD2(-/-) mice, which is mediated by increased neutrophil extracellular trap formation and citrullination of histone 3 through peptidylarginine deiminase activation. Recruitment of neutrophils to the lung is markedly increased in PLD2(-/-) mice. Furthermore, LPS-induced induction of G protein-coupled receptor kinase 2 (GRK2) and down-regulation of CXCR2 are markedly attenuated in PLD2(-/-) mice. A CXCR2-selective antagonist abolishes the protection conferred by PLD2 deficiency during experimental sepsis, suggesting that enhanced CXCR2 expression, likely driven by GRK2 down-regulation in neutrophils, promotes survival in PLD2(-/-) mice. Furthermore, adoptively transferred PLD2(-/-) neutrophils significantly protect WT recipients against sepsis-induced death compared with transferred WT neutrophils. We suggest that PLD2 in neutrophils is essential for the pathogenesis of experimental sepsis and that pharmaceutical agents that target PLD2 may prove beneficial for septic patients. PMID:26282875

  13. Gradient Infiltration of Neutrophil Extracellular Traps in Colon Cancer and Evidence for Their Involvement in Tumour Growth

    PubMed Central

    Kambas, Konstantinos; Papagoras, Charalampos; Miltiades, Paraskevi; Angelidou, Iliana; Mitsios, Alexandros; Kotsianidis, Ioannis; Skendros, Panagiotis; Sivridis, Efthimios; Maroulakou, Ioanna; Giatromanolaki, Alexandra; Ritis, Konstantinos

    2016-01-01

    Background The role of neutrophils in tumour biology is largely unresolved. Recently, independent studies indicated either neutrophil extracellular traps (NETs) or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, their individual or combined role in colonic adenocarcinoma is still unexplored. Methods Colectomy tissue specimens and variable number of draining lymph nodes were obtained from ten patients with adenocarcinoma of the colon. NETs deposition and neutrophil presence as well as TF expression were examined by immunostaining. The effect of NETs on cancer cell growth was studied in in vitro co-cultures of Caco-2 cell line and acute myeloid leukemia primary cells. Proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry. Results TF-bearing NETs and neutrophil localization were prominent in tumour sections and the respective metastatic lymph nodes. Interestingly, neutrophil infiltration and NETs concentration were gradually reduced from the tumour mass to the distal margin. The in vitro-generated NETs impeded growth of cancer cell cultures by inducing apoptosis and/or inhibiting proliferation. Conclusions These data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth. PMID:27136460

  14. The architecture of neutrophil extracellular traps investigated by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Pires, Ricardo H.; Felix, Stephan B.; Delcea, Mihaela

    2016-07-01

    Neutrophils are immune cells that engage in a suicidal pathway leading to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). NETs behave as a double edged sword; they can bind to pathogens thereby ensnaring them and limiting their spread during infection; however, they may bind to host circulating materials and trigger thrombotic events, and are associated with autoimmune disorders. Despite the fundamental role of NETs as part of an immune system response, there is currently a very poor understanding of how their nanoscale properties are reflected in their macroscopic impact. In this work, using a combination of fluorescence and atomic force microscopy, we show that NETs appear as a branching filament network that results in a substantially organized porous structure with openings with 0.03 +/- 0.04 μm2 on average and thus in the size range of small pathogens. Topological profiles typically up to 3 +/- 1 nm in height are compatible with a ``beads on a string'' model of nucleosome chromatin. Typical branch lengths of 153 +/- 103 nm appearing as rigid rods and height profiles of naked DNA in NETs of 1.2 +/- 0.5 nm are indicative of extensive DNA supercoiling throughout NETs. The presence of DNA duplexes could also be inferred from force spectroscopy and the occurrence of force plateaus that ranged from ~65 pN to 300 pN. Proteolytic digestion of NETs resulted in widespread disassembly of the network structure and considerable loss of mechanical properties. Our results suggest that the underlying structure of NETs is considerably organized and that part of its protein content plays an important role in maintaining its mesh architecture. We anticipate that NETs may work as microscopic mechanical sieves with elastic properties that stem from their DNA-protein composition, which is able to segregate particles also as a result of their size. Such a behavior may explain their participation in capturing pathogens and their association

  15. The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

    PubMed

    Jayaprakash, K; Demirel, I; Khalaf, H; Bengtsson, T

    2015-10-01

    Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria. PMID:25869817

  16. Carrageenan-induced inflammation promotes ROS generation and neutrophil extracellular trap formation in a mouse model of peritonitis.

    PubMed

    Barth, Cristiane R; Funchal, Giselle A; Luft, Carolina; de Oliveira, Jarbas R; Porto, Bárbara N; Donadio, Márcio V F

    2016-04-01

    Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular proteins, such as neutrophil elastase (NE). NETs are released in the extracellular space in response to different stimuli. Carrageenan is a sulfated polysaccharide extracted from Chondrus crispus, a marine algae, used for decades in research for its potential to induce inflammation in different animal models. In this study, we show for the first time that carrageenan injection can induce NET release in a mouse model of acute peritonitis. Carrageenan induced NET release by viable neutrophils with NE and myeloperoxidase (MPO) expressed on DNA fibers. Furthermore, although this polysaccharide was able to stimulate reactive oxygen species (ROS) generation by peritoneal neutrophils, NADPH oxidase derived ROS were dispensable for NET formation by carrageenan. In conclusion, our results show that carrageenan-induced inflammation in the peritoneum of mice can induce NET formation in an ROS-independent manner. These results may add important information to the field of inflammation and potentially lead to novel anti-inflammatory agents targeting the production of NETs. PMID:26786873

  17. Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites

    PubMed Central

    Rochael, Natalia C.; Guimarães-Costa, Anderson B.; Nascimento, Michelle T. C.; DeSouza-Vieira, Thiago S.; Oliveira, Matheus P.; Garcia e Souza, Luiz F.; Oliveira, Marcus F.; Saraiva, Elvira M.

    2015-01-01

    Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis. PMID:26673780

  18. Silver nanoparticles rapidly induce atypical human neutrophil cell death by a process involving inflammatory caspases and reactive oxygen species and induce neutrophil extracellular traps release upon cell adhesion.

    PubMed

    Liz, Rafael; Simard, Jean-Christophe; Leonardi, Laurien Bruna Araújo; Girard, Denis

    2015-09-01

    Inflammation is one of the major toxic effects reported in response to in vitro or in vivo nanoparticle (NP) exposure. Among engineered NPs, silver nanoparticles (AgNPs) are very attractive for the development of therapeutic strategies, especially because of their antimicrobial properties. In humans, neutrophils, key players in inflammation, are the most abundant blood leukocytes that spontaneously undergo apoptosis, a central cell death mechanism regulating inflammation. The aim of this study was to evaluate the effect of AgNPs on neutrophil apoptosis. Transmission electronic microscopy reveals that AgNPs rapidly penetrate inside neutrophils. AgNPs induced atypical cell death where the cell volume increased and the cell surface expression of CD16 remained unaltered unlike apoptotic neutrophils where cell shrinkage and loss of CD16 are typically observed. The AgNP-induced atypical cell death is distinct from necrosis and reversed by a pancaspase inhibitor or by inhibitors of the inflammatory caspase-1 and caspase-4. In addition, AgNPs induced IL-1β production inhibited by caspase-1 and caspase-4 inhibitors and also induced caspase-1 activity. Reactive oxygen species (ROS) production was increased by AgNPs and the atypical cell death was inhibited by the antioxidant n-acetylcysteine. Under similar experimental conditions, adhesion of neutrophils leads to neutrophil extracellular trap (NET) release induced by AgNPs. However, this process was not reversed by caspase inhibitors. We conclude that AgNPs rapidly induced an atypical cell death in neutrophils by a mechanism involving caspase-1, -4 and ROS. However, in adherent neutrophils, AgNPs induced NET release and, therefore, are novel agents able to trigger NET release. PMID:26241783

  19. Rab27a Is Essential for the Formation of Neutrophil Extracellular Traps (NETs) in Neutrophil-Like Differentiated HL60 Cells

    PubMed Central

    Morita, Hiroyuki; Yokoyama, Kunio; Kaji, Hiroaki; Tanaka, Chisato; Suemori, Shin-ichiro; Tohyama, Kaoru; Tohyama, Yumi

    2014-01-01

    Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs). NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS) is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA)- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an important role in

  20. New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation

    PubMed Central

    Krautgartner, Wolf-Dietrich; Klappacher, Michaela; Kofler, Barbara; Steinbacher, Peter; Vitkov, Ljubomir; Grabcanovic-Musija, Fikreta; Studnicka, Michael

    2014-01-01

    Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both

  1. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures

    PubMed Central

    Leshner, Marc; Wang, Shu; Lewis, Carrie; Zheng, Han; Chen, Xiangyun Amy; Santy, Lorraine; Wang, Yanming

    2012-01-01

    NETosis, the process wherein neutrophils release highly decondensed chromatin called neutrophil extracellular traps (NETs), has gained much attention as an alternative means of killing bacteria. In vivo, NETs are induced by bacteria and pro-inflammatory cytokines. We have reported that peptidylarginine deiminase 4 (PAD4), an enzyme that converts Arg or monomethyl-Arg to citrulline in histones, is essential for NET formation. The areas of extensive chromatin decondensation along the NETs were rich in histone citrullination. Here, upon investigating the effect of global citrullination in cultured cells, we discovered that PAD4 overexpression in osteosarcoma U2OS cells induces extensive chromatin decondensation independent of apoptosis. The highly decondensed chromatin is released to the extracellular space and stained strongly by a histone citrulline-specific antibody. The structure of the decondensed chromatin is reminiscent of NETs but is unique in that it occurs without stimulation of cells with pro-inflammatory cytokines and bacteria. Furthermore, histone citrullination during chromatin decondensation can dissociate heterochromatin protein 1 beta (HP1β) thereby offering a new molecular mechanism for understanding how citrullination regulates chromatin function. Taken together, our study suggests that PAD4 mediated citrullination induces chromatin decondensation, implicating its essential role in NET formation under physiological conditions in neutrophils. PMID:23060885

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

    PubMed Central

    Val, Stephanie; Poley, Marian; Brown, Kristy; Choi, Rachel; Jeong, Stephanie; Colberg-Poley, Annie; Rose, Mary C.; Panchapakesan, Karuna C.; Devaney, Joe C.; Perez-Losada, Marcos

    2016-01-01

    Background Chronic Otitis Media (COM) is characterized by middle ear effusion (MEE) and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited. Objective This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses. Method MEEs were collected from children (n = 49) with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis. Results 109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs) in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin. Conclusion Proteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions. PMID:27078692

  3. Selected mucolytic, anti-inflammatory and cardiovascular drugs change the ability of neutrophils to form extracellular traps (NETs).

    PubMed

    Zawrotniak, Marcin; Kozik, Andrzej; Rapala-Kozik, Maria

    2015-01-01

    Neutrophils form the first line of host defense against infections that combat pathogens using two major mechanisms, the phagocytosis or the release of neutrophil extracellular traps (NETs). The netosis (NET formation) exerts additional, unfavorable effects on the fitness of host cells and is also involved at the sites of lung infection, increasing the mucus viscosity and in the circulatory system where it can influence the intravascular clot formation. Although molecular mechanisms underlying the netosis are still incompletely understood, a role of NADPH oxidase that activates the production of reactive oxygen species (ROS) during the initiation of NETs has been well documented. Since several commonly used drugs can affects the netosis, our current study was aimed to determine the effects of selected mucolytic, anti-inflammatory and cardiovascular drugs on NET formation, with a special emphasis on ROS production and NADPH oxidase activity. The treatment of neutrophils with N-acetylcysteine, ketoprofen and ethamsylate reduced the production of ROS by these cells in a dose-dependent manner. NET formation was also modulated by selected drugs. N-acetylcysteine inhibited the netosis but in the presence of H2O2 this neutrophil ability was restored, indicating that N-acetylcysteine may influence the NET formation by modulating ROS productivity. The administration of ethamsylate led to a significant reduction in NET formation and this effect was not restored by H2O2 or S. aureus, suggesting the unexpected additional side effects of this drug. Ketoprofen seemed to promote ROS-independent NET release, simultaneously inhibiting ROS production. The results, obtained in this study strongly suggest that the therapeutic strategies applied in many neutrophil-mediated diseases should take into account the NET-associated effects. PMID:26291043

  4. Capsules of virulent pneumococcal serotypes enhance formation of neutrophil extracellular traps during in vivo pathogenesis of pneumonia

    PubMed Central

    Moorthy, Anandi Narayana; Rai, Prashant; Jiao, Huipeng; Wang, Shi; Tan, Kong Bing; Qin, Liang; Watanabe, Hiroshi; Zhang, Yongliang; Teluguakula, Narasaraju; Chow, Vincent Tak Kwong

    2016-01-01

    Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia. PMID:27034012

  5. Neutrophil Extracellular Trap-Associated Protein Activation of the NLRP3 Inflammasome Is Enhanced in Lupus Macrophages

    PubMed Central

    Kahlenberg, J. Michelle; Carmona-Rivera, Carmelo; Smith, Carolyne K.; Kaplan, Mariana J.

    2012-01-01

    Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus (SLE), while NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells and may participate in organ damage through incompletely characterized pathways. In order to better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on the NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1β and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2×7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines which further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage. PMID:23267025

  6. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease.

    PubMed

    Lood, Christian; Blanco, Luz P; Purmalek, Monica M; Carmona-Rivera, Carmelo; De Ravin, Suk S; Smith, Carolyne K; Malech, Harry L; Ledbetter, Jeffrey A; Elkon, Keith B; Kaplan, Mariana J

    2016-02-01

    Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases. PMID:26779811

  7. Neutrophil extracellular trap formation is increased in psoriasis and induces human β-defensin-2 production in epidermal keratinocytes.

    PubMed

    Hu, Stephen Chu-Sung; Yu, Hsin-Su; Yen, Feng-Lin; Lin, Chi-Ling; Chen, Gwo-Shing; Lan, Cheng-Che E

    2016-01-01

    Neutrophil extracellular traps (NETs) have been implicated in the development of certain immune-mediated diseases, but their role in psoriasis has not been clearly defined. Human β-defensin-2 (HBD-2) is an important antimicrobial peptide overexpressed in psoriasis epidermis. We evaluated whether the amount of NETs is increased in psoriasis and determined the effect of NETs on HBD-2 production in epidermal keratinocytes. Using fluorescent microscopy, we found that patients with psoriasis (n = 48) had higher amount of NETotic cells in their peripheral blood compared to healthy controls (n = 48) and patients with eczema (n = 35). Psoriasis sera showed increased ability to induce NET formation in control neutrophils but normal NET degradation ability. The amount of NETs in the peripheral blood correlated with psoriasis disease severity. NETosis was also observed in the majority (18 of 20) of psoriasis skin specimens. Furthermore, NETs induced HBD-2 mRNA and protein production in keratinocytes, and immunohistochemical analysis confirmed strong expression of HBD-2 in psoriasis lesional skin. In summary, NET formation is increased in peripheral blood and lesional skin of psoriasis patients and correlates with disease severity. Additionally, NET-induced HBD-2 production may provide a novel mechanism for the decreased susceptibility of psoriasis plaques to microbial infections. PMID:27493143

  8. Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro

    PubMed Central

    Della Coletta, Amanda Manoel; Bachiega, Tatiana Fernanda; de Quaglia e Silva, Juliana Carvalho; Soares, Ângela Maria Victoriano de Campos; De Faveri, Julio; Marques, Silvio Alencar; Marques, Mariângela Esther Alencar; Ximenes, Valdecir Farias; Dias-Melicio, Luciane Alarcão

    2015-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM. PMID:26327485

  9. Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro.

    PubMed

    Della Coletta, Amanda Manoel; Bachiega, Tatiana Fernanda; de Quaglia e Silva, Juliana Carvalho; Soares, Ângela Maria Victoriano de Campos; De Faveri, Julio; Marques, Silvio Alencar; Marques, Mariângela Esther Alencar; Ximenes, Valdecir Farias; Dias-Melicio, Luciane Alarcão

    2015-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM. PMID:26327485

  10. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

    PubMed Central

    Lood, Christian; Blanco, Luz P.; Purmalek, Monica M.; Carmona-Rivera, Carmelo; De Ravin, Suk S.; Smith, Carolyne K.; Malech, Harry L.; Ledbetter, Jeffrey A.; Elkon, Keith B.; Kaplan, Mariana J.

    2015-01-01

    Neutrophil extracellular traps (NETs) are implicated in autoimmunity but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes, inducers of NETosis, require mitochondrial ROS for maximal NET stimulation. During this process, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro and, when injected into mice, stimulates type-I interferon (IFN) signaling through a pathway dependent on the DNA sensor, STING. Mitochondrial ROS is also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus (SLE). This was also observed in individuals with chronic granulomatous disease (CGD), which lack NADPH-oxidase activity, but still develop autoimmunity and type I-IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type-I IFN responses in a mouse model of lupus. These findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases. PMID:26779811

  11. Autophagy and Reactive Oxygen Species Are Involved in Neutrophil Extracellular Traps Release Induced by C. albicans Morphotypes

    PubMed Central

    Kenno, Samyr; Perito, Stefano; Mosci, Paolo; Vecchiarelli, Anna; Monari, Claudia

    2016-01-01

    Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular enzymes, such as elastase and myeloperoxidase. In this study, we demonstrate that Candida albicans hyphal (CAH) cells and yeast (CAY) cells induce differential amounts, kinetics and mechanisms of NET release. CAH cells induced larger quantities of NET compared to CAY cells and can stimulate rapid NET formation up to 4 h of incubation. CAY cells are, also, able to induce rapid NET formation, but this ability was lost at 4 h. Both reactive oxygen species (ROS) and autophagy are implicated in NET induced by CAH and CAY cells, but with a time-different participation of these two mechanisms. In particular, in the early phase (15 min) CAH cells stimulate NET via autophagy, but not via ROS, while CAY cells induce NET via both autophagy and ROS. At 4 h, only CAH cells stimulate NET formation using autophagy as well as ROS. Finally, we demonstrate that NET release, in response to CAH cells, involves NF-κB activation and is strongly implicated in hyphal destruction. PMID:27375599

  12. Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

    PubMed Central

    Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

    2014-01-01

    Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

  13. Autophagy and Reactive Oxygen Species Are Involved in Neutrophil Extracellular Traps Release Induced by C. albicans Morphotypes.

    PubMed

    Kenno, Samyr; Perito, Stefano; Mosci, Paolo; Vecchiarelli, Anna; Monari, Claudia

    2016-01-01

    Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular enzymes, such as elastase and myeloperoxidase. In this study, we demonstrate that Candida albicans hyphal (CAH) cells and yeast (CAY) cells induce differential amounts, kinetics and mechanisms of NET release. CAH cells induced larger quantities of NET compared to CAY cells and can stimulate rapid NET formation up to 4 h of incubation. CAY cells are, also, able to induce rapid NET formation, but this ability was lost at 4 h. Both reactive oxygen species (ROS) and autophagy are implicated in NET induced by CAH and CAY cells, but with a time-different participation of these two mechanisms. In particular, in the early phase (15 min) CAH cells stimulate NET via autophagy, but not via ROS, while CAY cells induce NET via both autophagy and ROS. At 4 h, only CAH cells stimulate NET formation using autophagy as well as ROS. Finally, we demonstrate that NET release, in response to CAH cells, involves NF-κB activation and is strongly implicated in hyphal destruction. PMID:27375599

  14. Differential clearance mechanisms, neutrophil extracellular trap degradation and phagocytosis, are operative in systemic lupus erythematosus patients with distinct autoantibody specificities.

    PubMed

    Chauhan, Sudhir Kumar; Rai, Richa; Singh, Vikas Vikram; Rai, Madhukar; Rai, Geeta

    2015-12-01

    Systemic lupus erythematosus (SLE) patients are generally presented with autoantibodies against either dsDNA or RNA-associated antigens (also known as extractable nuclear antigens, ENA) or both. However, the mechanisms and processes that lead to this distinctive autoantibody profile are not well understood. Defects in clearance mechanism i.e. phagocytosis may lead to enhanced microbial and cellular debris of immunogenic potential. In addition to defective phagocytosis, impaired neutrophil extracellular trap (NET) degradation has been recently reported in SLE patients. However, the extent to which both these clearance processes (NET-degradation and phagocytosis) are operative in serologically distinguished subsets of SLE patients is not established. Therefore, in this report, we evaluated NET-degradation and phagocytosis efficiency among SLE patients with different autoantibody specificities. SLE patients were classified into three subsets based on their autoantibody profile (anti-dsDNA, anti-ENA or both) as determined by ELISA. NET-degradation by SLE and control sera was assessed by sytox orange-based fluorescence assay. Neutrophil-mediated phagocytosis in the presence of SLE and control sera was determined by flowcytometry. The segregation of SLE patients revealed significant differences in NET-degradation and phagocytosis in SLE patients with autoantibodies against dsDNA and ENA. We report that NET-degradation efficiency was significantly impaired in SLE patients with anti-dsDNA autoantibodies and not in those with anti-ENA autoantibodies. In contrast to NET-degradation, neutrophil-mediated phagocytosis was impaired in all three subsets independent of autoantibody specificity. These observations suggest that varying clearance mechanisms are operative in SLE subsets with anti-dsDNA or anti-ENA autoantibodies. The results outlined in this manuscript also suggest that sub-grouping of SLE patients could be useful in delineating the molecular and pathological

  15. Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation.

    PubMed

    Awasthi, Deepika; Nagarkoti, Sheela; Kumar, Amit; Dubey, Megha; Singh, Abhishek Kumar; Pathak, Priya; Chandra, Tulika; Barthwal, Manoj Kumar; Dikshit, Madhu

    2016-04-01

    Neutrophil extracellular traps (NETs) formation was initially linked with host defence and extracellular killing of pathogens. However, recent studies have highlighted their inflammatory potential. Oxidized low density lipoprotein (oxLDL) has been implicated as an independent risk factor in various acute or chronic inflammatory diseases including systemic inflammatory response syndrome (SIRS). In the present study we investigated effect of oxLDL on NETs formation and elucidated the underlying signalling mechanism. Treatment of oxLDL to adhered PMNs led to a time and concentration dependent ROS generation and NETs formation. OxLDL induced free radical formation and NETs release were significantly prevented in presence of NADPH oxidase (NOX) inhibitors suggesting role of NOX activation in oxLDL induced NETs release. Blocking of both toll like receptor (TLR)-2 and 6 significantly reduced oxLDL induced NETs formation indicating requirement of both the receptors. We further identified Protein kinase C (PKC), Interleukin-1 receptor associated kinase (IRAKs), mitogen-activated protein kinase (MAPK) pathway as downstream intracellular signalling mediators involved in oxLDL induced NETs formation. OxLDL components such as oxidized phospholipids (lysophosphatidylcholine (LPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC)) were most potent NETs inducers and might be crucial for oxLDL mediating NETs release. Other components like, oxysterols, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were however less potent as compared to oxidized phospholipids. This study thus demonstrates for the first time that treatment of human PMNs with oxLDL or its various oxidized phopholipid component mediated NETs release, implying their role in the pathogenesis of inflammatory diseases such as SIRS. PMID:26774674

  16. Cytokine-associated neutrophil extracellular traps and antinuclear antibodies in Plasmodium falciparum infected children under six years of age

    PubMed Central

    Baker, Virginia S; Imade, Godwin E; Molta, Norman B; Tawde, Pallavi; Pam, Sunday D; Obadofin, Michael O; Sagay, Soloman A; Egah, Daniel Z; Iya, Daniel; Afolabi, Bangmboye B; Baker, Murray; Ford, Karen; Ford, Robert; Roux, Kenneth H; Keller, Thomas CS

    2008-01-01

    Background In Plasmodium falciparum-infected children, the relationships between blood cell histopathology, blood plasma components, development of immunocompetence and disease severity remain poorly understood. Blood from Nigerian children with uncomplicated malaria was analysed to gain insight into these relationships. This investigation presents evidence for circulating neutrophil extracellular traps (NETs) and antinuclear IgG antibodies (ANA). The presence of NETs and ANA to double-stranded DNA along with the cytokine profiles found suggests autoimmune mechanisms that could produce pathogenesis in children, but immunoprotection in adults. Methods Peripheral blood smear slides and blood samples obtained from 21 Nigerian children under six years of age, presenting with uncomplicated malaria before and seven days after initiation of sulphadoxine-pyrimethamine (SP) treatment were analysed. The slides were stained with Giemsa and with DAPI. Levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF, CRP, and IL-6, select anti-inflammatory cytokines TGF-β and IL-10, and ANA were determined by immunoassay. Results The children exhibited circulating NETs with adherent parasites and erythrocytes, elevated ANA levels, a Th2 dominated cytokine profile, and left-shifted leukocyte differential counts. Nonspecific ANA levels were significant in 86% of the children pretreatment and in 100% of the children seven days after SP treatment, but in only 33% of age-matched control samples collected during the season of low parasite transmission. Levels of ANA specific for dsDNA were significant in 81% of the children both pre-treatment and post treatment. Conclusion The results of this investigation suggest that NET formation and ANA to dsDNA may induce pathology in falciparum-infected children, but activate a protective mechanism against falciparum malaria in adults. The significance of in vivo circulating chromatin in NETs and dsDNA ANA as a causative factor in the

  17. A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.

    PubMed

    Kraaij, Tineke; Tengström, Fredrik C; Kamerling, Sylvia W A; Pusey, Charles D; Scherer, H Ulrich; Toes, Rene E M; Rabelink, Ton J; van Kooten, Cees; Teng, Y K Onno

    2016-06-01

    A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases. PMID:26925759

  18. A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation.

    PubMed

    Azevedo, Estefania P; Rochael, Natalia C; Guimarães-Costa, Anderson B; de Souza-Vieira, Thiago S; Ganilho, Juliana; Saraiva, Elvira M; Palhano, Fernando L; Foguel, Debora

    2015-09-01

    Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

  19. A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

    PubMed Central

    Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

    2015-01-01

    Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

  20. Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

    PubMed

    Gogol, Mariusz; Ostrowska, Dominika; Klaga, Kinga; Bochenska, Oliwia; Wolak, Natalia; Aoki, Wataru; Ueda, Mitsuyoshi; Kozik, Andrzej; Rapala-Kozik, Maria

    2016-01-01

    Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense. PMID:26641639

  1. Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin-induced neutrophil extracellular trap-osis (NETosis)-like cell death in cultured human leukemia cells.

    PubMed

    Nakayama, Tomofumi; Saitoh, Noriko; Morotomi-Yano, Keiko; Yano, Ken-Ichi; Nakao, Mitsuyoshi; Saitoh, Hisato

    2016-05-01

    We previously reported that the nucleoside antibiotic tunicamycin (TN), a protein glycosylation inhibitor triggering unfolded protein response (UPR), induced neutrophil extracellular trap-osis (NETosis)-like cellular suicide and, thus, discharged genomic DNA fibers to extracellular spaces in a range of human myeloid cell lines under serum-free conditions. In this study, we further evaluated the effect of TN on human promyelocytic leukemia HL-60 cells using time-lapse microscopy. Our assay revealed a previously unappreciated early event induced by TN-exposure, in which, at 30-60 min after TN addition, the cells extruded their nuclei into the extracellular space, followed by discharge of DNA fibers to form NET-like structures. Intriguingly, neither nuclear extrusion nor DNA discharge was observed when cells were exposed to inducers of UPR, such as brefeldin A, thapsigargin, or dithiothreitol. Our findings revealed novel nuclear dynamics during TN-induced NETosis-like cellular suicide in HL-60 cells and suggested that the toxicological effect of TN on nuclear extrusion and DNA discharge was not a simple UPR. PMID:26888435

  2. Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

    PubMed Central

    Côté, Olivier; Clark, Mary Ellen; Viel, Laurent; Labbé, Geneviève; Seah, Stephen Y. K.; Khan, Meraj A.; Douda, David N.; Palaniyar, Nades; Bienzle, Dorothee

    2014-01-01

    Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases. PMID:24777050

  3. Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin

    PubMed Central

    Jann, Naja J.; Schmaler, Mathias; Kristian, Sascha A.; Radek, Katherine A.; Gallo, Richard L.; Nizet, Victor; Peschel, Andreas; Landmann, Regine

    2009-01-01

    Neutrophils kill invading pathogens by AMPs, including cathelicidins, ROS, and NETs. The human pathogen Staphylococcus aureus exhibits enhanced resistance to neutrophil AMPs, including the murine cathelicidin CRAMP, in part, as a result of alanylation of teichoic acids by the dlt operon. In this study, we took advantage of the hypersusceptible phenotype of S. aureus ΔdltA against cationic AMPs to study the impact of the murine cathelicidin CRAMP on staphylococcal killing and to identify its key site of action in murine neutrophils. We demonstrate that CRAMP remained intracellular during PMN exudation from blood and was secreted upon PMA stimulation. We show first evidence that CRAMP was recruited to phagolysosomes in infected neutrophils and exhibited intracellular activity against S. aureus. Later in infection, neutrophils produced NETs, and immunofluorescence revealed association of CRAMP with S. aureus in NETs, which similarly killed S. aureus wt and ΔdltA, indicating that CRAMP activity was reduced when associated with NETs. Indeed, the presence of DNA reduced the antimicrobial activity of CRAMP, and CRAMP localization in response to S. aureus was independent of the NADPH oxidase, whereas killing was partially dependent on a functional NADPH oxidase. Our study indicates that neutrophils use CRAMP in a timed and locally coordinated manner in defense against S. aureus. PMID:19638500

  4. Effect of High-Fat Diet on the Formation of Pulmonary Neutrophil Extracellular Traps during Influenza Pneumonia in BALB/c Mice.

    PubMed

    Moorthy, Anandi Narayana; Tan, Kong Bing; Wang, Shi; Narasaraju, Teluguakula; Chow, Vincent T

    2016-01-01

    Obesity is an independent risk factor for severe outcome of influenza infection. Higher dietary fat consumption has been linked to greater morbidity and severe influenza in mouse models. However, the extent of generation of neutrophil extracellular traps (NETs or NETosis) in obese individuals during influenza pneumonia is hitherto unknown. This study investigated pulmonary NETs generation in BALB/c mice fed with high-fat diet (HFD) and low-fat diet (LFD), during the course of influenza pneumonia. Clinical disease progression, histopathology, lung reactive oxygen species, and myeloperoxidase activity were also compared. Consumption of HFD over 18 weeks led to significantly higher body weight, body mass index, and adiposity in BALB/c mice compared with LFD. Lethal challenge of mice (on HFD and LFD) with influenza A/PR/8/34 (H1N1) virus led to similar body weight loss and histopathologic severity. However, NETs were formed at relatively higher levels in mice fed with HFD, despite the absence of significant difference in disease progression between HFD- and LFD-fed mice. PMID:27531997

  5. Effect of High-Fat Diet on the Formation of Pulmonary Neutrophil Extracellular Traps during Influenza Pneumonia in BALB/c Mice

    PubMed Central

    Moorthy, Anandi Narayana; Tan, Kong Bing; Wang, Shi; Narasaraju, Teluguakula; Chow, Vincent T.

    2016-01-01

    Obesity is an independent risk factor for severe outcome of influenza infection. Higher dietary fat consumption has been linked to greater morbidity and severe influenza in mouse models. However, the extent of generation of neutrophil extracellular traps (NETs or NETosis) in obese individuals during influenza pneumonia is hitherto unknown. This study investigated pulmonary NETs generation in BALB/c mice fed with high-fat diet (HFD) and low-fat diet (LFD), during the course of influenza pneumonia. Clinical disease progression, histopathology, lung reactive oxygen species, and myeloperoxidase activity were also compared. Consumption of HFD over 18 weeks led to significantly higher body weight, body mass index, and adiposity in BALB/c mice compared with LFD. Lethal challenge of mice (on HFD and LFD) with influenza A/PR/8/34 (H1N1) virus led to similar body weight loss and histopathologic severity. However, NETs were formed at relatively higher levels in mice fed with HFD, despite the absence of significant difference in disease progression between HFD- and LFD-fed mice. PMID:27531997

  6. Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation

    PubMed Central

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

    2016-01-01

    Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to

  7. Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

    PubMed

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

    2016-01-01

    Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis

  8. Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs) Formation and Arterial Thrombosis

    PubMed Central

    Mizurini, Daniella M.; Aslan, Jorgeane S.; Gomes, Tainá; Ma, Dongying; Francischetti, Ivo M. B.; Monteiro, Robson Q.

    2015-01-01

    Background The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs), a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability. Methodology/Principal Findings This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method. Conclusions/Significance Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host’s hemostatic responses and innate immune system. PMID:26110417

  9. Enhanced formation and impaired degradation of neutrophil extracellular traps in dermatomyositis and polymyositis: a potential contributor to interstitial lung disease complications

    PubMed Central

    Zhang, S; Shu, X; Tian, X; Chen, F; Lu, X; Wang, G

    2014-01-01

    Dermatomyositis (DM) and polymyosits (PM) are systemic autoimmune diseases whose pathogeneses remain unclear. Neutrophil extracellular traps (NETs) are reputed to play an important role in the pathogenesis of autoimmune diseases. This study tests the hypothesis that NETs may be pathogenic in DM/PM. Plasma samples from 97 DM/PM patients (72 DM, 25 PM) and 54 healthy controls were tested for the capacities to induce and degrade NETs. Plasma DNase I activity was tested to further explore possible reasons for the incomplete degradation of NETs. Results from 35 DM patients and seven PM patients with interstitial lung disease (ILD) were compared with results from DM/PM patients without ILD. Compared with control subjects, DM/PM patients exhibited a significantly enhanced capacity for inducing NETs, which was supported by elevated levels of plasma LL-37 and circulating cell-free DNA (cfDNA) in DM/PM. NETs degradation and DNase I activity were also decreased significantly in DM/PM patients and were correlated positively. Moreover, DM/PM patients with ILD exhibited the lowest NETs degradation in vitro due to the decrease in DNase I activity. DNase I activity in patients with anti-Jo-1 antibodies was significantly lower than in patients without. Glucocorticoid therapy seems to improve DNase I activity. Our findings demonstrate that excessively formed NETs cannot be degraded completely because of decreased DNase I activity in DM/PM patients, especially in patients with ILD, suggesting that abnormal regulation of NETs may be involved in the pathogenesis of DM/PM and could be one of the factors that initiate and aggravate ILD. PMID:24611519

  10. Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

    PubMed Central

    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. PMID:21960987

  11. Mycobacterium massiliense Induces Macrophage Extracellular Traps with Facilitating Bacterial Growth

    PubMed Central

    Yoon, Yina; Na, Yirang; Kim, Bum-Joon; Seok, Seung Hyeok

    2016-01-01

    Human neutrophils have been known to release neutrophil extracellular traps (NETs), antimicrobial DNA structures capable of capturing and killing microbes. Recently, a similar phenomenon has been reported in macrophages infected with various pathogens. However, a role for macrophages extracellular traps (METs) in host defense responses against Mycobacterium massiliense (M. mass) has yet to be described. In this study, we show that M. mass, a rapid growing mycobacterium (RGM), also induces the release of METs from PMA-differentiated THP-1 cells. Intriguingly, this process is not dependent on NADPH oxidase activity, which regulates NET formation. Instead, M. mass-induced MET formation partially depends on calcium influx and requires phagocytosis of high bacterial load. The METs consist of a DNA backbone embedded with microbicidal proteins such as histone, MPO and elastase. Released METs entrap M. mass and prevent their dissemination, but do not have bactericidal activity. Instead, they result in enhanced bacterial growth. In this regard, METs were considered to provide interaction of M. mass with cells and an environment for bacterial aggregation, which may facilitate mycobacterial survival and growth. In conclusion, our results demonstrate METs as an innate defense response against M. mass infection, and suggest that extracellular traps play a multifaceted role in the interplay between host and bacteria. PMID:27191593

  12. Mannheimia haemolytica and Its Leukotoxin Cause Macrophage Extracellular Trap Formation by Bovine Macrophages

    PubMed Central

    Aulik, Nicole A.; Hellenbrand, Katrina M.

    2012-01-01

    Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins. PMID:22354029

  13. Mycobacterium tuberculosis Exploits Human Interferon γ to Stimulate Macrophage Extracellular Trap Formation and Necrosis

    PubMed Central

    Wong, Ka-Wing; Jacobs, Williams R.

    2013-01-01

    Human neutrophils form extracellular traps during M. tuberculosis infection, but a similar phenomenon has not been reported in human macrophages. Here we demonstrate that M. tuberculosis induces release of extracellular traps from human macrophages. This process is regulated by elastase activity, previously shown to regulate formation of extracellular traps by neutrophils. Interestingly, formation of extracellular traps by macrophages during M. tuberculosis infection is inducible by interferon γ (IFN-γ). These traps are mainly produced by heavily infected macrophages. Accordingly, IFN-γ is found to stimulate M. tuberculosis aggregation in macrophages. Both IFN-γ–inducible events, extracellular trap formation and mycobacterial aggregation, require the ESX-1 secretion system. In addition, IFN-γ is found to enhance ESX-1–mediated macrophage necrosis. In the absence of ESX-1, IFN-γ does not restore any extracellular trap formation, mycobacterial aggregation, or macrophage necrosis. Thus, initial characterization of macrophage extracellular trap formation due to M. tuberculosis infection led to the uncovering of a novel role for IFN-γ in amplifying multiple effects of the mycobacterial ESX-1. PMID:23475311

  14. Inhibition of superoxide anion production by extracellular acidification in neutrophils.

    PubMed

    Murata, Naoya; Mogi, Chihiro; Tobo, Masayuki; Nakakura, Takashi; Sato, Koichi; Tomura, Hideaki; Okajima, Fumikazu

    2009-01-01

    Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O(2)(-)) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E(1), also inhibited the formyl peptide-induced O(2)(-) production. The inhibitory action on the O(2)(-) production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O(2)(-) production. PMID:19539899

  15. Extracellular Fibrils of Pathogenic Yeast Cryptococcus gattii Are Important for Ecological Niche, Murine Virulence and Human Neutrophil Interactions

    PubMed Central

    Springer, Deborah J.; Ren, Ping; Raina, Ramesh; Dong, Yimin; Behr, Melissa J.; McEwen, Bruce F.; Bowser, Samuel S.; Samsonoff, William A.; Chaturvedi, Sudha; Chaturvedi, Vishnu

    2010-01-01

    Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40–100 nm diameter ×500–3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12α mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing. PMID:20539754

  16. Extracellular Acidification Acts as a Key Modulator of Neutrophil Apoptosis and Functions

    PubMed Central

    Cao, Shannan; Liu, Peng; Zhu, Haiyan; Gong, Haiyan; Yao, Jianfeng; Sun, Yawei; Geng, Guangfeng; Wang, Tong; Feng, Sizhou; Han, Mingzhe; Zhou, Jiaxi; Xu, Yuanfu

    2015-01-01

    In human pathological conditions, the acidification of local environment is a frequent feature, such as tumor and inflammation. As the pH of microenvironment alters, the functions of immune cells are about to change. It makes the extracellular acidification a key modulator of innate immunity. Here we detected the impact of extracellular acidification on neutrophil apoptosis and functions, including cell death, respiratory burst, migration and phagocytosis. As a result, we found that under the acid environment, neutrophil apoptosis delayed, respiratory burst inhibited, polarization augmented, chemotaxis differed, endocytosis enhanced and bacteria killing suppressed. These findings suggested that extracellular acidification acts as a key regulator of neutrophil apoptosis and functions. PMID:26340269

  17. Antimicrobial Activity of Mast Cells: Role and Relevance of Extracellular DNA Traps

    PubMed Central

    Möllerherm, Helene; von Köckritz-Blickwede, Maren; Branitzki-Heinemann, Katja

    2016-01-01

    Mast cells (MCs) have been shown to release their nuclear DNA and subsequently form mast cell extracellular traps (MCETs) comparable to neutrophil extracellular traps, which are able to entrap and kill various microbes. The formation of extracellular traps is associated with the disruption of the nuclear membrane, which leads to mixing of nuclear compounds with granule components and causes the death of the cell, a process called ETosis. The question arises why do MCs release MCETs although they are very well known as multifunctional long-living sentinel cells? MCs are known to play a role during allergic reactions and certain parasitic infections. Nonetheless, they are also critical components of the early host innate immune response to bacterial and fungal pathogens: MCs contribute to the initiation of the early immune response by recruiting effector cells including neutrophils and macrophages by locally releasing inflammatory mediators, such as TNF-α. Moreover, various studies demonstrate that MCs are able to eliminate microbes through intracellular as well as extracellular antimicrobial mechanisms, including MCET formation similar to that of professional phagocytes. Recent literature leads to the suggestion that MCET formation is not the result of a passive release of DNA and granule proteins during cellular disintegration, but rather an active and controlled process in response to specific stimulation, which contributes to the innate host defense. This review will discuss the different known aspects of the antimicrobial activities of MCs with a special focus on MCETs, and their role and relevance during infection and inflammation. PMID:27486458

  18. Priming of Human Neutrophils Is Necessary for Their Activation by Extracellular DNA.

    PubMed

    Prikhodko, A S; Vitushkina, M V; Zinovkina, L A; Popova, E N; Zinovkin, R A

    2016-06-01

    Extracellular plasma DNA is thought to act as a damage-associated molecular pattern causing activation of immune cells. However, purified preparations of mitochondrial and nuclear DNA were unable to induce neutrophil activation in vitro. Thus, we examined whether granulocyte-macrophage colony-stimulating factor (GM-CSF) acting as a neutrophil priming agent can promote the activation of neutrophils by different types of extracellular DNA. GM-CSF pretreatment greatly increased p38 MAPK phosphorylation and promoted CD11b/CD66b expression in human neutrophils treated with mitochondrial and, to a lesser extent, with nuclear DNA. Our experiments clearly indicate that GM-CSF-induced priming of human neutrophils is necessary for their subsequent activation by extracellular DNA. PMID:27301289

  19. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

    PubMed

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-06-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  20. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    PubMed Central

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-01-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  1. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice

    PubMed Central

    Thomas, Grace M.; Carbo, Carla; Curtis, Brian R.; Martinod, Kimberly; Mazo, Irina B.; Schatzberg, Daphne; Cifuni, Stephen M.; Fuchs, Tobias A.; von Andrian, Ulrich H.; Hartwig, John H.; Aster, Richard H.

    2012-01-01

    Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biologic processes contributing to TRALI are poorly understood. All blood products can cause TRALI, and no specific treatment is available. A “2-event model” has been proposed as the trigger. The first event may include surgery, trauma, or infection; the second involves the transfusion of antileukocyte antibodies or bioactive lipids within the blood product. Together, these events induce neutrophil activation in the lungs, causing endothelial damage and capillary leakage. Neutrophils, in response to pathogens or under stress, can release their chromatin coated with granule contents, thus forming neutrophil extracellular traps (NETs). Although protective against infection, these NETs are injurious to tissue. Here we show that NET biomarkers are present in TRALI patients' blood and that NETs are produced in vitro by primed human neutrophils when challenged with anti–HNA-3a antibodies previously implicated in TRALI. NETs are found in alveoli of mice experiencing antibody-mediated TRALI. DNase 1 inhalation prevents their alveolar accumulation and improves arterial oxygen saturation even when administered 90 minutes after TRALI onset. We suggest that NETs form in the lungs during TRALI, contribute to the disease process, and thus could be targeted to prevent or treat TRALI. PMID:22596262

  2. Basophils exhibit antibacterial activity through extracellular trap formation.

    PubMed

    Yousefi, S; Morshed, M; Amini, P; Stojkov, D; Simon, D; von Gunten, S; Kaufmann, T; Simon, H-U

    2015-09-01

    Basophils are primarily associated with immunomodulatory functions in allergic diseases and parasitic infections. Recently, it has been demonstrated that both activated human and mouse basophils can form extracellular DNA traps (BETs) containing mitochondrial DNA and granule proteins. In this report, we provide evidence that, in spite of an apparent lack of phagocytic activity, basophils can kill bacteria through BET formation. PMID:26043360

  3. A canine model of Cohen syndrome: Trapped Neutrophil Syndrome

    PubMed Central

    2011-01-01

    Background Trapped Neutrophil Syndrome (TNS) is a common autosomal recessive neutropenia in Border collie dogs. Results We used a candidate gene approach and linkage analysis to show that the causative gene for TNS is VPS13B. We chose VPS13B as a candidate because of similarities in clinical signs between TNS and Cohen syndrome, in human, such as neutropenia and a typical facial dysmorphism. Linkage analysis using microsatellites close to VPS13B showed positive linkage of the region to TNS. We sequenced each of the 63 exons of VPS13B in affected and control dogs and found that the causative mutation in Border collies is a 4 bp deletion in exon 19 of the largest transcript that results in premature truncation of the protein. Cohen syndrome patients present with mental retardation in 99% of cases, but learning disabilities featured in less than half of TNS affected dogs. It has been implied that loss of the alternate transcript of VPS13B in the human brain utilising an alternate exon, 28, may cause mental retardation. Mice cannot be used to test this hypothesis as they do not express the alternate exon. We show that dogs do express alternate transcripts in the brain utilising an alternate exon homologous to human exon 28. Conclusion Dogs can be used as a model organism to explore the function of the alternately spliced transcript of VPS13B in the brain. TNS in Border collies is the first animal model for Cohen syndrome and can be used to study the disease aetiology. PMID:21605373

  4. Neutrophil elastase processing of Gelatinase A is mediated by extracellular matrix

    SciTech Connect

    Rice, A.; Banda, M.J.

    1995-07-18

    Gelatinase A (72-kDa type IV collagenase) is a metalloproteinase that is expressed by many cells in culture and is overexpressed by some tumor cells. It has been suggested that the serine proteinase neutrophil elastase might play a role iii the posttranslational processing of gelatinase A and that noncatalytic interactions between gelatinase A and components of the extracellular matrix might alter potential processing pathways. These questions were addressed with the use of gelatin substrate zymography, gelatinolytic activity assays, and amino acid sequence analysis. We found that neutrophil elastase does proteolytically modify gelatinase A by cleaving at a number of sites within gelatinase A. Sequential treatment of gelatinase A with 4-aminophenylmercuric acetate (APMA) and neutrophil elastase yielded an active gelatinase with a 4-fold increase in gelatinolytic activity. The increased gelatinolytic activity correlated with that of a 40-kDa fragment of gelatinase A. Matrix components altered the proteolytic modifications in gelatinase A that were mediated by neutrophil elastase. In the absence of gelatin, neutrophil elastase destructively degraded gelatinase A by hydrolyzing at least two bonds within the fibronectin-like gelatin-binding domain of gelatinase A. In the presence of gelatin, these two inactivating cleavage sites were protected, and cleavage at a site within the hemopexin-like carboxyl-terminal domain resulted in a truncated yet active gelatinase. The results suggest a regulatory role for extracellular matrix molecules in stabilizing gelatinase A fragments and in altering the availability of sites susceptible to destructive proteolysis by neutrophil elastase. 32 refs., 10 figs.

  5. Presentation and management of trapped neutrophil syndrome (TNS) in UK Border collies.

    PubMed

    Mason, S L; Jepson, R; Maltman, M; Batchelor, D J

    2014-01-01

    Three UK bred Border collie puppies were presented for investigation of pyrexia and severe lameness with associated joint swelling. Investigations revealed neutropenia, radiographic findings suggesting metaphyseal osteopathy, and polyarthritis and all dogs were subsequently confirmed with trapped neutrophil syndrome. Clinical improvement was seen after treatment with prednisolone and antibiotics and the dogs all survived to adulthood with a good short- to medium-term outcome. Trapped neutrophil syndrome is an important differential diagnosis for young Border collie dogs in the UK presenting with pyrexia, neutropenia and musculoskeletal signs. PMID:24032537

  6. The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans

    PubMed Central

    Stoiber, Walter; Obermayer, Astrid; Steinbacher, Peter; Krautgartner, Wolf-Dietrich

    2015-01-01

    Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects. PMID:25946076

  7. Adhesiveness for extracellular matrices and lysosomal enzyme release from normal and beta 2 integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Noda, H; Tamoto, K; Kuwabara, M

    1996-01-01

    The adhesiveness of control and CD18-deficient bovine neutrophils on culture plates precoated with collagen I, collagen IV, fibronectin and laminin was measured to evaluate the possible factors for adherence to extracellular matrices. The release of N-acetyl-beta-D-glucosaminidase (NAGase) from control and CD18-deficient neutrophils stimulated with complement receptor type 3 (CR3) or Fc receptor dependent stimuli was also evaluated. The adhesive activities of CD18-deficient neutrophils to collagen I, collagen IV and fibronectin were significantly diminished (P < 0.05); however, similar adhesion to laminin was observed in CD18-deficient neutrophils and control neutrophils. The adhesive activity of control neutrophils on uncoated plates increased 2.5 times (P < 0.05) with the presence of PMA. The mean activities for NAGase release from CD18-deficient neutrophils stimulated with opsonized zymosan and aggregated bovine immunoglobulin G (Agg-IgG) were 46.7 and 82.7% that of the control neutrophils, respectively. The Agg-IgG-induced NAGase release from control and CD18-deficient neutrophils was eliminated by H7, a protein kinase C inhibitor. These results support that an association between CR3 and Fc receptors on neutrophils appears to play an essential role in neutrophil functions. PMID:8981354

  8. Neutrophil's weapons in atherosclerosis.

    PubMed

    Chistiakov, Dimitry A; Bobryshev, Yuri V; Orekhov, Alexander N

    2015-12-01

    Neutrophils are important components of immunity associated with inflammatory responses against a broad spectrum of pathogens. These cells could be rapidly activated by proinflammatory stimuli and migrate to the inflamed and infected sites where they release a variety of cytotoxic molecules with antimicrobial activity. Neutrophil antibacterial factors include extracellular proteases, redox enzymes, antimicrobial peptides, and small bioactive molecules. In resting neutrophils, these factors are stored in granules and released upon activation during degranulation. These factors could be also secreted in a neutrophil-derived microparticle-dependent fashion. Neutrophils exhibit a unique property to produce neutrophil extracellular traps (NETs) composed of decondensed chromatin and granular proteins to catch and kill bacteria. Neutrophil-released factors are efficient in inactivation and elimination of pathogens through oxidation-dependent or independent damage of bacterial cells, inactivation and neutralization of virulence factors and other mechanisms. However, in chronic atherosclerosis-associated inflammation, protective function of neutrophils could be impaired and misdirected against own cells. This could lead to deleterious effects and progressive vascular injury. In atherogenesis, a pathogenic role of neutrophils could be especially seen in early stages associated with endothelial dysfunction and induction of vascular inflammation and in late atherosclerosis associated with plaque rupture and atherothrombosis. Assuming a prominent impact of neutrophils in cardiovascular pathology, developing therapeutic strategies targeting neutrophil-specific antigens could have a promising clinical potential. PMID:26551083

  9. Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species.

    PubMed

    Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

    2016-01-01

    The successful treatment of bacterial infections is the achievement of a synergy between the host's immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host's immune defences and antibiotic interactions in microbial infections. PMID:26778774

  10. Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species

    PubMed Central

    Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

    2016-01-01

    The successful treatment of bacterial infections is the achievement of a synergy between the host’s immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host’s immune defences and antibiotic interactions in microbial infections. PMID:26778774

  11. Dimethylfumarate Impairs Neutrophil Functions.

    PubMed

    Müller, Susen; Behnen, Martina; Bieber, Katja; Möller, Sonja; Hellberg, Lars; Witte, Mareike; Hänsel, Martin; Zillikens, Detlef; Solbach, Werner; Laskay, Tamás; Ludwig, Ralf J

    2016-01-01

    Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases. PMID:26763431

  12. The Multifaceted Functions of Neutrophils

    PubMed Central

    Mayadas, Tanya N.; Cullere, Xavier; Lowell, Clifford A.

    2014-01-01

    Neutrophils and neutrophil-like cells are the major pathogen-fighting immune cells in organisms ranging from slime molds to mammals. Central to their function is their ability to be recruited to sites of infection, to recognize and phagocytose microbes, and then to kill pathogens through a combination of cytotoxic mechanisms. These include the production of reactive oxygen species, the release of antimicrobial peptides, and the recently discovered expulsion of their nuclear contents to form neutrophil extracellular traps. Here we discuss these primordial neutrophil functions, which also play key roles in tissue injury, by providing details of neutrophil cytotoxic functions and congenital disorders of neutrophils. In addition, we present more recent evidence that interactions between neutrophils and adaptive immune cells establish a feed-forward mechanism that amplifies pathologic inflammation. These newly appreciated contributions of neutrophils are described in the setting of several inflammatory and autoimmune diseases. PMID:24050624

  13. Characteristics of Mononuclear Extracellular Traps in the Offspring of Female Rats with Drug-Induced Hepatitis.

    PubMed

    Bryukhin, G V; Shopova, A V

    2015-08-01

    We studied the effect of experimental tetracycline-induced liver injury in mothers on the capacity of macrophages from various compartments to form traps and on activity of extracellular macrophage traps in the offspring. Trap-forming capacity was evaluated by the number of traps. We found reduction in the number and suppression of activity of the macrophage extracellular traps in the offspring of females with experimental liver injury. The findings suggest that mothers with drug-induced liver injury produce physiologically immature offspring with reduced unspecific resistance. PMID:26388577

  14. Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes

    SciTech Connect

    Ramos, C.L.; Pou, S.; Britigan, B.E.; Cohen, M.S.; Rosen, G.M. )

    1992-04-25

    Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.

  15. Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion

    PubMed Central

    Pruenster, Monika; Kurz, Angela R. M.; Chung, Kyoung-Jin; Cao-Ehlker, Xiao; Bieber, Stephanie; Nussbaum, Claudia F.; Bierschenk, Susanne; Eggersmann, Tanja K.; Rohwedder, Ina; Heinig, Kristina; Immler, Roland; Moser, Markus; Koedel, Uwe; Gran, Sandra; McEver, Rodger P.; Vestweber, Dietmar; Verschoor, Admar; Leanderson, Tomas; Chavakis, Triantafyllos; Roth, Johannes; Vogl, Thomas; Sperandio, Markus

    2015-01-01

    Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin–PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid β2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo. PMID:25892652

  16. Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion.

    PubMed

    Pruenster, Monika; Kurz, Angela R M; Chung, Kyoung-Jin; Cao-Ehlker, Xiao; Bieber, Stephanie; Nussbaum, Claudia F; Bierschenk, Susanne; Eggersmann, Tanja K; Rohwedder, Ina; Heinig, Kristina; Immler, Roland; Moser, Markus; Koedel, Uwe; Gran, Sandra; McEver, Rodger P; Vestweber, Dietmar; Verschoor, Admar; Leanderson, Tomas; Chavakis, Triantafyllos; Roth, Johannes; Vogl, Thomas; Sperandio, Markus

    2015-01-01

    Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid β2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo. PMID:25892652

  17. Of Amoebae and Men: Extracellular DNA Traps as an Ancient Cell-Intrinsic Defense Mechanism

    PubMed Central

    Zhang, Xuezhi; Soldati, Thierry

    2016-01-01

    Since the discovery of the formation of DNA-based extracellular traps (ETs) by neutrophils as an innate immune defense mechanism (1), hundreds of articles describe the involvement of ETs in physiological and pathological human and animal conditions [reviewed in Ref. (2), and the previous Frontiers Research Topic on NETosis: http://www.frontiersin.org/books/NETosis_At_the_Intersection_of_Cell_Biology_Microbiology_and_Immunology/195]. Interestingly, a few reports reveal that ETs can be formed by immune cells of more ancient organisms, as far back as the common ancestor of vertebrates and invertebrates (3). Recently, we reported that the Sentinel cells of the multicellular slug of the social amoeba Dictyostelium discoideum also produce ETs to trap and kill slug-invading bacteria [see Box 1; and Figure 1 Ref. (4)]. This is a strong evidence that DNA-based cell-intrinsic defense mechanisms emerged much earlier than thought, about 1.3 billion years ago. Amazingly, using extrusion of DNA as a weapon to capture and kill uningestable microbes has its rationale. During the emergence of multicellularity, a primitive innate immune system developed in the form of a dedicated set of specialized phagocytic cells. This professionalization of immunity allowed the evolution of sophisticated defense mechanisms including the sacrifice of a small set of cells by a mechanism related to NETosis. This altruistic behavior likely emerged in steps, starting from the release of “dispensable” mitochondrial DNA by D. discoideum Sentinel cells. Grounded in this realization, one can anticipate that in the near future, many more examples of the invention and fine-tuning of ETs by early metazoan ancestors will be identified. Consequently, it can be expected that this more complete picture of the evolution of ETs will impact our views of the involvement and pathologies linked to ETs in human and animals. PMID:27458458

  18. Of Amoebae and Men: Extracellular DNA Traps as an Ancient Cell-Intrinsic Defense Mechanism.

    PubMed

    Zhang, Xuezhi; Soldati, Thierry

    2016-01-01

    Since the discovery of the formation of DNA-based extracellular traps (ETs) by neutrophils as an innate immune defense mechanism (1), hundreds of articles describe the involvement of ETs in physiological and pathological human and animal conditions [reviewed in Ref. (2), and the previous Frontiers Research Topic on NETosis: http://www.frontiersin.org/books/NETosis_At_the_Intersection_of_Cell_Biology_Microbiology_and_Immunology/195]. Interestingly, a few reports reveal that ETs can be formed by immune cells of more ancient organisms, as far back as the common ancestor of vertebrates and invertebrates (3). Recently, we reported that the Sentinel cells of the multicellular slug of the social amoeba Dictyostelium discoideum also produce ETs to trap and kill slug-invading bacteria [see Box 1; and Figure 1 Ref. (4)]. This is a strong evidence that DNA-based cell-intrinsic defense mechanisms emerged much earlier than thought, about 1.3 billion years ago. Amazingly, using extrusion of DNA as a weapon to capture and kill uningestable microbes has its rationale. During the emergence of multicellularity, a primitive innate immune system developed in the form of a dedicated set of specialized phagocytic cells. This professionalization of immunity allowed the evolution of sophisticated defense mechanisms including the sacrifice of a small set of cells by a mechanism related to NETosis. This altruistic behavior likely emerged in steps, starting from the release of "dispensable" mitochondrial DNA by D. discoideum Sentinel cells. Grounded in this realization, one can anticipate that in the near future, many more examples of the invention and fine-tuning of ETs by early metazoan ancestors will be identified. Consequently, it can be expected that this more complete picture of the evolution of ETs will impact our views of the involvement and pathologies linked to ETs in human and animals. PMID:27458458

  19. Cis-urocanic acid inhibits bovine neutrophil generation of extracellular superoxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neutrophils play a fundamental role in the host innate immune response during mastitis and other bacterial-mediated diseases of cattle through their ability to phagocytose and kill bacteria. The ability of neutrophils to kill bacteria is mediated through the generation of reactive oxygen species (R...

  20. Different Leishmania Species Drive Distinct Neutrophil Functions.

    PubMed

    Hurrell, Benjamin P; Regli, Ivo B; Tacchini-Cottier, Fabienne

    2016-05-01

    Leishmaniases are vector-borne diseases of serious public health importance. During a sand fly blood meal, Leishmania parasites are deposited in the host dermis where neutrophils are rapidly recruited. Neutrophils are the first line of defense and can kill pathogens by an array of mechanisms. They can also form web-like structures called neutrophil extracellular traps (NETs) that can trap and/or kill microbes. The function of neutrophils in leishmaniasis was reported to be either beneficial by contributing to parasite killing or detrimental by impairing immune response development and control of parasite load. Here we review recent data showing that different Leishmania species elicit distinct neutrophil functions thereby influencing disease outcomes. Emerging evidence suggests that neutrophils should be considered important modulators of leishmaniasis. PMID:26944469

  1. Far beyond Phagocytosis: Phagocyte-Derived Extracellular Traps Act Efficiently against Protozoan Parasites In Vitro and In Vivo.

    PubMed

    Silva, Liliana M R; Muñoz-Caro, Tamara; Burgos, Rafael A; Hidalgo, Maria A; Taubert, Anja; Hermosilla, Carlos

    2016-01-01

    Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages are considered as the first line of defence against invasive pathogens. The formation of extracellular traps (ETs) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections. Recent investigations showed evidence that ETosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria, fungi, viruses, and protozoan parasites. ETs are released in response to intact protozoan parasites or to parasite-specific antigens in a controlled cell death process. Released ETs consist of nuclear DNA as backbone adorned with histones, antimicrobial peptides, and phagocyte-specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens. This review summarizes recent data on protozoa-induced ETosis. Special attention will be given to molecular mechanisms of protozoa-induced ETosis and on its consequences for the parasites successful reproduction and life cycle accomplishment. PMID:27445437

  2. Far beyond Phagocytosis: Phagocyte-Derived Extracellular Traps Act Efficiently against Protozoan Parasites In Vitro and In Vivo

    PubMed Central

    Muñoz-Caro, Tamara; Hidalgo, Maria A.; Taubert, Anja; Hermosilla, Carlos

    2016-01-01

    Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages are considered as the first line of defence against invasive pathogens. The formation of extracellular traps (ETs) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections. Recent investigations showed evidence that ETosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria, fungi, viruses, and protozoan parasites. ETs are released in response to intact protozoan parasites or to parasite-specific antigens in a controlled cell death process. Released ETs consist of nuclear DNA as backbone adorned with histones, antimicrobial peptides, and phagocyte-specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens. This review summarizes recent data on protozoa-induced ETosis. Special attention will be given to molecular mechanisms of protozoa-induced ETosis and on its consequences for the parasites successful reproduction and life cycle accomplishment. PMID:27445437

  3. Extracellular traps and macrophages: new roles for the versatile phagocyte

    PubMed Central

    Boe, Devin M.; Curtis, Brenda J.; Chen, Michael M.; Ippolito, Jill A.; Kovacs, Elizabeth J.

    2015-01-01

    MΦ are multipurpose phagocytes with a large repertoire of well-characterized abilities and functions, including regulation of inflammation, wound healing, maintenance of tissue homeostasis, as well as serving as an integral component of the innate-immune defense against microbial pathogens. Working along with neutrophils and dendritic cells, the other myeloid-derived professional phagocytes, MΦ are one of the key effector cells initiating and directing the host reaction to pathogenic organisms and resolving subsequent responses once the threat has been cleared. ETs are a relatively novel strategy of host defense involving expulsion of nuclear material and embedded proteins from immune cells to immobilize and kill bacteria, fungi, and viruses. As research on ETs expands, it has begun to encompass many immune cell types in unexpected ways, including various types of MΦ, which are not only capable of generating METs in response to various stimuli, but recent preclinical data suggest that they are an important agent in clearing ETs and limiting ET-mediated inflammation and tissue damage. This review aims to summarize historical and recent findings of biologic research regarding ET formation and function and discuss the role of MΦ in ET physiology and associated pathologies. PMID:25877927

  4. Neutrophil ageing is regulated by the microbiome.

    PubMed

    Zhang, Dachuan; Chen, Grace; Manwani, Deepa; Mortha, Arthur; Xu, Chunliang; Faith, Jeremiah J; Burk, Robert D; Kunisaki, Yuya; Jang, Jung-Eun; Scheiermann, Christoph; Merad, Miriam; Frenette, Paul S

    2015-09-24

    Blood polymorphonuclear neutrophils provide immune protection against pathogens, but may also promote tissue injury in inflammatory diseases. Although neutrophils are generally considered to be a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and replenishment by newly released neutrophils from the bone marrow. Aged neutrophils upregulate CXCR4, a receptor allowing their clearance in the bone marrow, with feedback inhibition of neutrophil production via the IL-17/G-CSF axis, and rhythmic modulation of the haematopoietic stem-cell niche. The aged subset also expresses low levels of L-selectin. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties. Here, using in vivo ageing analyses in mice, we show that neutrophil pro-inflammatory activity correlates positively with their ageing whilst in circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMβ2 integrin activation and neutrophil extracellular trap formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptor and myeloid differentiation factor 88-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle-cell disease or endotoxin-induced septic shock. These results identify a role for the microbiota in regulating a disease-promoting neutrophil subset. PMID:26374999

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

    PubMed Central

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

    1994-01-01

    BACKGROUND--Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. METHODS--The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. RESULTS--Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. CONCLUSIONS--These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue. PMID:8202875

  6. Trapped neutrophil syndrome in a Border Collie dog: clinical, clinico-pathologic, and molecular findings.

    PubMed

    Mizukami, Keijiro; Shoubudani, Tomoaki; Nishimoto, Seira; Kawamura, Ryuta; Yabuki, Akira; Yamato, Osamu

    2012-06-01

    Trapped neutrophil syndrome (TNS) is an autosomal recessive inherited neutropenia known in Border Collies since the 1990's. Recently, the causative mutation has been identified in the canine VPS13B gene and a DNA-based diagnosis has now become available. The present paper describes clinical and clinico-pathologic findings in a Border Collie with TNS that was molecularly diagnosed for the first time in Japan. In a 10-week-old male Border Collie with microgenesis and symptoms related to recurrent infections, a hematological examination revealed severe leukopenia due to neutropenia, suggesting the dog to be affected by inherited neutropenic immunodeficiency. Direct DNA sequencing demonstrated that the dog was homozygous for the causative mutation of TNS and both its parents were heterozygous carriers. In addition, a simple and rapid polymerase chain reaction-based length polymorphism analysis coupled with microchip electrophoresis was developed for the genotyping of TNS. This assay could discriminate clearly all genotypes, suggesting that it was suitable for both individual diagnosis and large-scale surveys for prevention. PMID:22240985

  7. Contributions of neutrophils to the adaptive immune response in autoimmune disease

    PubMed Central

    Pietrosimone, Kathryn M; Liu, Peng

    2016-01-01

    Neutrophils are granulocytic cytotoxic leukocytes of the innate immune system that activate during acute inflammation. Neutrophils can also persist beyond the acute phase of inflammation to impact the adaptive immune response during chronic inflammation. In the context of the autoimmune disease, neutrophils modulating T and B cell functions by producing cytokines and chemokines, forming neutrophil extracellular traps, and acting as or priming antigen presentation cells. Thus, neutrophils are actively involved in chronic inflammation and tissue damage in autoimmune disease. Using rheumatoid arthritis as an example, this review focuses on functions of neutrophils in adaptive immunity and the therapeutic potential of these cells in the treatment of autoimmune disease and chronic inflammation. PMID:27042404

  8. Neutrophil Responses to Sterile Implant Materials

    PubMed Central

    Jhunjhunwala, Siddharth; Aresta-DaSilva, Stephanie; Tang, Katherine; Alvarez, David; Webber, Matthew J.; Tang, Benjamin C.; Lavin, Danya M.; Veiseh, Omid; Doloff, Joshua C.; Bose, Suman; Vegas, Arturo; Ma, Minglin; Sahay, Gaurav; Chiu, Alan; Bader, Andrew; Langan, Erin; Siebert, Sean; Li, Jie; Greiner, Dale L.; Newburger, Peter E.; von Andrian, Ulrich H.; Langer, Robert; Anderson, Daniel G.

    2015-01-01

    In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30–500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs) on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices. PMID:26355958

  9. Neutrophil biology: an update

    PubMed Central

    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 response are not completely clarified, as compared with the response after removal of either apoptotic or necrotic neutrophils by phagocytes. How neutrophil density in peripheral blood is kept within a certain range is important for health and disease. Although the studies on severe congenital neutropenia and benign ethnic neutropenia have provided unbiased views on it, the studies are rather limited to human neutropenia, and mice with a mutation of mouse counterpart gene often fail to exhibit neutropenia. Degranulation plays a critical role in bactericidal action. The recent studies revealed that it is also involved in immunomodulation, pain control and estrous cycle control. N1 and N2 are representative of neutrophil subpopulations. The dichotomy holds true in patients or mice with severe trauma or cancer, providing the basis of differential roles of neutrophils in diseases. PMID:26600743

  10. The site of the bite: Leishmania interaction with macrophages, neutrophils and the extracellular matrix in the dermis.

    PubMed

    de Menezes, Juliana Perrone; Saraiva, Elvira M; da Rocha-Azevedo, Bruno

    2016-01-01

    Leishmania spp., the causative agents of leishmaniasis, are intracellular parasites, transmitted to humans via the bite of their sand fly vectors. Once inoculated, the promastigotes are exposed to the dermis, which is composed of extracellular matrix (ECM), growth factors and its resident cells. Promastigote forms are phagocytosed by macrophages recruited to the site of the sand fly bite, either directly or after interaction with neutrophils. Since Leishmania is an intracellular parasite, its interaction with the host ECM has been neglected as well as the immediate steps after the sand fly bite. However, promastigotes must overcome the obstacles presented by the dermis ECM in order to establish the infection. Thus, the study of the interaction between Leishmania promastigotes and ECM components as well as the earliest stages of infection are important steps to understand the establishment of the disease, and could contribute in the future to new drug developments towards leishmaniasis. PMID:27146515

  11. Neutrophils: Between Host Defence, Immune Modulation, and Tissue Injury

    PubMed Central

    Kruger, Philipp; Saffarzadeh, Mona; Weber, Alexander N. R.; Rieber, Nikolaus; Radsak, Markus; von Bernuth, Horst; Benarafa, Charaf; Roos, Dirk; Skokowa, Julia; Hartl, Dominik

    2015-01-01

    Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage. PMID:25764063

  12. Comparative Proteomic Analysis of Extracellular Vesicles Isolated by Acoustic Trapping or Differential Centrifugation.

    PubMed

    Rezeli, Melinda; Gidlöf, Olof; Evander, Mikael; Bryl-Górecka, Paulina; Sathanoori, Ramasri; Gilje, Patrik; Pawłowski, Krzysztof; Horvatovich, Péter; Erlinge, David; Marko-Varga, György; Laurell, Thomas

    2016-09-01

    Extracellular vesicles (ECVs), including microparticles and exosomes, are submicrometer membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared to their source, blood plasma, which makes ECV-based biomarker studies more promising. Proteomic profiling of ECVs is important not only to discover new diagnostic or prognostic markers but also to understand their roles in biological function. In the current study, we investigated the protein composition of plasma-derived ECVs isolated by acoustic seed trapping. Additionally, the protein composition of ECVs isolated with acoustic trapping was compared to that isolated with a conventional differential centrifugation protocol. Finally, the proteome of ECVs originating from ST-elevation myocardial infarction patients was compared with that of healthy controls using label-free LC-MS quantification. The acoustic trapping platform allows rapid and automated preparation of ECVs from small sample volumes, which are therefore well-suited for biobank repositories. We found that the protein composition of trapped ECVs is very similar to that isolated by the conventional differential centrifugation method. PMID:27487081

  13. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

    PubMed

    Homa, Joanna; Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  14. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    PubMed Central

    Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  15. Role of membrane depolarization and extracellular calcium in increased complement receptor expression during neutrophil (PMN) activation

    SciTech Connect

    Berger, M.; Wetzler, E.; Birx, D.L.

    1986-03-05

    During PMN activation the surface expression of receptors (R) for C3b and C3bi increases rapidly. This is necessary for optimal cell adhesion, migration, and phagocytosis. Following stimulation with fMLP or LTB-4, the increased expression of C3bR depends only on the Ca/sup + +/ released from intracellular stores and is not inhibited by 5mM EDTA, while the increase in C3biR also requires extracellular Ca/sup + +/. CR expression also increases when the PMN are depolarized with 140 mM K/sup +/, but with this stimulus, EDTA inhibits C3bR by 67% and C3biR 100%, suggesting that intracellular Ca/sup + +/ stores may not be released. Pertussis toxin caused dose-dependent inhibition of both CR responses to fMLP and also inhibited the increases in both CR induced by K/sup +/. Membrane depolarization (monitored by di-O-C5 fluorescence) due to fMLP was similarly inhibited by toxin but the depolarization due to K/sup +/ was not. The dose of phorbol myristate acetate that maximally increased CR expression, 0.1 ng/ml, did not depolarize the membrane. These results suggest that membrane depolarization is neither necessary nor sufficient for increased CR expression. A Ca/sup + +/ and GTP binding protein-dependent enzyme such as phospholipase C is necessary to the amplify initial signals generated either by release of Ca/sup + +/ stores or by opening voltage dependent Ca/sup + +/ channels following membrane depolarization.

  16. Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment.

    PubMed

    Bou Ghanem, Elsa N; Clark, Stacie; Roggensack, Sara E; McIver, Sally R; Alcaide, Pilar; Haydon, Philip G; Leong, John M

    2015-08-01

    An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73-/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73-/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters

  17. NET amyloidogenic backbone in human activated neutrophils.

    PubMed

    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. PMID:26462606

  18. Neutrophil ageing is regulated by the microbiome

    PubMed Central

    Zhang, Dachuan; Chen, Grace; Manwani, Deepa; Mortha, Arthur; Xu, Chunliang; Faith, Jeremiah J.; Burk, Robert D.; Kunisaki, Yuya; Jang, Jung-Eun; Scheiermann, Christoph; Merad, Miriam; Frenette, Paul S.

    2015-01-01

    Blood polymorphonuclear neutrophils provide immune protection against pathogens but also may promote tissue injury in inflammatory diseases1,2. Although neutrophils are generally considered as a relatively homogeneous population, evidence for heterogeneity is emerging3,4. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and the replenishment by newly released neutrophils from the bone marrow5. Aged neutrophils up-regulate CXCR4, a receptor allowing their clearance in the bone marrow6,7, with feedback inhibition of neutrophil production via the IL17/G-CSF axis8, and rhythmic modulation of the haematopoietic stem cell niche5. The aged subset also expresses low levels of L-selectin (CD62L)5,9. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties6,10. Here, we show using in vivo ageing analyses that the neutrophil pro-inflammatory activity correlates positively with their ageing in the circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMβ2 integrin (Mac-1) activation and neutrophil extracellular trap (NET) formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptors (TLRs)- and myeloid differentiation factor 88 (Myd88)-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle cell disease or endotoxin-induced septic shock. These results thus identify an unprecedented role for the microbiota in regulating a disease-promoting neutrophil subset. PMID:26374999

  19. Characterization of neutrophil function in Papillon-Lefèvre syndrome.

    PubMed

    Roberts, Helen; White, Phillipa; Dias, Irundika; McKaig, Sarah; Veeramachaneni, Ratna; Thakker, Nalin; Grant, Melissa; Chapple, Iain

    2016-08-01

    Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal

  20. Escherichia coli and Candida albicans Induced Macrophage Extracellular Trap-Like Structures with Limited Microbicidal Activity

    PubMed Central

    Liao, Chengshui; Liu, Xiaolei; Du, Jing; Shi, Haining; Wang, Xuelin; Bai, Xue; Peng, Peng; Yu, Lu; Wang, Feng; Zhao, Ying; Liu, Mingyuan

    2014-01-01

    The formation of extracellular traps (ETs) has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs) in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17%) and peritoneal macrophages (8.5% to 15%) form METs-like structures (METs-LS) in response to Escherichia coli and Candida albicans challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped E. coli but not C. albicans. Fluorescence and scanning electron microscopy analyses revealed that, in vitro, both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them. PMID:24587206

  1. Dynamic interactions of neutrophils and biofilms

    PubMed Central

    Hirschfeld, Josefine

    2014-01-01

    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. PMID:25523872

  2. Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.

    PubMed Central

    Matzner, Y; Bar-Ner, M; Yahalom, J; Ishai-Michaeli, R; Fuks, Z; Vlodavsky, I

    1985-01-01

    Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ. PMID:2997275

  3. Differential expression of pentraxin 3 in neutrophils.

    PubMed

    Razvina, Olga; Jiang, Shuying; Matsubara, Koichi; Ohashi, Riuko; Hasegawa, Go; Aoyama, Takashi; Daigo, Kenji; Kodama, Tatsuhiko; Hamakubo, Takao; Naito, Makoto

    2015-02-01

    Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense. PMID:25449330

  4. [The efficiency of macrophage extracellular trap formation induced by different inducers in vitro].

    PubMed

    Su, Chengcheng; Xiang, Guoan; Ma, Yongqiang; Zhang, Yidan; Zhou, Xin; Peng, Shouchun; Wei, Luqing; Ji, Wenjie

    2016-04-01

    Objective To compare the different methods of inducing the formation of macrophage extracellular trap (MET) in vitro. Methods MET release was initiated by culturing RAW264.7 cells with 0.5, 1, 5, 10 μg/mL lipopolysaccharide (LPS), or 10, 25, 50, 80 μmol/L phorbolmyristate acetate (PMA), or 50, 100, 150 μg/mL silicon dioxide (SiO2). Three and 6 hours later, MET were validated by immunofluorescence staining, followed by immunofluorescence-based semi-quantitative analysis. Results Immunofluorescence staining showed that the network structures were mainly composed of DNA and histones. RAW264.7 cells treated with 1 μg/mL LPS for 6 hours produced the highest percent of MET [(37.04±10.02)%], which was statistically higher compared with control group [(7.90±2.71)%]. RAW264.7 cells treated with 80 μmol/L PMA for 6 hours also produced the higher percent of MET [(22.40±1.83)%] compared with control group [(10.11±1.13)%]. However, there was no significantly increased MET formation in cells treated with SiO2 compared with control group. Conclusion LPS and PMA can induce MET formation in vitro, while SiO2 was not efficient inducer. PMID:27053611

  5. Central role of neutrophil in the pathogenesis of severe acute pancreatitis

    PubMed Central

    Yang, Zhi-wen; Meng, Xiao-xiao; Xu, Ping

    2015-01-01

    Severe acute pancreatitis (SAP) is an acute abdominal disease with the strong systemic inflammatory response, and rapidly progresses from a local pancreatic damage into multiple organ dysfunction. For many decades, the contributions of neutrophils to the pathology of SAP were traditionally thought to be the chemokine and cytokine cascades that accompany inflammation. In this review, we focus mainly on those recently recognized aspects of neutrophils in SAP processes. First, emerging evidence suggests that therapeutic interventions targeting neutrophils significantly lower tissue damage and protect against the occurrence of pancreatitis. Second, trypsin activation promotes the initial neutrophils recruitment into local pancreas, and subsequently neutrophils infiltration in turn triggers trypsin production. Finally, neutrophils have the unique ability to release neutrophil extracellular traps even in the absence of pathogens. PMID:26249268

  6. Central role of neutrophil in the pathogenesis of severe acute pancreatitis.

    PubMed

    Yang, Zhi-Wen; Meng, Xiao-Xiao; Xu, Ping

    2015-11-01

    Severe acute pancreatitis (SAP) is an acute abdominal disease with the strong systemic inflammatory response, and rapidly progresses from a local pancreatic damage into multiple organ dysfunction. For many decades, the contributions of neutrophils to the pathology of SAP were traditionally thought to be the chemokine and cytokine cascades that accompany inflammation. In this review, we focus mainly on those recently recognized aspects of neutrophils in SAP processes. First, emerging evidence suggests that therapeutic interventions targeting neutrophils significantly lower tissue damage and protect against the occurrence of pancreatitis. Second, trypsin activation promotes the initial neutrophils recruitment into local pancreas, and subsequently neutrophils infiltration in turn triggers trypsin production. Finally, neutrophils have the unique ability to release neutrophil extracellular traps even in the absence of pathogens. PMID:26249268

  7. Matters of life and death. How neutrophils die or survive along NET release and is "NETosis" = necroptosis?

    PubMed

    Desai, Jyaysi; Mulay, Shrikant R; Nakazawa, Daigo; Anders, Hans-Joachim

    2016-06-01

    Neutrophil extracellular trap (NET) formation is a hallmark of many disorders that involve neutrophil recruitment, tissue damage, and inflammation. As NET formation is often associated with neutrophil death, the term "NETosis" has become popular. Upon discovery that neutrophils may survive NET release, apparent misnomers, such as "vital NETosis," have been proposed. Meanwhile, it has become obvious that certain stimuli can trigger neutrophil necroptosis, a process associated with NET-like chromatin release. Here, we discuss the relationship between NET release and neutrophil death in view highlighting that many assays used in the field do not properly distinguish between the two. An updated nomenclature is needed replacing the term "NETosis" to meet the growing variety of settings leading to chromatin release with and without neutrophil death. Dissecting which triggers of NET release involve which signaling pathway will help to define drugable molecular targets that inhibit NET release and/or neutrophil necrosis in specific disorders. PMID:27048811

  8. Eimeria ninakohlyakimovae induces NADPH oxidase-dependent monocyte extracellular trap formation and upregulates IL-12 and TNF-α, IL-6 and CCL2 gene transcription.

    PubMed

    Pérez, D; Muñoz, M C; Molina, J M; Muñoz-Caro, T; Silva, L M R; Taubert, A; Hermosilla, C; Ruiz, A

    2016-08-30

    Extracellular trap (ET) formation has been demonstrated as novel effector mechanism against diverse pathogens in polymorphonuclear neutrophils (PMN), eosinophils, mast cells, macrophages and recently also in monocytes. In the current study, we show that E. ninakohlyakimovae triggers the deliverance of monocyte-derived ETs in vitro. Fluorescence illustrations as well as scanning electron microscopy (SEM) analyses showed that monocyte-derived ET formation was rapidly induced upon exposure to viable sporozoites, sporocysts and oocysts of E. ninakohlyakimovae. Classical features of monocyte-released ETs were confirmed by the co-localization of extracellular DNA adorned with myeloperoxidase (MPO) and histones (H3) in parasite-entrapping structures. The treatment of caprine monocyte ET structures with NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced ETosis confirming the essential role of reactive oxygen species (ROS) in monocyte mediated ETs formation. Additionally, co-culture of monocytes with viable sporozoites and soluble oocyst antigen (SOA) induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IL-12 and TNF-α was significantly upregulated after sporozoite encounter. In contrast IL-6 and CCL2 gene transcripts were rather weakly induced by parasites. Conversely, SOA only induced the up-regulation of IL-6 and CCL2 gene transcription, and failed to enhance transcripts of IL-12 and TNF-α in vitro. We here report on monocyte-triggered ETs as novel effector mechanism against E. ninakohlyakimovae. Our results strongly suggest that monocyte-mediated innate immune reactions might play an important role in early host immune reactions against E. ninakohlyakimovae in goats. PMID:27523951

  9. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

    PubMed Central

    Ueki, Shigeharu; Melo, Rossana C. N.; Ghiran, Ionita; Spencer, Lisa A.; Dvorak, Ann M.; Weller, Peter F.

    2013-01-01

    Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets. PMID:23303825

  10. Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

    PubMed

    Ramadass, Mahalakshmi; Catz, Sergio D

    2016-09-01

    Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion. PMID:27558339

  11. Neutrophils in the tumor microenvironment: trying to heal the wound that cannot heal.

    PubMed

    Singel, Kelly L; Segal, Brahm H

    2016-09-01

    Neutrophils are the first responders to infection and injury and are critical for antimicrobial host defense. Through the generation of reactive oxidants, activation of granular constituents and neutrophil extracellular traps, neutrophils target microbes and prevent their dissemination. While these pathways are beneficial in the context of trauma and infection, their off-target effects in the context of tumor are variable. Tumor-derived factors have been shown to reprogram the marrow, skewing toward the expansion of myelopoiesis. This can result in stimulation of both neutrophilic leukocytosis and the release of immature granulocytic populations that accumulate in circulation and in the tumor microenvironment. While activated neutrophils have been shown to kill tumor cells, there is growing evidence for neutrophil activation driving tumor progression and metastasis through a number of pathways, including stimulation of thrombosis and angiogenesis, stromal remodeling, and impairment of T cell-dependent anti-tumor immunity. There is also growing appreciation of neutrophil heterogeneity in cancer, with distinct neutrophil populations promoting cancer control or progression. In addition to the effects of tumor on neutrophil responses, anti-neoplastic treatment, including surgery, chemotherapy, and growth factors, can influence neutrophil responses. Future directions for research are expected to result in more mechanistic knowledge of neutrophil biology in the tumor microenvironment that may be exploited as prognostic biomarkers and therapeutic targets. PMID:27558344

  12. Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.

    PubMed

    Andzinski, Lisa; Kasnitz, Nadine; Stahnke, Stephanie; Wu, Ching-Fang; Gereke, Marcus; von Köckritz-Blickwede, Maren; Schilling, Bastian; Brandau, Sven; Weiss, Siegfried; Jablonska, Jadwiga

    2016-04-15

    The importance of tumor associated neutrophils (TANs) in cancer development is in the meantime well established. Numerous of clinical data document the adverse prognostic effects of neutrophil infiltration in solid tumors. However, certain tumor therapies need functional neutrophils to be effective, suggesting altered neutrophil polarization associated with different outcomes for cancer patients. Therefore, modulation of neutrophilic phenotypes represents a potent therapeutic option, but factors mediating neutrophil polarization are still poorly defined. In this manuscript we provide evidence that type I IFNs alter neutrophilic phenotype into anti-tumor, both in mice and human. In the absence of IFN-β, pro-tumor properties, such as reduced tumor cytotoxicity with low neutrophil extracellular traps (NETs) expression, low ICAM1 and TNF-α expression, dominated neutrophil phenotypes in primary lesion and premetastatic lung. Interestingly, such neutrophils have significantly prolonged life-span. Notably, interferon therapy in mice altered TAN polarization towards anti-tumor N1. Similar changes in neutrophil activation could be observed in melanoma patients undergoing type I IFN therapy. Altogether, these data highlight the therapeutic potential of interferons, suggesting optimization of its clinical use as potent anti-tumor agent. PMID:26619320

  13. Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin.

    PubMed

    Zenaro, Elena; Pietronigro, Enrica; Della Bianca, Vittorina; Piacentino, Gennj; Marongiu, Laura; Budui, Simona; Turano, Ermanna; Rossi, Barbara; Angiari, Stefano; Dusi, Silvia; Montresor, Alessio; Carlucci, Tommaso; Nanì, Sara; Tosadori, Gabriele; Calciano, Lucia; Catalucci, Daniele; Berton, Giorgio; Bonetti, Bruno; Constantin, Gabriela

    2015-08-01

    Inflammation is a pathological hallmark of Alzheimer's disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimer's disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimer's disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimer's disease-like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimer's disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimer's disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimer's disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimer's disease. PMID:26214837

  14. Tamoxifen augments the innate immune function of neutrophils through modulation of intracellular ceramide.

    PubMed

    Corriden, Ross; Hollands, Andrew; Olson, Joshua; Derieux, Jaclyn; Lopez, Justine; Chang, John T; Gonzalez, David J; Nizet, Victor

    2015-01-01

    Tamoxifen is a selective oestrogen receptor modulator widely used for the treatment of breast cancer. In addition to its activity as an oestrogen receptor agonist/antagonist, tamoxifen also modulates sphingolipid biosynthesis, which has been shown to play an important role in the regulation of neutrophil activity. Here, we find that tamoxifen stimulation enhances several pro-inflammatory pathways in human neutrophils, including chemotaxis, phagocytosis and neutrophil extracellular trap (NET) formation. The enhancement of NET production occurs via a ceramide/PKCζ-mediated pathway, and treatment with synthetic ceramide is sufficient to promote NET formation. Pretreatment of human neutrophils with tamoxifen boosts neutrophil bactericidal capacity against a variety of pathogens in vitro and enhances clearance of the leading human pathogen methicillin-resistant Staphylococcus aureus in vivo. Our results suggest that tamoxifen, and the lipid signalling pathways it modulates, merit further exploration as targets for boosting host innate immune function. PMID:26458291

  15. Tamoxifen Augments the Innate Immune Function of Neutrophils Through Modulation of Intracellular Ceramide

    PubMed Central

    Corriden, Ross; Hollands, Andrew; Olson, Joshua; Derieux, Jaclyn; Lopez, Justine; Chang, John T.; Gonzalez, David J.; Nizet, Victor

    2015-01-01

    Tamoxifen is a selective estrogen receptor modulator widely used for the treatment of breast cancer. In addition to its activity as an estrogen receptor agonist/antagonist, tamoxifen also modulates sphingolipid biosynthesis, which has been shown to play an important role in the regulation of neutrophil activity. Here, we find that tamoxifen stimulation enhances several pro-inflammatory pathways in human neutrophils, including chemotaxis, phagocytosis and neutrophil extracellular trap (NET) formation. The enhancement of NET production occurs via a ceramide/PKCζ-mediated pathway, and treatment with synthetic ceramide is sufficient to promote NET formation. Pretreatment of human neutrophils with tamoxifen boosts neutrophil bactericidal capacity against a variety of pathogens in vitro and enhances clearance of the leading human pathogen methicillin-resistant Staphylococcus aureus in vivo. Our results suggest that tamoxifen, and the lipid signaling pathways it modulates, merit further exploration as targets for boosting host innate immune function. PMID:26458291

  16. The role of extracellular histones in haematological disorders.

    PubMed

    Alhamdi, Yasir; Toh, Cheng-Hock

    2016-06-01

    Over the past decades, chromosomal alterations have been extensively investigated for their pathophysiological relevance in haematological malignancies. In particular, epigenetic modifications of intra-nuclear histones are now known as key regulators of healthy cell cycles that have also evolved into novel therapeutic targets for certain blood cancers. Thus, for most haematologists, histones are DNA-chained proteins that are buried deep within chromatin. However, the plot has deepened with recent revelations on the function of histones when unchained and released extracellularly upon cell death or from activated neutrophils as part of neutrophil extracellular traps (NETs). Extracellular histones and NETs are increasingly recognized for profound cytotoxicity and pro-coagulant effects. This article highlights the importance of recognizing this new paradigm of extracellular histones as a key player in host defence through its damage-associated molecular patterns, which could translate into novel diagnostic and therapeutic biomarkers in various haematological and critical disorders. PMID:27062156

  17. Immunoreceptors on neutrophils.

    PubMed

    van Rees, Dieke J; Szilagyi, Katka; Kuijpers, Taco W; Matlung, Hanke L; van den Berg, Timo K

    2016-04-01

    Neutrophils play a critical role in the host defense against infection, and they are able to perform a variety of effector mechanisms for this purpose. However, there are also a number of pathological conditions, including autoimmunity and cancer, in which the activities of neutrophils can be harmful to the host. Thus the activities of neutrophils need to be tightly controlled. As in the case of other immune cells, many of the neutrophil effector functions are regulated by a series of immunoreceptors on the plasma membrane. Here, we review what is currently known about the functions of the various individual immunoreceptors and their signaling in neutrophils. While these immunoreceptors allow for the recognition of a diverse range of extracellular ligands, such as cell surface structures (like proteins, glycans and lipids) and extracellular matrix components, they commonly signal via conserved ITAM or ITIM motifs and their associated downstream pathways that depend on the phosphorylation of tyrosine residues in proteins and/or inositol lipids. This allows for a balanced homeostatic regulation of neutrophil effector functions. Given the number of available immunoreceptors and their fundamental importance for neutrophil behavior, it is perhaps not surprising that pathogens have evolved means to evade immune responses through some of these pathways. Inversely, some of these receptors evolved to specifically recognize these pathogens. Finally, some interactions mediated by immunoreceptors in neutrophils have been identified as promising targets for therapeutic intervention. PMID:26976825

  18. Invertebrate extracellular phagocyte traps show that chromatin is an ancient defence weapon

    PubMed Central

    Robb, Calum T.; Dyrynda, Elisabeth A.; Gray, Robert D.; Rossi, Adriano G.; Smith, Valerie J.

    2014-01-01

    Controlled release of chromatin from the nuclei of inflammatory cells is a process that entraps and kills microorganisms in the extracellular environment. Now termed ETosis, it is important for innate immunity in vertebrates. Paradoxically, however, in mammals, it can also contribute to certain pathologies. Here we show that ETosis occurs in several invertebrate species, including, remarkably, an acoelomate. Our findings reveal that the phenomenon is primordial and predates the evolution of the coelom. In invertebrates, the released chromatin participates in defence not only by ensnaring microorganisms and externalizing antibacterial histones together with other haemocyte-derived defence factors, but crucially, also provides the scaffold on which intact haemocytes assemble during encapsulation; a response that sequesters and kills potential pathogens infecting the body cavity. This insight into the early origin of ETosis identifies it as a very ancient process that helps explain some of its detrimental effects in mammals. PMID:25115909

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

    PubMed

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

    2015-05-01

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

  20. Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity

    PubMed Central

    Xu, Ding; Olson, Joshua; Cole, Jason N.; van Wijk, Xander M.; Brinkmann, Volker; Zychlinsky, Arturo; Nizet, Victor

    2015-01-01

    Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity. PMID:26150541

  1. Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity.

    PubMed

    Xu, Ding; Olson, Joshua; Cole, Jason N; van Wijk, Xander M; Brinkmann, Volker; Zychlinsky, Arturo; Nizet, Victor; Esko, Jeffrey D; Chang, Yung-Chi

    2015-09-01

    Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity. PMID:26150541

  2. Myeloperoxidase Stimulates Neutrophil Degranulation.

    PubMed

    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. PMID:27597056

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

    PubMed Central

    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

  4. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis.

    PubMed

    Leppkes, Moritz; Maueröder, Christian; Hirth, Sebastian; Nowecki, Stefanie; Günther, Claudia; Billmeier, Ulrike; Paulus, Susanne; Biermann, Mona; Munoz, Luis E; Hoffmann, Markus; Wildner, Dane; Croxford, Andrew L; Waisman, Ari; Mowen, Kerri; Jenne, Dieter E; Krenn, Veit; Mayerle, Julia; Lerch, Markus M; Schett, Georg; Wirtz, Stefan; Neurath, Markus F; Herrmann, Martin; Becker, Christoph

    2016-01-01

    Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts. PMID:26964500

  5. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis

    PubMed Central

    Leppkes, Moritz; Maueröder, Christian; Hirth, Sebastian; Nowecki, Stefanie; Günther, Claudia; Billmeier, Ulrike; Paulus, Susanne; Biermann, Mona; Munoz, Luis E.; Hoffmann, Markus; Wildner, Dane; Croxford, Andrew L.; Waisman, Ari; Mowen, Kerri; Jenne, Dieter E.; Krenn, Veit; Mayerle, Julia; Lerch, Markus M.; Schett, Georg; Wirtz, Stefan; Neurath, Markus F.; Herrmann, Martin; Becker, Christoph

    2016-01-01

    Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts. PMID:26964500

  6. Neutrophil migration into the placenta: Good, bad or deadly?

    PubMed Central

    Giaglis, Stavros; Stoikou, Maria; Grimolizzi, Franco; Subramanian, Bibin Y.; van Breda, Shane V.; Hoesli, Irene; Lapaire, Olav; Hasler, Paul; Than, Nandor Gabor; Hahn, Sinuhe

    2016-01-01

    ABSTRACT Almost 2 decades have passed since the discovery that pregnancy is associated with a basal inflammatory state involving neutrophil activation, and that this is more overt in cases with preeclampsia, than in instances with sepsis. This pivotal observation paved the way for our report, made almost a decade ago, describing the first involvement of neutrophil extracellular traps (NETs) in a non-infectious human pathology, namely preeclampsia, where an abundance of these structures were detected directly in the placental intervillous space. Despite these remarkable findings, there remains a paucity of interest among reproductive biologists in further exploring the role or involvement of neutrophils in pregnancy and related pathologies. In this review we attempt to redress this deficit by highlighting novel recent findings including the discovery of a novel neutrophil subset in the decidua, the interaction of placental protein 13 (PP13) and neutrophils in modulating spiral artery modification, as well as the use of animal model systems to elucidate neutrophil function in implantation, gestation and parturition. These model systems have been particularly useful in identifying key components implicated in recurrent fetal loss, preeclampsia or new signaling molecules such as sphingolipids. Finally, the recent discovery that anti-phospolipid antibodies can trigger NETosis, supports our hypothesis that these structures may contribute to placental dysfunction in pertinent cases with recurrent fetal loss. PMID:26933824

  7. Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

    PubMed

    Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

    2016-01-01

    We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

  8. Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells

    PubMed Central

    Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

    2016-01-01

    We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

  9. Neutrophil roles in left ventricular remodeling following myocardial infarction

    PubMed Central

    2013-01-01

    Polymorphonuclear granulocytes (PMNs; neutrophils) serve as key effector cells in the innate immune system and provide the first line of defense against invading microorganisms. In addition to producing inflammatory cytokines and chemokines and undergoing a respiratory burst that stimulates the release of reactive oxygen species, PMNs also degranulate to release components that kill pathogens. Recently, neutrophil extracellular traps have been shown to be an alternative way to trap microorganisms and contain infection. PMN-derived granule components are also involved in multiple non-infectious inflammatory processes, including the response to myocardial infarction (MI). In this review, we will discuss the biological characteristics, recruitment, activation, and removal of PMNs, as well as the roles of PMN-derived granule proteins in inflammation and innate immunity, focusing on the MI setting when applicable. We also discuss future perspectives that will direct research in PMN biology. PMID:23731794

  10. Trace element landscape of resting and activated human neutrophils on the sub-micrometer level.

    PubMed

    Niemiec, M J; De Samber, B; Garrevoet, J; Vergucht, E; Vekemans, B; De Rycke, R; Björn, E; Sandblad, L; Wellenreuther, G; Falkenberg, G; Cloetens, P; Vincze, L; Urban, C F

    2015-06-01

    Every infection is a battle for trace elements. Neutrophils migrate first to the infection site and accumulate quickly to high numbers. They fight pathogens by phagocytosis and intracellular toxication. Additionally, neutrophils form neutrophil extracellular traps (NETs) to inhibit extracellular microbes. Yet, neutrophil trace element characteristics are largely unexplored. We investigated unstimulated and phorbol myristate acetate-stimulated neutrophils using synchrotron radiation X-ray fluorescence (SR-XRF) on the sub-micron spatial resolution level. PMA activates pinocytosis, cytoskeletal rearrangements and the release of NETs, all mechanisms deployed by neutrophils to combat infection. By analyzing Zn, Fe, Cu, Mn, P, S, and Ca, not only the nucleus but also vesicular granules were identifiable in the elemental maps. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) revealed a neutrophil-specific composition of Zn, Fe, Cu, and Mn in comparison with J774 and HeLa cells, indicating a neutrophil-specific metallome complying with their designated functions. When investigating PMA-activated neutrophils, the SR-XRF analysis depicted typical subcellular morphological changes: the transformation of nucleus and granules and the emergence of void vacuoles. Mature NETs were evenly composed of Fe, P, S, and Ca with occasional hot spots containing Zn, Fe, and Ca. An ICP-MS-based quantification of NET supernatants revealed a NETosis-induced decrease of soluble Zn, whereas Fe, Cu, and Mn concentrations were only slightly affected. In summary, we present a combination of SR-XRF and ICP-MS as a powerful tool to analyze trace elements in human neutrophils. The approach will be applicable and valuable to numerous aspects of nutritional immunity. PMID:25832493

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

    PubMed

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

    2015-09-01

    Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCβII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug. PMID:26071217

  12. Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection.

    PubMed

    Clark, Heather L; Jhingran, Anupam; Sun, Yan; Vareechon, Chairut; de Jesus Carrion, Steven; Skaar, Eric P; Chazin, Walter J; Calera, José Antonio; Hohl, Tobias M; Pearlman, Eric

    2016-01-01

    Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared with wild-type. Collectively, these studies demonstrate a novel stage-specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin. PMID:26582948

  13. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice

    PubMed Central

    Martinod, Kimberly; Demers, Melanie; Fuchs, Tobias A.; Wong, Siu Ling; Brill, Alexander; Gallant, Maureen; Hu, Jing; Wang, Yanming; Wagner, Denisa D.

    2013-01-01

    Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4−/− mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4−/− mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis. PMID:23650392

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

    PubMed

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

    2016-02-01

    Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response. PMID:26718340

  15. Entamoeba histolytica induces human neutrophils to form NETs.

    PubMed

    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. PMID:27138813

  16. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.

    PubMed

    Wong, Siu Ling; Demers, Melanie; Martinod, Kimberly; Gallant, Maureen; Wang, Yanming; Goldfine, Allison B; Kahn, C Ronald; Wagner, Denisa D

    2015-07-01

    Wound healing is impaired in diabetes, resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed NETosis). Expression of peptidylarginine deiminase 4 (PAD4, encoded by Padi4 in mice), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4(-/-) mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds than in normoglycemic mice and healing was delayed. Wound healing was accelerated in Padi4(-/-) mice as compared to WT mice, and it was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes, in which neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes. PMID:26076037

  17. Streptolysin O Rapidly Impairs Neutrophil Oxidative Burst and Antibacterial Responses to Group A Streptococcus

    PubMed Central

    Uchiyama, Satoshi; Döhrmann, Simon; Timmer, Anjuli M.; Dixit, Neha; Ghochani, Mariam; Bhandari, Tamara; Timmer, John C.; Sprague, Kimberly; Bubeck-Wardenburg, Juliane; Simon, Scott I.; Nizet, Victor

    2015-01-01

    Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance. PMID:26635795

  18. Streptolysin O Rapidly Impairs Neutrophil Oxidative Burst and Antibacterial Responses to Group A Streptococcus.

    PubMed

    Uchiyama, Satoshi; Döhrmann, Simon; Timmer, Anjuli M; Dixit, Neha; Ghochani, Mariam; Bhandari, Tamara; Timmer, John C; Sprague, Kimberly; Bubeck-Wardenburg, Juliane; Simon, Scott I; Nizet, Victor

    2015-01-01

    Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance. PMID:26635795

  19. Warifteine, an Alkaloid Purified from Cissampelos sympodialis, Inhibits Neutrophil Migration In Vitro and In Vivo

    PubMed Central

    Lima, Thaline F. A.; Rocha, Juliana D. B.; Guimarães-Costa, Anderson B.; Barbosa-Filho, José M.; Decoté-Ricardo, Débora; Saraiva, Elvira M.; Arruda, Luciana B.; Piuvezam, Marcia R.; Peçanha, Ligia M. T.

    2014-01-01

    Cissampelos sympodialis Eichl is a plant from the Northeast and Southeast of Brazil. Its root infusion is popularly used for treatment of inflammatory and allergic diseases. We investigated whether warifteine, its main alkaloid, would have anti-inflammatory effect due to a blockage of neutrophil function. In vivo warifteine treatment inhibited casein-induced neutrophil migration to the peritoneal cavity but did not inhibit neutrophil mobilization from the bone marrow. Analysis of the direct effect of warifteine upon neutrophil adherence and migration in vitro demonstrated that the alkaloid decreased cell adhesion to P and E-selectin-transfected cells. In addition, fLMP-induced neutrophil migration in a transwell system was blocked by warifteine; this effect was mimicked by cAMP mimetic/inducing substances, and warifteine increased intracellular cAMP levels in neutrophils. The production of DNA extracellular traps (NETs) was also blocked by warifteine but there was no alteration on PMA-induced oxidative burst or LPS-stimulated TNFα secretion. Taken together, our data indicate that the alkaloid warifteine is a potent anti-inflammatory substance and that it has an effect on neutrophil migration through a decrease in both cell adhesion and migration. PMID:24995347

  20. Enhanced human neutrophil vitamin C status, chemotaxis and oxidant generation following dietary supplementation with vitamin C-rich SunGold kiwifruit.

    PubMed

    Bozonet, Stephanie M; Carr, Anitra C; Pullar, Juliet M; Vissers, Margreet C M

    2015-04-01

    Neutrophils are the body's primary defenders against invading pathogens. These cells migrate to loci of infection where they engulf micro-organisms and subject them to an array of reactive oxygen species and antimicrobial proteins to effect killing. Spent neutrophils subsequently undergo apoptosis and are cleared by macrophages, thereby resolving the inflammatory episode. Neutrophils contain high concentrations of vitamin C (ascorbate) and this is thought to be essential for their function. This may be one mechanism whereby vitamin C enhances immune function. The aim of our study was to assess the effect of dietary supplementation with vitamin C-rich SunGold kiwifruit on four important functions of neutrophils: chemotaxis, oxidant generation, extracellular trap formation, and apoptosis. Fourteen young men (aged 18-30 years) with suboptimal plasma vitamin C status (<50 μmol/L) were supplemented for four weeks with two SunGold kiwifruit/day. Plasma vitamin C status was monitored weekly and neutrophil vitamin C levels were assessed at baseline and post-intervention. Neutrophil function assays were carried out on cells isolated at baseline and post-intervention. Plasma vitamin C levels increased to >70 μmol/L (p < 0.001) within one week of supplementation and there was a significant increase in neutrophil vitamin C status following four weeks' intervention (p = 0.016). We observed a significant 20% increase in neutrophil chemotaxis post-intervention (p = 0.041) and also a comparable increase in oxidant generation (p = 0.031). Supplementation did not affect neutrophil extracellular trap formation or spontaneous apoptosis. Our data indicate that supplementation with vitamin C-rich kiwifruit is associated with improvement of important neutrophil functions, which would be expected to translate into enhanced immunity. PMID:25912037

  1. Enhanced Human Neutrophil Vitamin C Status, Chemotaxis and Oxidant Generation Following Dietary Supplementation with Vitamin C-Rich SunGold Kiwifruit

    PubMed Central

    Bozonet, Stephanie M.; Carr, Anitra C.; Pullar, Juliet M.; Vissers, Margreet C. M.

    2015-01-01

    Neutrophils are the body’s primary defenders against invading pathogens. These cells migrate to loci of infection where they engulf micro-organisms and subject them to an array of reactive oxygen species and antimicrobial proteins to effect killing. Spent neutrophils subsequently undergo apoptosis and are cleared by macrophages, thereby resolving the inflammatory episode. Neutrophils contain high concentrations of vitamin C (ascorbate) and this is thought to be essential for their function. This may be one mechanism whereby vitamin C enhances immune function. The aim of our study was to assess the effect of dietary supplementation with vitamin C-rich SunGold kiwifruit on four important functions of neutrophils: chemotaxis, oxidant generation, extracellular trap formation, and apoptosis. Fourteen young men (aged 18–30 years) with suboptimal plasma vitamin C status (<50 μmol/L) were supplemented for four weeks with two SunGold kiwifruit/day. Plasma vitamin C status was monitored weekly and neutrophil vitamin C levels were assessed at baseline and post-intervention. Neutrophil function assays were carried out on cells isolated at baseline and post-intervention. Plasma vitamin C levels increased to >70 μmol/L (p < 0.001) within one week of supplementation and there was a significant increase in neutrophil vitamin C status following four weeks’ intervention (p = 0.016). We observed a significant 20% increase in neutrophil chemotaxis post-intervention (p = 0.041) and also a comparable increase in oxidant generation (p = 0.031). Supplementation did not affect neutrophil extracellular trap formation or spontaneous apoptosis. Our data indicate that supplementation with vitamin C-rich kiwifruit is associated with improvement of important neutrophil functions, which would be expected to translate into enhanced immunity. PMID:25912037

  2. Coagulation induced by C3aR-dependent NETosis drives protumorigenic neutrophils during small intestinal tumorigenesis.

    PubMed

    Guglietta, Silvia; Chiavelli, Andrea; Zagato, Elena; Krieg, Carsten; Gandini, Sara; Ravenda, Paola Simona; Bazolli, Barbara; Lu, Bao; Penna, Giuseppe; Rescigno, Maria

    2016-01-01

    Excessive activation of blood coagulation and neutrophil accumulation have been described in several human cancers. However, whether hypercoagulation and neutrophilia are linked and involved in cancer development is currently unknown. Here we show that spontaneous intestinal tumorigenesis correlates with the accumulation of low-density neutrophils with a pro-tumorigenic N2 phenotype and unprompted neutrophil extracellular traps (NET) formation. We find that increased circulating lipopolysaccharide induces upregulation of complement C3a receptor on neutrophils and activation of the complement cascade. This leads to NETosis, induction of coagulation and N2 polarization, which prompts tumorigenesis, showing a novel link between coagulation, neutrophilia and complement activation. Finally, in a cohort of patients with small but not large intestinal cancer, we find a correlation between neutrophilia and hypercoagulation. This study provides a mechanistic explanation for the tumour-promoting effects of hypercoagulation, which could be used as a new biomarker or as a therapeutic target. PMID:26996437

  3. Coagulation induced by C3aR-dependent NETosis drives protumorigenic neutrophils during small intestinal tumorigenesis

    PubMed Central

    Guglietta, Silvia; Chiavelli, Andrea; Zagato, Elena; Krieg, Carsten; Gandini, Sara; Ravenda, Paola Simona; Bazolli, Barbara; Lu, Bao; Penna, Giuseppe; Rescigno, Maria

    2016-01-01

    Excessive activation of blood coagulation and neutrophil accumulation have been described in several human cancers. However, whether hypercoagulation and neutrophilia are linked and involved in cancer development is currently unknown. Here we show that spontaneous intestinal tumorigenesis correlates with the accumulation of low-density neutrophils with a pro-tumorigenic N2 phenotype and unprompted neutrophil extracellular traps (NET) formation. We find that increased circulating lipopolysaccharide induces upregulation of complement C3a receptor on neutrophils and activation of the complement cascade. This leads to NETosis, induction of coagulation and N2 polarization, which prompts tumorigenesis, showing a novel link between coagulation, neutrophilia and complement activation. Finally, in a cohort of patients with small but not large intestinal cancer, we find a correlation between neutrophilia and hypercoagulation. This study provides a mechanistic explanation for the tumour-promoting effects of hypercoagulation, which could be used as a new biomarker or as a therapeutic target. PMID:26996437

  4. Extracellular entrapment and degradation of single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Farrera, Consol; Bhattacharya, Kunal; Lazzaretto, Beatrice; Andón, Fernando T.; Hultenby, Kjell; Kotchey, Gregg P.; Star, Alexander; Fadeel, Bengt

    2014-05-01

    Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials.Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials. Electronic supplementary information (ESI) available: Suppl. Fig. 1 - length distribution of SWCNTs; suppl. Fig. 2 - characterization of pristine vs. oxidized SWCNTs; suppl. Fig. 3 - endotoxin evaluation; suppl. Fig. 4 - NET characterization; suppl. Fig. 5 - UV-Vis/NIR analysis of biodegradation of oxidized SWCNTs; suppl. Fig. 6 - cytotoxicity of partially degraded SWCNTs. See DOI: 10.1039/c3nr06047k

  5. Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response

    PubMed Central

    Carlin, Aaron F.; Uchiyama, Satoshi; Chang, Yung-Chi; Lewis, Amanda L.; Nizet, Victor

    2009-01-01

    Human neutrophil Siglec-9 is a lectin that recognizes sialic acids (Sias) via an amino-terminal V-set Ig domain and possesses tyrosine-based inhibitory motifs in its cytoplasmic tail. We hypothesized that Siglec-9 recognizes host Sias as “self,” including in cis interactions with Sias on the neutrophil's own surface, thereby dampening unwanted neutrophil reactivity. Here we show that neutrophils presented with immobilized multimerized Siaα2-3Galβ1-4GlcNAc units engage them in trans via Siglec-9. The sialylated capsular polysaccharide of group B Streptococcus (GBS) also presents terminal Siaα2-3Galβ1-4GlcNAc units, and similarly engages neutrophil Siglec-9, dampening neutrophil responses in a Sia- and Siglec-9–dependent manner. Reduction in the neutrophil oxidative burst, diminished formation of neutrophil extracellular DNA traps, and increased bacterial survival are also facilitated by GBS sialylated capsular polysaccharide interactions with Siglec-9. Thus, GBS can impair neutrophil defense functions by coopting a host inhibitory receptor via sialoglycan molecular mimicry, a novel mechanism of bacterial immune evasion. PMID:19196661

  6. Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor.

    PubMed

    Carretta, M D; Hidalgo, A I; Burgos, J; Opazo, L; Castro, L; Hidalgo, M A; Figueroa, C D; Taubert, A; Hermosilla, C; Burgos, R A

    2016-08-01

    Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants. PMID:27288853

  7. Intracellular signalling during neutrophil recruitment.

    PubMed

    Mócsai, Attila; Walzog, Barbara; Lowell, Clifford A

    2015-08-01

    Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage. PMID:25998986

  8. Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

    PubMed

    Schaffer, Jessica N; Norsworthy, Allison N; Sun, Tung-Tien; Pearson, Melanie M

    2016-04-19

    The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder. PMID:27044107

  9. [PHENOTYPE OF PERIPHERAL BLOOD NEUTROPHILS IN THE INITIAL STAGE OF ENDOMETRIAL CANCER].

    PubMed

    Abakumova, T V; Antoneeva, I I; Gening, T P; Dolgova, D R; Gening, S O

    2016-01-01

    We have examined peripheral blood neutrophils from 123 patients with primary endometrial cancer at stage Ia. Receptor system and the ability of neutrophils to form extracellular traps were assessed by fluorescence microscopy, the spontaneous production of cytokines IL-2, IFN-γ, g-CSF, matrix metalloproteinases-1,9,13 by the method of enzyme-linked immunosorbent assay, phagocytic activity, myeloperoxidase activity, the level of cationic proteis activity in NBT-test were evaluated by cytochemical methods, activity of neutrophils in the spontaneous NBT-test was used to evaluate the oxygen-dependent bactericidal action of neutrophils. The topology and the rigidity of the membrane of neutrophils were assessed by scanning probe microscopy. We have shown that the increase in the relative number of neutrophils lead to a change in their receptor system, aerobic and anaerobic cytotoxicity and ability to phagocytosis are enchanced while reducing NET-activity. We have observed a change in the secretory activity of neutrophils, which is characterized by increased level of MMP-1, possibly initiated by enhanced production of reactive oxygen species, by a reduction in the IL-2 level (inductor of cytotoxic activity) and a sharp increase in the level of the G-CSF. Architectonics of neutrophils in the case of endonetrial cancer at stage Ia is characterized by changing the shape and loss of grit. The rigidity of the cell membrane decreased. Changes in the morphology of neutrophils on the background of the continuing hyperactivity suggests that a state of balance between the immune system and the tumor is already in stage Ia endometrial cancer. PMID:27220248

  10. Neutrophil Elastase Enhances Sputum Solubilization in Cystic Fibrosis Patients Receiving DNase Therapy

    PubMed Central

    Papayannopoulos, Venizelos; Staab, Doris; Zychlinsky, Arturo

    2011-01-01

    Cystic fibrosis patients suffer from chronic lung infection and inflammation due to the secretion of viscous sputum. Sputum viscosity is caused by extracellular DNA, some of which originates from the release of neutrophil extracellular traps (NETs). During NET formation neutrophil elastase (NE) partially processes histones to decondense chromatin. NE is abundant in CF sputum and is thought to contribute to tissue damage. Exogenous nucleases are a palliative treatment in CF as they promote sputum solubilization. We show that in a process reminiscent of NET formation, NE enhances sputum solubilization by cleaving histones to enhance the access of exogenous nucleases to DNA. In addition, we find that in Cf sputum NE is predominantly bound to DNA, which is known to downregulate its proteolytic activity and may restrict host tissue damage. The beneficial role of NE in CF sputum solubilization may have important implications for the development of CF therapies targeting NE. PMID:22174830

  11. TLR2 and neutrophils potentiate endothelial stress, apoptosis and detachment: implications for superficial erosion

    PubMed Central

    Quillard, Thibaut; Araújo, Haniel Alves; Franck, Gregory; Shvartz, Eugenia; Sukhova, Galina; Libby, Peter

    2015-01-01

    Aims Superficial erosion of atheromata causes many acute coronary syndromes, but arises from unknown mechanisms. This study tested the hypothesis that Toll-like receptor-2 (TLR2) activation contributes to endothelial apoptosis and denudation and thus contributes to the pathogenesis of superficial erosion. Methods and results Toll-like receptor-2 and neutrophils localized at sites of superficially eroded human plaques. In vitro, TLR2 ligands (including hyaluronan, a matrix macromolecule abundant in eroded lesions) induced endothelial stress, characterized by reactive oxygen species production, endoplasmic reticulum (ER) stress, and apoptosis. Co-incubation of neutrophils with endothelial cells (ECs) potentiated these effects and induced EC apoptosis and detachment. We then categorized human atherosclerotic plaques (n = 56) based on morphologic features associated with superficial erosion, ‘stable’ fibrotic, or ‘vulnerable’ lesions. Morphometric analyses of the human atheromata localized neutrophils and neutrophil extracellular traps (NETs) near clusters of apoptotic ECs in smooth muscle cell (SMC)-rich plaques. The number of luminal apoptotic ECs correlated with neutrophil accumulation, amount of NETs, and TLR2 staining in SMC-rich plaques, but not in ‘vulnerable’ atheromata. Conclusion These in vitro observations and analyses of human plaques indicate that TLR2 stimulation followed by neutrophil participation may render smooth muscle cell-rich plaques susceptible to superficial erosion and thrombotic complications by inducing ER stress, apoptosis, and favouring detachment of EC. PMID:25755115

  12. Dynamic NETosis is Carried Out by Live Neutrophils in Human and Mouse Bacterial Abscesses and During Severe Gram-Positive Infection

    PubMed Central

    Yipp, Bryan G.; Petri, Björn; Salina, Davide; Jenne, Craig N.; Scott, Brittney N. V.; Zbytnuik, Lori D.; Pittman, Keir; Asaduzzaman, Muhammad; Wu, Kaiyu; Meijndert, H. Christopher; Malawista, Stephen E.; de Boisfleury Chevance, Anne; Zhang, Kunyan; Conly, John; Kubes, Paul

    2013-01-01

    Neutrophil extracellular traps (NETs) are released, as neutrophils die in vitro, in a process requiring hours, leaving a temporal gap for invasive microbes to exploit. Functional neutrophils undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live PMN in vivo rapidly releasing NETs, which prevented bacterial dissemination. NETosis occurred during crawling thereby casting large areas of NETs. NET-releasing PMN developed diffuse decondensed nuclei ultimately becoming devoid of DNA. Cells with abnormal nuclei displayed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A combined requirement of Tlr2 and complement mediated opsonization tightly regulated NET release. Additionally live human PMN developed decondensed nuclei and formed NETS in vivo and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection, non-cell death NETosis occurs in vivo during Gram-positive infection in mice and humans. PMID:22922410

  13. Neutrophils trigger a NF-κB dependent polarization of tumor-supportive stromal cells in germinal center B-cell lymphomas.

    PubMed

    Grégoire, Murielle; Guilloton, Fabien; Pangault, Céline; Mourcin, Frédéric; Sok, Phaktra; Latour, Maelle; Amé-Thomas, Patricia; Flecher, Erwan; Fest, Thierry; Tarte, Karin

    2015-06-30

    Both tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific phenotypic and functional features and contribute to tumor cell niche. However, their bidirectional crosstalk has been poorly studied, in particular in the context of hematological malignancies. Follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL) are two germinal center-derived lymphomas where various cell components of infiltrating microenvironment, including TAN and CAFs, have been demonstrated to favor directly and indirectly malignant B-cell survival, growth, and drug resistance. We show here that, besides a direct and contact-dependent supportive effect of neutrophils on DLBCL B-cell survival, mediated through the BAFF/APRIL pathway, neutrophils and stromal cells cooperate to sustain FL B-cell growth. This cooperation relies on an overexpression of IL-8 by lymphoma-infiltrating stromal cells that could thereafter efficiently promote neutrophil survival and prime them to neutrophil extracellular trap. Conversely, neutrophils are able to activate stromal cells in a NF-κB-dependent manner, inducing their commitment towards an inflammatory lymphoid stroma phenotype associated with an increased capacity to trigger malignant B-cell survival, and to recruit additional monocytes and neutrophils through the release of CCL2 and IL-8, respectively. Altogether, a better understanding of the lymphoma-supporting effects of neutrophils could be helpful to design new anti-tumor therapeutic strategies. PMID:26158216

  14. Participation of dectin-1 receptor on NETs release against Paracoccidioides brasiliensis: Role on extracellular killing.

    PubMed

    Bachiega, Tatiana Fernanda; Dias-Melicio, Luciane Alarcão; Fernandes, Reginaldo Keller; de Almeida Balderramas, Helanderson; Rodrigues, Daniela Ramos; Ximenes, Valdecir Farias; de Campos Soares, Ângela Maria Victoriano

    2016-02-01

    Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse. PMID:26416210

  15. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

    PubMed Central

    von Brühl, Marie-Luise; Stark, Konstantin; Steinhart, Alexander; Chandraratne, Sue; Konrad, Ildiko; Lorenz, Michael; Khandoga, Alexander; Tirniceriu, Anca; Coletti, Raffaele; Köllnberger, Maria; Byrne, Robert A.; Laitinen, Iina; Walch, Axel; Brill, Alexander; Pfeiler, Susanne; Manukyan, Davit; Braun, Siegmund; Lange, Philipp; Riegger, Julia; Ware, Jerry; Eckart, Annekathrin; Haidari, Selgai; Rudelius, Martina; Schulz, Christian; Echtler, Katrin; Brinkmann, Volker; Schwaiger, Markus; Preissner, Klaus T.; Wagner, Denisa D.; Mackman, Nigel; Engelmann, Bernd

    2012-01-01

    Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features. PMID:22451716

  16. NETosis-associated serum biomarkers are reduced in type 1 diabetes in association with neutrophil count.

    PubMed

    Qin, J; Fu, S; Speake, C; Greenbaum, C J; Odegard, J M

    2016-06-01

    As the immune pathways involved in the pathogenesis of type 1 diabetes (T1D) are not fully understood, biomarkers implicating novel mechanisms of disease are of great interest and call for independent evaluation. Recently, it was reported that individuals with T1D display dramatic elevations in circulating components of neutrophil extracellular traps (NETs), indicating a potential role for NETosis in T1D. Our aim was to evaluate further the potential of NET-associated proteins as novel circulating biomarkers in T1D. We tested serum from subjects with T1D (n = 44) with a median age of 26·5 years and a median duration of 2·2 years, along with 38 age-matched controls. T1D subjects did not show elevations in either neutrophil elastase (NE) or proteinase 3 (PR3), as reported previously. In fact, both NE and PR3 levels were reduced significantly in T1D subjects, particularly in subjects within 3 years of diagnosis, consistent with the known reduction in neutrophil counts in recent-onset T1D. Indeed, levels of both NE and PR3 correlated with absolute neutrophil counts. Therefore, while not ruling out potential local or transient spikes in NETosis activity, the levels of these serum markers do not support a role for systemically elevated NETosis in the T1D population we studied. Rather, a modest reduction in these markers may reflect other important aspects of disease activity associated with reduced neutrophil numbers. PMID:26939803

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

    PubMed Central

    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

  18. Regulators and Effectors of Arf GTPases in Neutrophils

    PubMed Central

    Gamara, Jouda; Chouinard, François; Davis, Lynn; Aoudjit, Fawzi; Bourgoin, Sylvain G.

    2015-01-01

    Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology. PMID:26609537

  19. Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.

    PubMed

    Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J; Parker, Heather; Winterbourn, Christine C; Salvesen, Guy S; Drag, Marcin

    2014-02-18

    The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1-S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

  20. Neutrophil MiRNA-128-3p is Decreased During Active Phase of Granulo-matosis with Polyangiitis

    PubMed Central

    Surmiak, Marcin; Hubalewska-Mazgaj, Magdalena; Wawrzycka-Adamczyk, Katarzyna; Musiał, Jacek; Sanak, Marek

    2015-01-01

    Granulomatosis with polyangiitis is a rare chronic inflammatory disease. In this multisystem autoimmune disorder neutrophils cause small vessels necrosis and infiltrate perivascular tissue to form granulomas. Progression of the disease is evaluated by the symptoms score and by a titer of anti-neutrophil cytoplasm antibodies. Despite glucocorticoid and immunosuppressive therapy, prognosis is complicated by chronic renal insufficiency, hearing loss and skin ulceration. In this preliminary study we tested the hypothesis that altered neutrophil expression of miRNAs can contribute to the cell activation, extracellular traps formation and decreased apoptosis. First we compared a profile of 728 miRNAs expressed in circulating neutrophils of patients with active disease and matched healthy donors. Subsequently, candidate miRNAs were quantified in neutrophils from 16 subjects with active disease, 16 asymptomatic patients at the remission and in 16 healthy controls. Out of 11 candidate miRNAs, only miR-128-3p was both biologically (relative quantity < 30% control or remission patients) and statistically (p<0.01) decreased in the cells during active stage of the disease. This miRNA correlated with a clinical score of the disease well. A set of 10 transcripts involved in the mechanism of the disease was quantified from the same neutrophils RNA. Relative expression of MMP9 was higher in neutrophils from the patients with active disease and correlated negatively with miR-128-3p. The opposite finding was present for MTA1 transcripts. Despite surprisingly scarce changes in the expression of neutrophil miRNAs, miR-128-3p is the best candidate for deciphering etiology of granulomatosis with polyangiitis. PMID:27047256

  1. Neutrophil MiRNA-128-3p is Decreased During Active Phase of Granulo-matosis with Polyangiitis.

    PubMed

    Surmiak, Marcin; Hubalewska-Mazgaj, Magdalena; Wawrzycka-Adamczyk, Katarzyna; Musiał, Jacek; Sanak, Marek

    2015-10-01

    Granulomatosis with polyangiitis is a rare chronic inflammatory disease. In this multisystem autoimmune disorder neutrophils cause small vessels necrosis and infiltrate perivascular tissue to form granulomas. Progression of the disease is evaluated by the symptoms score and by a titer of anti-neutrophil cytoplasm antibodies. Despite glucocorticoid and immunosuppressive therapy, prognosis is complicated by chronic renal insufficiency, hearing loss and skin ulceration. In this preliminary study we tested the hypothesis that altered neutrophil expression of miRNAs can contribute to the cell activation, extracellular traps formation and decreased apoptosis. First we compared a profile of 728 miRNAs expressed in circulating neutrophils of patients with active disease and matched healthy donors. Subsequently, candidate miRNAs were quantified in neutrophils from 16 subjects with active disease, 16 asymptomatic patients at the remission and in 16 healthy controls. Out of 11 candidate miRNAs, only miR-128-3p was both biologically (relative quantity < 30% control or remission patients) and statistically (p<0.01) decreased in the cells during active stage of the disease. This miRNA correlated with a clinical score of the disease well. A set of 10 transcripts involved in the mechanism of the disease was quantified from the same neutrophils RNA. Relative expression of MMP9 was higher in neutrophils from the patients with active disease and correlated negatively with miR-128-3p. The opposite finding was present for MTA1 transcripts. Despite surprisingly scarce changes in the expression of neutrophil miRNAs, miR-128-3p is the best candidate for deciphering etiology of granulomatosis with polyangiitis. PMID:27047256

  2. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

    PubMed Central

    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

  3. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

    PubMed

    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

  4. Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry

    PubMed Central

    Björnsdottir, Halla; Welin, Amanda; Dahlgren, Claes; Karlsson, Anna; Bylund, Johan

    2015-01-01

    Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015) [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils. PMID:26862586

  5. A review of the proposed role of neutrophils in rodent amebic liver abscess models.

    PubMed

    Campos-Rodríguez, Rafael; Gutiérrez-Meza, Manuel; Jarillo-Luna, Rosa Adriana; Drago-Serrano, María Elisa; Abarca-Rojano, Edgar; Ventura-Juárez, Javier; Cárdenas-Jaramillo, Luz María; Pacheco-Yepez, Judith

    2016-01-01

    Host invasion by Entamoeba histolytica, the pathogenic agent of amebiasis, can lead to the development of amebic liver abscess (ALA). Due to the difficulty of exploring host and amebic factors involved in the pathogenesis of ALA in humans, most studies have been conducted with animal models (e.g., mice, gerbils, and hamsters). Histopathological findings reveal that the chronic phase of ALA in humans corresponds to lytic or liquefactive necrosis, whereas in rodent models there is granulomatous inflammation. However, the use of animal models has provided important information on molecules and mechanisms of the host/parasite interaction. Hence, the present review discusses the possible role of neutrophils in the effector immune response in ALA in rodents. Properly activated neutrophils are probably successful in eliminating amebas through oxidative and non-oxidative mechanisms, including neutrophil degranulation, the generation of free radicals (O2(-), H2O2, HOCl) and peroxynitrite, the activation of NADPH-oxidase and myeloperoxidase (MPO) enzymes, and the formation of neutrophil extracellular traps (NETs). On the other hand, if amebas are not eliminated in the early stages of infection, they trigger a prolonged and exaggerated inflammatory response that apparently causes ALAs. Genetic differences in animals and humans are likely to be key to a successful host immune response. PMID:26880421

  6. The role of neutrophils and NETosis in autoimmune and renal diseases.

    PubMed

    Gupta, Sarthak; Kaplan, Mariana J

    2016-07-01

    Systemic autoimmune diseases are a group of disorders characterized by a failure in self-tolerance to a wide variety of autoantigens. In genetically predisposed individuals, these diseases occur as a multistep process in which environmental factors have key roles in the development of abnormal innate and adaptive immune responses. Experimental evidence collected in the past decade suggests that neutrophils - the most abundant type of white blood cell - might have an important role in the pathogenesis of these diseases by contributing to the initiation and perpetuation of immune dysregulation through the formation of neutrophil extracellular traps (NETs), synthesis of proinflammatory cytokines and direct tissue damage. Many of the molecules externalized through NET formation are considered to be key autoantigens and might be involved in the generation of autoimmune responses in predisposed individuals. In several systemic autoimmune diseases, the imbalance between NET formation and degradation might increase the half-life of these lattices, which could enhance the exposure of the immune system to modified autoantigens and increase the capacity for NET-induced organ damage. This Review details the role of neutrophils and NETs in the pathophysiology of systemic autoimmune diseases, including their effect on renal damage, and discusses neutrophil targets as potential novel therapies for these diseases. PMID:27241241

  7. A review of the proposed role of neutrophils in rodent amebic liver abscess models

    PubMed Central

    Campos-Rodríguez, Rafael; Gutiérrez-Meza, Manuel; Jarillo-Luna, Rosa Adriana; Drago-Serrano, María Elisa; Abarca-Rojano, Edgar; Ventura-Juárez, Javier; Cárdenas-Jaramillo, Luz María; Pacheco-Yepez, Judith

    2016-01-01

    Host invasion by Entamoeba histolytica, the pathogenic agent of amebiasis, can lead to the development of amebic liver abscess (ALA). Due to the difficulty of exploring host and amebic factors involved in the pathogenesis of ALA in humans, most studies have been conducted with animal models (e.g., mice, gerbils, and hamsters). Histopathological findings reveal that the chronic phase of ALA in humans corresponds to lytic or liquefactive necrosis, whereas in rodent models there is granulomatous inflammation. However, the use of animal models has provided important information on molecules and mechanisms of the host/parasite interaction. Hence, the present review discusses the possible role of neutrophils in the effector immune response in ALA in rodents. Properly activated neutrophils are probably successful in eliminating amebas through oxidative and non-oxidative mechanisms, including neutrophil degranulation, the generation of free radicals (O2−, H2O2, HOCl) and peroxynitrite, the activation of NADPH-oxidase and myeloperoxidase (MPO) enzymes, and the formation of neutrophil extracellular traps (NETs). On the other hand, if amebas are not eliminated in the early stages of infection, they trigger a prolonged and exaggerated inflammatory response that apparently causes ALAs. Genetic differences in animals and humans are likely to be key to a successful host immune response. PMID:26880421

  8. CXCL1 Contributes to Host Defense in Polymicrobial Sepsis via Modulating T cell and Neutrophil Functions

    PubMed Central

    Liliang, Jin; Batra, Sanjay; Douda, David Nobuhiro; Palaniyar, Nades; Jeyaseelan, Samithamby

    2014-01-01

    Severe bacterial sepsis leads to a pro-inflammatory condition that can manifest as septic shock, multiple organ failure, and death. Neutrophils are critical for the rapid elimination of bacteria, however, the role of neutrophil chemoattractant CXCL1 in bacterial clearance during sepsis remains elusive. To test the hypothesis that CXCL1 is critical to host defense during sepsis. We used CXCL1-deficient mice and bone marrow chimeras to demonstrate the importance of this molecule in sepsis. We demonstrate that CXCL1 plays a pivotal role in mediating host defense to polymicrobial sepsis following cecal ligation and puncture (CLP) in gene-deficient mice. CXCL1 appears to be essential for restricting bacterial outgrowth and death in mice. CXCL1 derived from both hematopoietic and resident cells contributed to bacterial clearance. Moreover, CXCL1 is essential for neutrophil migration, expression of pro-inflammatory mediators, activation of Nuclear-Factor-κ-B (NF-κB) and Mitogen-Activated Protein (MAP) kinases and upregulation of adhesion molecule Intercellular Adhesion Molecule-1 (ICAM-1). Recombinant interleukin 17 (IL-17) rescued impaired host defenses in cxcl1−/− mice. CXCL1 is important for IL-17A production via Th17 differentiation. CXCL1 is essential for Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase-mediated reactive oxygen species production and neutrophil extracellular trap (NET) formation. This study reveals a novel role for CXCL1 in neutrophil recruitment via modulating T cell function and neutrophil-related bactericidal functions. These studies suggest that modulation of CXCL1 levels in tissues and blood could reduce bacterial burden in sepsis. PMID:25172493

  9. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

    SciTech Connect

    Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François

    2013-07-15

    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion

  10. Mechanotransduction in neutrophil activation and deactivation.

    PubMed

    Ekpenyong, Andrew E; Toepfner, Nicole; Chilvers, Edwin R; Guck, Jochen

    2015-11-01

    Mechanotransduction refers to the processes through which cells sense mechanical stimuli by converting them to biochemical signals and, thus, eliciting specific cellular responses. Cells sense mechanical stimuli from their 3D environment, including the extracellular matrix, neighboring cells and other mechanical forces. Incidentally, the emerging concept of mechanical homeostasis,long term or chronic regulation of mechanical properties, seems to apply to neutrophils in a peculiar manner, owing to neutrophils' ability to dynamically switch between the activated/primed and deactivated/deprimed states. While neutrophil activation has been known for over a century, its deactivation is a relatively recent discovery. Even more intriguing is the reversibility of neutrophil activation and deactivation. We review and critically evaluate recent findings that suggest physiological roles for neutrophil activation and deactivation and discuss possible mechanisms by which mechanical stimuli can drive the oscillation of neutrophils between the activated and resting states. We highlight several molecules that have been identified in neutrophil mechanotransduction, including cell adhesion and transmembrane receptors, cytoskeletal and ion channel molecules. The physiological and pathophysiological implications of such mechanically induced signal transduction in neutrophils are highlighted as a basis for future work. This article is part of a Special Issue entitled: Mechanobiology. PMID:26211453

  11. Hyperbaric Oxygen Reduces Production of Reactive Oxygen Species in Neutrophils from Polytraumatized Patients Yielding in the Inhibition of p38 MAP Kinase and Downstream Pathways

    PubMed Central

    Windolf, Joachim; Wahlers, Thorsten

    2016-01-01

    Trauma represents the leading cause of death among young people in western countries. Among the beneficial role of neutrophils in host defence, excessive priming and activation of neutrophils after major trauma lead to an overwhelming inflammatory response and secondary host tissue injury due to the release of toxic metabolites and enzymes. Hyperbaric oxygen (HBO) therapy has been proposed to possess antiinflammatory effects and might represent an appropriate therapeutic option to lower inflammation in a broad range of patients. Here, we studied the effects of HBO on the activity of neutrophils isolated from severely injured patients (days 1–2 after trauma), in fact on the production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs). We found exposure to HBO therapy to significantly diminish phorbol-12-myristate-13-acetate (PMA)-induced ROS production in neutrophils isolated from patients and healthy volunteers. At the same time, marked decrease in NETs release was found in control cells and a less pronounced reduction in patient neutrophils. Impaired ability to produce ROS following exposure to HBO was demonstrated to be linked to a strong downregulation of the activity of p38 MAPK. Only slight suppression of ERK activity could be found. In addition, HBO did not influence neutrophil chemotaxis or apoptosis, respectively. Collectively, this study shows for the first time that HBO therapy suppresses ROS production in inflammatory human neutrophils, and thus might impair ROS-dependent pathways, e.g. kinases activation and NETs release. Thus, HBO might represent a feasible therapy for patients suffering from systemic inflammation, including those with multiple trauma. PMID:27529549

  12. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae

    PubMed Central

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine

    2016-01-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  13. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

    PubMed

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine; Bachelerie, Françoise; Martin, Coralie

    2016-04-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  14. Hyperbaric Oxygen Reduces Production of Reactive Oxygen Species in Neutrophils from Polytraumatized Patients Yielding in the Inhibition of p38 MAP Kinase and Downstream Pathways.

    PubMed

    Grimberg-Peters, Deborah; Büren, Carina; Windolf, Joachim; Wahlers, Thorsten; Paunel-Görgülü, Adnana

    2016-01-01

    Trauma represents the leading cause of death among young people in western countries. Among the beneficial role of neutrophils in host defence, excessive priming and activation of neutrophils after major trauma lead to an overwhelming inflammatory response and secondary host tissue injury due to the release of toxic metabolites and enzymes. Hyperbaric oxygen (HBO) therapy has been proposed to possess antiinflammatory effects and might represent an appropriate therapeutic option to lower inflammation in a broad range of patients. Here, we studied the effects of HBO on the activity of neutrophils isolated from severely injured patients (days 1-2 after trauma), in fact on the production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs). We found exposure to HBO therapy to significantly diminish phorbol-12-myristate-13-acetate (PMA)-induced ROS production in neutrophils isolated from patients and healthy volunteers. At the same time, marked decrease in NETs release was found in control cells and a less pronounced reduction in patient neutrophils. Impaired ability to produce ROS following exposure to HBO was demonstrated to be linked to a strong downregulation of the activity of p38 MAPK. Only slight suppression of ERK activity could be found. In addition, HBO did not influence neutrophil chemotaxis or apoptosis, respectively. Collectively, this study shows for the first time that HBO therapy suppresses ROS production in inflammatory human neutrophils, and thus might impair ROS-dependent pathways, e.g. kinases activation and NETs release. Thus, HBO might represent a feasible therapy for patients suffering from systemic inflammation, including those with multiple trauma. PMID:27529549

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

    PubMed Central

    Paoliello-Paschoalato, Adriana Balbina; Marchi, Larissa Fávaro; de Andrade, Micássio Fernandes; Kabeya, Luciana Mariko; Donadi, Eduardo Antônio; Lucisano-Valim, Yara Maria

    2015-01-01

    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. PMID:26346244

  16. Presence of Citrullinated Histone H3-Positive Neutrophils in Microscopic Polyangiitis from the Early Phase: An Autopsy Proven Case.

    PubMed

    Matsuda, Yoko; Hamayasu, Hideki; Seki, Atsuko; Nonaka, Keisuke; Wang, Tan; Matsumoto, Takumi; Hamano, Yoshitomo; Sumikura, Hiroyuki; Kumasaka, Toshio; Murayama, Shigeo; Ishizu, Akihiko; Shimizu, Akira; Sugihara, Takahiko; Arai, Tomio

    2016-08-01

    A 76-year-old man was admitted with general fatigue, weight loss, fever, headache, renal failure, and a high serum level of myeloperoxidase-antineutrophil cytoplasmic antibody. Biopsy revealed citrullinated histone H3 (citH3)-positive neutrophils adherent to the temporal artery endothelium. Three days after completing pulse steroid therapy, he suffered from a sudden disturbance of consciousness and died. On autopsy, the kidneys showed the most severe vasculitis with dense infiltration of citH3-positive neutrophils. The lungs showed intra-alveolar hemorrhage due to capillaritis. Severe brain hemorrhage was found in the left frontal lobe and putamen with uncal herniation. No vasculitis or thrombi was observed in the brain. The right dura mater was thickened due to fibrosis and inflammation. In conclusion, autopsy revealed systemic vasculitis with infiltration of abundant citH3-positive neutrophils, suggesting that the neutrophil extracellular trap formation and citH3 might play important roles in the early phases and development of microscopic polyangiitis. PMID:27427341

  17. NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

    PubMed Central

    Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

    2016-01-01

    Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

  18. NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways.

    PubMed

    Yuen, Joshua; Pluthero, Fred G; Douda, David N; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H A; Palaniyar, Nades; Licht, Christoph

    2016-01-01

    Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b-9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their "AP tool kit" to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

  19. Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

    PubMed Central

    Dang, Guanghui; Cao, Jun; Cui, Yingying; Song, Ningning; Chen, Liping; Pang, Hai; Liu, Siguo

    2016-01-01

    Bacterial extracellular nucleases play important roles in virulence, biofilm formation, utilization of extracellular DNA as a nutrient, and degradation of neutrophil DNA extracellular traps. However, there is no current data available for extracellular nucleases derived from M. tuberculosis. Herein, we have identified and characterized Rv0888, an extracellular nuclease in M. tuberculosis. The protein was overexpressed in E. coli, and the purified Rv0888 protein was found to require divalent cations for activity, with an optimal temperature and pH of 41 °C and 6.5, respectively. Further results demonstrated that Rv0888 nuclease activity could be inhibited by four Chinese medicine monomers. Based on sequence analysis, Rv0888 nuclease exhibited no homology with any known extracellular nucleases, indicating that Rv0888 is a novel nuclease. Site-directed mutagenesis studies revealed that the H353, D387, and D438 residues play catalytic roles in Rv0888. In vivo infection studies confirmed that Rv0888 is required for infection and is related to pathogenicity, as the persistent ability of recombinant Mycobacterium smegmatis (rMS) Rv0888NS/MS and Rv0888S/MS is significantly higher than pMV262/MS in the lung tissue, and the Rv0888NS/MS and Rv0888S/MS could produce pathological changes in the mice lung. These results show that Rv0888 is relevant to pathogenicity of M. tuberculosis. PMID:26742696

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

    PubMed Central

    Hahn, Sinuhe; Giaglis, Stavros; Hoesli, Irene; Hasler, Paul

    2012-01-01

    The intention of this review is to provide an overview of the potential role of neutrophil extracellular traps (NETs) in mammalian reproduction. Neutrophil NETs appear to be involved in various stages of the reproductive cycle, starting with fertility and possibly ending with fetal loss. The first suggestion that NETs may play a role in pregnancy-related disorders was in preeclampsia, where vast numbers were detected in the intervillous space of affected placentae. The induction of NETosis involved an auto-inflammatory component, mediated by the increased release of placental micro-debris in preeclampsia. This report was the first indicating that NETs may be associated with a human pathology not involving infection. Subsequently, NETs have since then been implicated in bovine or equine infertility, in that semen may become entrapped in the female reproductive tract during their passage to the oocyte. In this instance interesting species-specific differences are apparent, in that equine sperm evade entrapment via expression of a DNAse-like molecule, whereas highly motile bovine sperm, once free from seminal plasma (SP) that promotes interaction with neutrophils, appear impervious to NETs entrapment. Although still in the realm of speculation it is plausible that NETs may be involved in recurrent fetal loss mediated by anti-phospholipid antibodies, or perhaps even in fetal abortion triggered by infections with microorganisms such as L. monocytogenes or B. abortus. PMID:23205021

  1. Reactive Oxygen Species and Neutrophil Function.

    PubMed

    Winterbourn, Christine C; Kettle, Anthony J; Hampton, Mark B

    2016-06-01

    Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling. PMID:27050287

  2. How neutrophils kill fungi.

    PubMed

    Gazendam, Roel P; van de Geer, Annemarie; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2016-09-01

    Neutrophils play a critical role in the prevention of invasive fungal infections. Whereas mouse studies have demonstrated the role of various neutrophil pathogen recognition receptors (PRRs), signal transduction pathways, and cytotoxicity in the murine antifungal immune response, much less is known about the killing of fungi by human neutrophils. Recently, novel primary immunodeficiencies have been identified in patients with a susceptibility to fungal infections. These human 'knock-out' neutrophils expand our knowledge to understand the role of PRRs and signaling in human fungal killing. From the studies with these patients it is becoming clear that neutrophils employ fundamentally distinct mechanisms to kill Candida albicans or Aspergillus fumigatus. PMID:27558342

  3. Granulopoiesis and granules of human neutrophils.

    PubMed

    Cowland, Jack B; Borregaard, Niels

    2016-09-01

    Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules and how degranulation of granule subsets is regulated. PMID:27558325

  4. ISOLATION OF MOUSE NEUTROPHILS

    PubMed Central

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

    2015-01-01

    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 19.6. 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. PMID:26237011

  5. Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis.

    PubMed

    Majumdar, Ritankar; Tavakoli Tameh, Aidin; Parent, Carole A

    2016-01-01

    Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments. PMID:26741884

  6. Cytoprotection against neutrophil-delivered oxidant attack by antibiotics.

    PubMed

    Ottonello, L; Dallegri, F; Dapino, P; Pastorino, G; Sacchetti, C

    1991-11-27

    In the present study we have investigated the effect of six antibiotics (penicillin G, ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) on the neutrophil cytolytic activity by using a system constituted of phorbol-12-myristate-13-acetate-triggered neutrophils and 51Cr-labelled lymphoblastoid Daudi target cells. The results demonstrate that five of these drugs (ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) are capable of inhibiting the neutrophil cytolytic activity by inactivating the hypochlorous acid (HOCl) generated extracellularly by the myeloperoxidase pathway and crucial to the target cell lysis. Penicillin G had no effect on neutrophil-mediated cytolysis. Thus, these data demonstrate that ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin lower the neutrophil-mediated target cell damage by a HOCl-scavenging mechanism, suggesting a possible cytoprotective role for these drugs during infections. PMID:1662510

  7. Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis

    PubMed Central

    Majumdar, Ritankar; Tavakoli Tameh, Aidin; Parent, Carole A.

    2016-01-01

    Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments. PMID:26741884

  8. Neutrophil lipoxygenase metabolism and adhesive function following acute thermal injury.

    PubMed

    Damtew, B; Marino, J A; Fratianne, R B; Spagnuolo, P J

    1993-02-01

    Leukotrienes, especially leukotriene B4, are important modulators of various neutrophil functions including adherence and chemotaxis. In previous work, we demonstrated that neutrophil adherence to extracellular matrixes was diminished in the acute stages of burn injury. In this study, we demonstrated that neutrophil adhesion to human and bovine endothelium in the baseline state and after stimulation with leukotriene B4 is depressed markedly after burn injury. The defect in stimulated adherence to endothelium was not specific to leukotriene B4 because impaired adhesion was observed with n-formyl-methionyl-leucyl-phenylalanine and ionophore A23187 as well. Moreover, the adherence defect correlated with 95% and 81% decreases in the release of leukotriene B4 and 5-hydroxy-(6E,87,117,147)-eicosatetraenoic acid, respectively, from burn PMN treated with A23187. Burn neutrophils also released proportionately more byproducts of leukotriene B4 omega oxidation, particularly 20-COOH-leukotriene B4, than did control neutrophils. When examined 3 1/2 weeks after injury, abnormalities in neutrophil leukotriene B4 generation and the adherence of burn neutrophils had recovered to near normal values. To determine whether the decreased release of leukotriene B4 from burn neutrophils was due to increased degradation or diminished synthesis of leukotriene B4, we examined the degradation of exogenous tritiated leukotriene B4 as well as the production of leukotriene B4 from tritiated arachidonic acid in neutrophils. Burn neutrophils converted significantly greater quantities of tritiated leukotriene B4 to tritiated 20-COOH-leukotriene B4 and synthesized markedly less tritiated leukotriene B4 from tritiated arachidonic acid than did control neutrophils, suggesting that decreased leukotriene B4 release by burn neutrophils was the result of both enhanced degradation and decreased synthesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8381849

  9. Neutrophilic dermatoses in children.

    PubMed

    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). PMID:18950391

  10. Plasmodium falciparum Infection Induces Expression of a Mosquito Salivary Protein (Agaphelin) That Targets Neutrophil Function and Inhibits Thrombosis without Impairing Hemostasis

    PubMed Central

    Waisberg, Michael; Molina-Cruz, Alvaro; Mizurini, Daniella M.; Gera, Nidhi; Sousa, Beatriz C.; Ma, Dongying; Leal, Ana C.; Gomes, Tainá; Kotsyfakis, Michalis; Ribeiro, José M. C.; Lukszo, Jan; Reiter, Karine; Porcella, Stephen F.; Oliveira, Carlo J.; Monteiro, Robson Q.; Barillas-Mury, Carolina; Pierce, Susan K.; Francischetti, Ivo M. B.

    2014-01-01

    Background Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear. Principal Findings Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (KD∼10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis. Conclusions Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in

  11. Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques

    PubMed Central

    Mattila, Joshua T.; Maiello, Pauline; Sun, Tao; Via, Laura E.; Flynn, JoAnne L.

    2015-01-01

    Summary The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis-infected cynomolgus macaques and humans to determine whether mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB-expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M. tuberculosis bacilli, M. tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme-mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme-linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro-inflammatory environment of granulomas up-regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB. PMID:25653138

  12. Correlation of trimethoprim and brodimoprim physicochemical and lipid membrane interaction properties with their accumulation in human neutrophils.

    PubMed Central

    Fresta, M; Furneri, P M; Mezzasalma, E; Nicolosi, V M; Puglisi, G

    1996-01-01

    Dipalmitoylphosphatidylcholine vesicles were used as a biological membrane model to investigate the interaction and the permeation properties of trimethoprim and brodimoprim as a function of drug protonation. The drug-membrane interaction was studied by differential scanning calorimetry. Both drugs interacted with the hydrophilic phospholipid head groups when in a protonated form. An experiment on the permeation of the two drugs through dipalmitoylphosphatidylcholine biomembranes showed higher diffusion rate constants when the two drugs were in the uncharged form; lowering of the pH (formation of protonated species) caused a reduction of permeation. Drug uptake by human neutrophil cells was also investigated. Both drugs may accumulate within neutrophils; however, brodimoprim does so to a greater extent. This accumulation is probably due to a pH gradient driving force, which allows the two drugs to move easily from the extracellular medium (pH approximately 7.3) into the internal cell compartments (acid pH). Once protonated, both drugs are less able to permeate and can be trapped by the neutrophils. This investigation showed the importance of the physicochemical properties of brodimoprim and trimethoprim in determining drug accumulation and membrane permeation pathways. PMID:9124856

  13. Neutrophils in cancer.

    PubMed

    Treffers, Louise W; Hiemstra, Ida H; Kuijpers, Taco W; van den Berg, Timo K; Matlung, Hanke L

    2016-09-01

    Neutrophils play an important role in cancer. This does not only relate to the well-established prognostic value of the presence of neutrophils, either in the blood or in tumor tissue, in the context of cancer progression or for the monitoring of therapy, but also to their active role in the progression of cancer. In the current review, we describe what is known in general about the role of neutrophils in cancer. What is emerging is a complex, rather heterogeneous picture with both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage. Furthermore, we will discuss the well-known role of neutrophils as myeloid-derived suppressor cells (MDSC), and also on the role of neutrophils as important effector cells during antibody therapy in cancer. It is clear that neutrophils contribute substantially to cancer progression in multiple ways, and this includes both direct effects on the cancer cells and indirect effect on the tumor microenvironment. While in many cases neutrophils have been shown to promote tumor progression, for instance by acting as MDSC, there are also protective effects, particularly when antibody immunotherapy is performed. A better understanding of the role of neutrophils is likely to provide opportunities for immunomodulation and for improving the treatment of cancer patients. PMID:27558343

  14. Neutrophil Elastase Modulates Cytokine Expression

    PubMed Central

    Benabid, Rym; Wartelle, Julien; Malleret, Laurette; Guyot, Nicolas; Gangloff, Sophie; Lebargy, François; Belaaouaj, Azzaq

    2012-01-01

    There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme. PMID:22927440

  15. Salmonella Transiently Reside in Luminal Neutrophils in the Inflamed Gut

    PubMed Central

    Loetscher, Yvonne; Wieser, Andreas; Lengefeld, Jette; Kaiser, Patrick; Schubert, Sören; Heikenwalder, Mathias; Hardt, Wolf-Dietrich; Stecher, Bärbel

    2012-01-01

    Background Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model. Results Upon S. Tmwt infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This “fast cycling” through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut. Conclusion In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut. PMID:22493718

  16. [Neutrophilic functional heterogeneity].

    PubMed

    2006-02-01

    Blood neutrophilic functional heterogeneity is under discussion. The neutrophils of one subpopulation, namely killer neutrophils (Nk), potential phagocytes, constitute a marginal pool and a part of the circulating pool, intensively produce active oxygen forms (AOF) and they are adherent to the substrate. The neutrophils of another subpopulation, cager neutrophils (Nc), seem to perform a transport function of delivering foreign particles to the competent organs, to form about half of the circulating pool, to produce APC to a lesser extent, exclusively for self-defense and, probably, in usual conditions, to fail to interact with substrate. Analysis of the experimental findings suggests that the phylogenetic age of Nk is older than that of Nc and Nk has predominantly a tendency to spontaneous apoptosis under physiological conditions. PMID:16610631

  17. Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils.

    PubMed

    Kuckleburg, Christopher J; Tilkens, Sarah B; Santoso, Sentot; Newman, Peter J

    2012-03-01

    Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population. PMID:22266279

  18. Neutrophil kinetics in man.

    PubMed Central

    Dancey, J T; Deubelbeiss, K A; Harker, L A; Finch, C A

    1976-01-01

    A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5 +/- 0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07 +/- 0.84 X 10(9) cells/kg. Total marrow neutrophils (X 10(9) cells/kg) were 7.70 +/- 1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59 +/- 0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11 +/- 0.36. Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59 +/- 0.90 X 10(9) neutrophils/kg, and the transit time was 6.60 +/- 0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10(9) cells/kg per day. Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87 +/- 0.13 X 10(9) cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62 +/- 0.46 X 10(9) cells/kg per day was obtained when diisopropylfluorophosphate-32P was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-32P demonstrated a lower recovery and shorter t1/2 of the 32P label. Images PMID:956397

  19. Chlorination of Taurine by Human Neutrophils

    PubMed Central

    Weiss, Stephen J.; Klein, Roger; Slivka, Adam; Wei, Maria

    1982-01-01

    The model hydrogen peroxide-myeloperoxidase-chloride system is capable of generating the powerful oxidant hypochlorous acid, which can be quantitated by trapping the generated species with the β-amino acid, taurine. The resultant stable product, taurine chloramine, can be quantitated by its ability to oxidize the sulfhydryl compound, 5-thio-2-nitro-benzoic acid to the disulfide, 5,5′-dithiobis(2-nitroben-zoic acid) or to oxidize iodide to iodine. Using this system, purified myeloperoxidase in the presence of chloride and taurine converted stoichiometric quantities of hydrogen peroxide to taurine chloramine. Chloramine generation was absolutely dependent on hydrogen peroxide, myeloperoxidase, and chloride and could be inhibited by catalase, myeloperoxidase inhibitors, or chloride-free conditions. In the presence of taurine, intact human neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan particles generated a stable species capable of oxidizing 5-thio-2-nitrobenzoic acid or iodide. Resting cells did not form this species. The oxidant formed by the stimulated neutrophils was identified as taurine chloramine by both ultraviolet spectrophotometry and electrophoresis. Taurine chloramine formation by the neutrophil was dependent on the taurine concentration, time, and cell number. Neutrophil-dependent chloramine generation was inhibited by catalase, the myeloperoxidase inhibitors, azide, cyanide, or aminotriazole and by chloride-free conditions, but not by superoxide dismutase or hydroxyl radical scavengers. Thus, it appears that stimulated human neutrophils can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate taurine chloramine. Based on the demonstrated ability of the myeloperoxidase system to generate free hypochlorous acid we conclude that neutrophils chlorinate taurine by producing this powerful oxidant. The biologic reactivity and cytotoxic potential of hypochlorous acid and its chloramine derivatives

  20. Social amoebae trap and kill bacteria by casting DNA nets.

    PubMed

    Zhang, Xuezhi; Zhuchenko, Olga; Kuspa, Adam; Soldati, Thierry

    2016-01-01

    Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans. PMID:26927887

  1. Social amoebae trap and kill bacteria by casting DNA nets

    PubMed Central

    Zhang, Xuezhi; Zhuchenko, Olga; Kuspa, Adam; Soldati, Thierry

    2016-01-01

    Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans. PMID:26927887

  2. The lymph node neutrophil.

    PubMed

    Hampton, Henry R; Chtanova, Tatyana

    2016-04-01

    Secondary lymphoid organs provide a specialized microenvironment tailored to foster communication between cells of the innate and adaptive immune systems. These interactions allow immune cells to coordinate multilayered defense against pathogens. Until recently dendritic cells and macrophages were thought to comprise the main innate immune cell subsets responsible for delivering signals that drive the adaptive immune response, while the function of neutrophils was largely confined to the innate immune system. However, the discovery of neutrophils in lymph nodes has raised the question of whether neutrophils might play a more extensive role not only in innate immunity per se, but also in coordinating the interactions between innate and adaptive immune responses. In this review we discuss the mechanisms and consequences of neutrophil recruitment to lymph nodes and how this recruitment influences subsequent immune responses both in situ and at distant sites. PMID:27025975

  3. Neutrophils in cystic fibrosis.

    PubMed

    Laval, Julie; Ralhan, Anjali; Hartl, Dominik

    2016-06-01

    Cystic fibrosis (CF) lung disease is characterized by chronic infection and inflammation. Among inflammatory cells, neutrophils represent the major cell population accumulating in the airways of CF patients. While neutrophils provide the first defensive cellular shield against bacterial and fungal pathogens, in chronic disease conditions such as CF these short-lived immune cells release their toxic granule contents that cause tissue remodeling and irreversible structural damage to the host. A variety of human and murine studies have analyzed neutrophils and their products in the context of CF, yet their precise functional role and therapeutic potential remain controversial and incompletely understood. Here, we summarize the current evidence in this field to shed light on the complex and multi-faceted role of neutrophils in CF lung disease. PMID:26854289

  4. Neutrophil swarming: an essential process of the neutrophil tissue response.

    PubMed

    Kienle, Korbinian; Lämmermann, Tim

    2016-09-01

    Neutrophil infiltration into inflamed and infected tissues is a fundamental process of the innate immune response. While neutrophil interactions with the blood vessel wall have been intensely studied over the last decades, neutrophil dynamics beyond the vasculature have for a long time remained poorly investigated. Recent intravital microscopy studies of neutrophil populations directly at the site of tissue damage or microbial invasion have changed our perspective on neutrophil responses within tissues. Swarm-like migration patterns of neutrophils, referred to as 'neutrophil swarming', have been detected in diverse tissues under conditions of sterile inflammation and infection with various pathogens, including bacteria, fungi, and parasites. Current work has begun to unravel the molecular pathways choreographing the sequential phases of highly coordinated chemotaxis followed by neutrophil accumulation and the formation of substantial neutrophil clusters. It is now clear that intercellular communication among neutrophils amplifies their recruitment in a feed-forward manner, which provides them with a level of self-organization during neutrophil swarming. This review will summarize recent developments and current concepts on neutrophil swarming, an important process of the neutrophil tissue response with a critical role in maintaining the balance between host protection and inflammation-driven tissue destruction. PMID:27558329

  5. The Essential Role of Neutrophils during Infection with the Intracellular Bacterial Pathogen Listeria monocytogenes.

    PubMed

    Witter, Alexandra R; Okunnu, Busola M; Berg, Rance E

    2016-09-01

    Neutrophils have historically been characterized as first responder cells vital to host survival because of their ability to contain and eliminate bacterial and fungal pathogens. However, recent studies have shown that neutrophils participate in both protective and detrimental responses to a diverse array of inflammatory and infectious diseases. Although the contribution of neutrophils to extracellular infections has been investigated for decades, their specific role during intracellular bacterial infections has only recently been appreciated. During infection with the Gram-positive intracellular pathogen Listeria monocytogenes, neutrophils are recruited from the bone marrow to sites of infection where they use novel bacterial-sensing pathways leading to phagocytosis and production of bactericidal factors. This review summarizes the requirement of neutrophils during L. monocytogenes infection by examining both neutrophil trafficking and function during primary and secondary infection. PMID:27543669

  6. On the pharmacology of oxidative burst of human neutrophils.

    PubMed

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

    2015-01-01

    The effect of three therapeutically used drugs and five polyphenolic compounds on the mechanism of oxidative burst was compared in whole blood and isolated neutrophils at cellular and molecular level. In 10 microM concentration, the compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin (N-f-5HT) > curcumin (CUR) > quercetin (QUER) > arbutin (ARB) > resveratrol (RES) > dithiaden (DIT) > carvedilol (CARV) > brompheniramine (BPA). The ratio between the percentage inhibition of extracellular versus intracellular chemiluminescence (CL) followed the rank order QUER > N-f-5HT > RES > CUR > DIT and is indicative of the positive effect of the compounds tested against oxidative burst of neutrophils, demonstrating suppression of reactive oxygen species extracellularly with minimal alteration of intracellular reactive oxygen species (ROS). Activation of protein kinase C was significantly decreased by DIT, CUR, QUER and N-f-5HT. CARV, DIT, QUER and ARB reduced activated neutrophil myeloperoxidase release more significantly compared with the effect on superoxide anion generation. All compounds tested increased the activity of caspase-3 in cell-free system. It is suggested that other regulatory mechanisms than protein kinase C might participate in the inhibition of neutrophil activation with the compounds tested. Different mechanisms are concerned in controlling the assembly of NADPH oxidase and the regulatory role of calcium ions is suggested. Compounds decreasing the amount of extracellular ROS generation, yet affecting but minimally intracellular ROS generation, are promising for further investigation in vivo. PMID:26681073

  7. Role of extracellular calcium in in vitro uptake and intraphagocytic location of macrolides.

    PubMed Central

    Mtairag, E M; Abdelghaffar, H; Douhet, C; Labro, M T

    1995-01-01

    We compared the uptakes and intracellular locations of four 14-membered-ring macrolides (roxithromycin, dirithromycin, erythromycin, and erythromycylamine) in human polymorphonuclear neutrophils (PMNs) in vitro. Intracellular location was assessed by cell fractionation and uptake kinetics in cytoplasts (granule-poor PMNs). Trapping of dirithromycin within PMN granules (up to 80% at 30 min) was significantly more marked than the intracellular trapping of the other drugs (erythromycylamine, 45% +/- 5.1%; erythromycin, 42% +/- 3.7%; roxithromycin, 35% +/- 3.0%). A new finding was that, in the absence of extracellular calcium, the uptakes of all of the macrolides by PMNs and cytoplasts were significantly impaired, by about 50% (PMN) and 90% (cytoplasts). Furthermore, inorganic Ca2+ channel blockers inhibited macrolide uptake in a concentration-dependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 microM, respectively, for Ni2+ and La3+. The intracellular distributions of the drugs were unchanged in the presence of Ni2+ and La3+ and in Ca(2+)-free medium supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The organic Ca2+ channel blocker nifedipine had no effect on macrolide uptake, whereas verapamil inhibited it in a time- and concentration-dependent manner. These data show the importance of extracellular Ca2+ in macrolide uptake by phagocytes and suggest a link with Ca2+ channels or a Ca2+ channel-operated mechanism. PMID:7486899

  8. Extracellular respiration

    PubMed Central

    Gralnick, Jeffrey A.; Newman, Dianne K.

    2009-01-01

    Summary Although it has long been known that microbes can generate energy using diverse strategies, only recently has it become clear that a growing number involve electron transfer to or from extracellular substrates. The best-known example of what we will term ‘extracellular respiration’ is electron transfer between microbes and minerals, such as iron and manganese (hydr)oxides. This makes sense, given that these minerals are sparingly soluble. What is perhaps surprising, however, is that a number of substrates that might typically be classified as ‘soluble’ are also respired at the cell surface. There are several reasons why this might be the case: the substrate, in its ecological context, might be associated with a solid surface and thus effectively insoluble; the substrate, while soluble, might simply be too large to transport inside the cell; or the substrate, while benign in one redox state, might become toxic after it is metabolized. In this review, we discuss various examples of extracellular respiration, paying particular attention to what is known about the molecular mechanisms underlying these processes. As will become clear, much remains to be learned about the biochemistry, cell biology and regulation of extracellular respiration, making it a rich field of study for molecular microbiologists. PMID:17581115

  9. Platelets enhance neutrophil transendothelial migration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflame...

  10. The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion

    PubMed Central

    Fiddler, Christine A.; Parfrey, Helen; Cowburn, Andrew S.; Luo, Ding; Nash, Gerard B.; Murphy, Gillian; Chilvers, Edwin R.

    2016-01-01

    Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo. PMID:27467268

  11. Stimulus-dependent secretion of plasma proteins from human neutrophils.

    PubMed Central

    Borregaard, N; Kjeldsen, L; Rygaard, K; Bastholm, L; Nielsen, M H; Sengeløv, H; Bjerrum, O W; Johnsen, A H

    1992-01-01

    In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli. Images PMID:1378856

  12. Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment

    PubMed Central

    Kim, Jaehong; Bae, Jong-Sup

    2016-01-01

    Distinct tumor microenvironment forms in each progression step of cancer and has diverse capacities to induce both adverse and beneficial consequences for tumorigenesis. It is now known that immune cells can be activated to favor tumor growth and progression, most probably influenced by the tumor microenvironment. Tumor-associated macrophages and tumor-associated neutrophils can exert protumoral functions, enhancing tumor cell invasion and metastasis, angiogenesis, and extracellular matrix remodeling, while inhibiting the antitumoral immune surveillance. Considering that neutrophils in inflammatory environments recruit macrophages and that recruited macrophages affect neutrophil functions, there may be various degrees of interaction between tumor-associated macrophages and tumor-associated neutrophils. Platelets also play an important role in the recruitment and regulation of monocytic and granulocytic cells in the tumor tissues, suggesting that platelet function may be essential for generation of tumor-associated macrophages and tumor-associated neutrophils. In this review, we will explore the biology of tumor-associated macrophages and tumor-associated neutrophils and their possible interactions in the tumor microenvironment. Special attention will be given to the recruitment and activation of these tumor-associated cells and to the roles they play in maintenance of the tumor microenvironment and progression of tumors. PMID:26966341

  13. [Leukemic neutrophilic dermatosis].

    PubMed

    Török, L; Kirschner, A; Gurzó, M; Krenács, L

    1999-03-28

    A case of a 67 year-old female patient with acute myeloid leukemia is presented. As the first manifestation of the disease, the patient had symptoms of Sweet's syndrome, later signs of gangrenous pyoderma have developed. This transient form is termed as a "leukemic neutrophilic dermatosis". The authors focus on the important diagnostic and prognostic value of this entity. PMID:10349319

  14. DAMP and DIC: The role of extracellular DNA and DNA-binding proteins in the pathogenesis of DIC.

    PubMed

    Liaw, Patricia C; Ito, Takashi; Iba, Toshiaki; Thachil, Jecko; Zeerleder, Sacha

    2016-07-01

    Disseminated intravascular coagulation (DIC) is a heterogeneous group of disorders, which manifest as a spectrum of haemorrhage and thrombosis complicating many primary conditions including sepsis, trauma and malignancies. The pathophysiology of this condition is complex. In the recent years there is growing evidence that damage associated molecular patterns (DAMPs) play a crucial role in the pathogenesis of DIC. Upon cell-death and/or cell activation of hematopoietic and parenchymal cells extracellular cell-free DNA as well as DNA binding proteins (e.g. histones and high mobility group box 1 protein [HMGB1]) are released into circulation. This release is a highly regulated process mediated among others by serine proteases, such as factor VII-activating protease (FSAP) and DNase1. Circulating cell-free DNA has been demonstrated to influence primary and secondary hemostasis by inducing platelet aggregation, promoting coagulation activation, inhibition of fibrinolysis and directly interfering with clot stability. In this respect cell-free DNA in tissue as well as released into the circulation after neutrophil activation in the form of neutrophil extracellular traps (NETs) has been shown to be cytotoxic and highly procoagulant. DNA-binding proteins such as histones and HMGB1 are also strongly procoagulant and are involved in the pathogenesis of DIC. The present review gives an overview on how extracellular DNA is released into circulation and the structure of circulating DNA. In addition it summarizes the effect of extracellular DNA and DNA-binding proteins on platelet activation, plasmatic coagulation as well as fibrinolysis. PMID:26776504

  15. Lundep, a Sand Fly Salivary Endonuclease Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma

    PubMed Central

    Chagas, Andrezza C.; Oliveira, Fabiano; Debrabant, Alain; Valenzuela, Jesus G.; Ribeiro, José M. C.; Calvo, Eric

    2014-01-01

    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. PMID:24516388

  16. Human neutrophil elastase: mediator and therapeutic target in atherosclerosis.

    PubMed

    Henriksen, Peter A; Sallenave, Jean-Michel

    2008-01-01

    Human neutrophil elastase (HNE) is present within atherosclerotic plaques where it contributes to matrix degradation and weakening of the vessel wall associated with the complications of aneurysm formation and plaque rupture. It is joined by other extracellular proteases in these actions but the broad range of substrates and potency of HNE coupled with the potential for rapid increases in HNE activity associated with neutrophil degranulation in acute coronary syndromes single this disruptive protease out as therapeutic target in atherosclerotic disease. This review summarises the role of HNE in neutrophil-mediated endothelial injury and the evidence for HNE as a mediator of atherosclerotic plaque development. The therapeutic potential of HNE neutralising antiproteases, alpha-1-antitrypsin and elafin, in atherosclerosis, is discussed. PMID:18289916

  17. Sulphonamides as anti-inflammatory agents: old drugs for new therapeutic strategies in neutrophilic inflammation?

    PubMed

    Ottonello, L; Dapino, P; Scirocco, M C; Balbi, A; Bevilacqua, M; Dallegri, F

    1995-03-01

    1. It is well known that neutrophils act as mediators of tissue injury in a variety of inflammatory diseases. Their histotoxic activity is presently thought to involve proteinases and oxidants, primarily hypochlorous acid (HOCl). This oxidant is also capable of inactivating the specific inhibitor of neutrophil elastase (alpha 1-antitrypsin), thereby favouring digestion of the connective matrix. 2. In the present work, we found that sulphanilamide and some sulphanilamide-related anti-inflammatory drugs such as dapsone, nimesulide and sulphapyridine reduce the availability of HOCl in the extracellular microenvironment of activated neutrophils and prevent the inactivation of alpha 1-antitrypsin by these cells in a dose-dependent manner. The ability of each drug to prevent alpha 1-antitrypsin from inactivation by neutrophils correlates significantly with its capacity to reduce the recovery of HOCl from neutrophils. Five other non-steroidal anti-inflammatory drugs were completely ineffective. 3. Therefore, sulphanilamide-related drugs, i.e. dapsone, nimesulide and sulphapyridine, have the potential to reduce the bioavailability of neutrophil-derived HOCl and, in turn, to favour the alpha 1-antitrypsin-dependent control of neutrophil elastolytic activity. These drugs appear as a well-defined group of agents which are particularly prone to attenuate neutrophil histotoxicity. They can also be viewed as a previously unrecognized starting point for the development of new compounds in order to plan rational therapeutic strategies for controlling tissue injury during neutrophilic inflammation. PMID:7736703

  18. Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase.

    PubMed Central

    English, D; Martin, M; Harvey, K A; Akard, L P; Allen, R; Widlanski, T S; Garcia, J G; Siddiqui, R A

    1997-01-01

    Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 degrees C and pH7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1. 4x10(-3) M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of

  19. TREM-like transcript 2 is stored in human neutrophil primary granules and is up-regulated in response to inflammatory mediators.

    PubMed

    Thomas, Kimberly A; King, R Glenn; Sestero, Christine M; Justement, Louis B

    2016-07-01

    The triggering receptor expressed on myeloid cell locus encodes a family of receptors that is emerging as an important class of molecules involved in modulating the innate immune response and inflammation. Of the 4 conserved members, including triggering receptor expressed on myeloid cells 1 and 2 and triggering receptor expressed on myeloid cell-like transcripts 1 and 2, relatively little is known about triggering receptor expressed on myeloid cell-like transcript 2 expression and function, particularly in humans. In this study, experiments were performed to determine if triggering receptor expressed on myeloid cell-like transcript 2 expression is conserved between mouse and human, demonstrating that human triggering receptor expressed on myeloid cell-like transcript 2 is expressed on cells of the lymphoid, as well as myeloid/granuloid lineages, similar to murine triggering receptor expressed on myeloid cell-like transcript 2. Consistent with studies in the mouse, triggering receptor expressed on myeloid cell-like transcript 2 expression is up-regulated in response to inflammatory mediators on human neutrophils. Importantly, it was shown that triggering receptor expressed on myeloid cell-like transcript 2, in resting human neutrophils, is predominantly localized to intracellular vesicles, including secretory vesicles and primary granules; with the majority of triggering receptor expressed on myeloid cell-like transcript 2 stored in primary granules. In contrast to other primary granule proteins, triggering receptor expressed on myeloid cell-like transcript 2 is not expelled on neutrophil extracellular traps but is retained in the plasma membrane following primary granule exocytosis. In summary, these findings establish that triggering receptor expressed on myeloid cell-like transcript 2 expression is conserved between species and is likely to be important in regulating neutrophil antimicrobial function following primary granule exocytosis. PMID:26753760

  20. Transformation of lupus-inducing drugs to cytotoxic products by activated neutrophils.

    PubMed

    Jiang, X; Khursigara, G; Rubin, R L

    1994-11-01

    Drug-induced lupus is a serious side effect of certain medications, but the chemical features that confer this property and the underlying pathogenesis are puzzling. Prototypes of all six therapeutic classes of lupus-inducing drugs were highly cytotoxic only in the presence of activated neutrophils. Removal of extracellular hydrogen peroxide before, but not after, exposure of the drug to activated neutrophils prevented cytotoxicity. Neutrophil-dependent cytotoxicity required the enzymatic action of myeloperoxidase, resulting in the chemical transformation of the drug to a reactive product. The capacity of drugs to serve as myeloperoxidase substrates in vitro was associated with the ability to induce lupus in vivo. PMID:7973636

  1. Osmotically induced cytosolic free Ca(2+) changes in human neutrophils.

    PubMed

    Morris, M R; Doull, I J; Hallett, M B

    2001-02-01

    Cytosolic free Ca(2+) concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300-600 mM) or sucrose (600-1200 mM), caused a rise in cytosolic free Ca(2+) (Delta(max) approximately equal to 600 nM). This was not due to cell lysis as the cytosolic free Ca(2+) concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca(2+) could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca(2+) concentration occurred in the absence of extracellular Ca(2+), demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca(2+) was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca(2+) may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca(2+) (Delta(max) approximately equal to 600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca(2+) to physiologically important levels. PMID:11341979

  2. Neutrophil-derived resistin release induced by Aggregatibacter actinomycetemcomitans.

    PubMed

    Furugen, Reiko; Hayashida, Hideaki; Yoshii, Yumiko; Saito, Toshiyuki

    2011-08-01

    Resistin is an adipokine that induces insulin resistance in mice. In humans, resistin is not produced in adipocytes, but in various leukocytes instead, and it acts as a proinflammatory molecule. The present investigation demonstrated high levels of resistin in culture supernatants of neutrophils that are stimulated by a highly leukotoxic strain of Aggregatibacter actinomycetemcomitans. In contrast, the level of resistin was remarkably low when neutrophils were exposed to two other strains that produce minimal levels of leukotoxin and a further isogenic mutant strain incapable of producing leukotoxin. Pretreatment of neutrophils with a monoclonal antibody to CD18, β chain of lymphocyte function-associated molecule 1 (LFA-1), or an Src family tyrosine kinase inhibitor before incubation with the highly leukotoxic strain inhibited the release of resistin. These results show that A. actinomycetemcomitans-expressed leukotoxin induces extracellular release of human neutrophil-derived resistin by interacting with LFA-1 on the surface of neutrophils and, consequently, activating Src family tyrosine kinases. PMID:21658109

  3. Sulforaphane Restores Cellular Glutathione Levels and Reduces Chronic Periodontitis Neutrophil Hyperactivity In Vitro

    PubMed Central

    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. PMID:23826097

  4. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis.

    PubMed

    Gonzalez, Anjelica L; El-Bjeirami, Wafa; West, Jennifer L; McIntire, Larry V; Smith, C Wayne

    2007-03-01

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination. RGDS is a bioactive sequence found in a number of proteins, and P2 is a membrane-activated complex-1 (Mac-1) ligand located in the gamma-chain of the fibrinogen protein. We evaluated, via video microscopy, cell motility by measuring cell displacement from origin and total accumulated distance traveled and then calculated average velocity. Results indicate that although adhesion and shape change were supported by hydrogels containing RGD alone, motility was not. Mac-1-dependent motility was supported on hydrogels containing P2 alone. Motility was enhanced through combined presentation of RGD and P2, engaging Mac-1, alpha(V)beta(3), and beta(1) integrins. Naïve neutrophil motility on combined peptide substrates was dependent on Mac-1, and alpha(4)beta(1) while alpha(6)beta(1) contributed to speed and linear movement. Transmigrated neutrophil motility was dependent on alpha(v)beta(3) and alpha(5)beta(1), and alpha(4)beta(1), alpha(6)beta(1), and Mac-1 contributed to speed and linear motion. Together, the data demonstrate that efficient neutrophil migration, dependent on multi-integrin interaction, is enhanced after transendothelial migration. PMID:17164427

  5. Endothelin-1 downregulates sperm phagocytosis by neutrophils in vitro: A physiological implication in bovine oviduct immunity

    PubMed Central

    MAREY, Mohamed Ali; YOUSEF, Mohamed Samy; LIU, Jinghui; MORITA, Kazuhiro; SASAKI, Motoki; HAYAKAWA, Hiroyuki; SHIMIZU, Takashi; ELSHAHAWY, Ibrahim I.; MIYAMOTO, Akio

    2016-01-01

    The oviduct is an active contractile tube that provides the proper environment for sperm transport, capacitation and survival. Oviductal contractions are regulated by autocrine/paracrine secretion of several factors, such as prostaglandins (PGs) and endothelin-1 (EDN-1). We have previously shown that during the preovulatory stage, sperm are exposed to polymorphonuclear neutrophils (PMNs) in the bovine oviduct, and the bovine oviduct epithelial cells (BOECs) secrete molecules including PGE2 that suppress sperm phagocytosis by PMNs in vitro. In this study, we investigated the possible effects of EDN-1 on the phagocytic activity of PMNs toward sperm. The local concentrations of EDN-1 in oviduct fluid and BOEC culture medium ranged from 10–10 to 10–11 M as determined by EIA. Phagocytosis and superoxide production were assayed by co-incubation of sperm pretreated to induce capacitation with PMNs exposed to EDN-1 (0, 10–11, 10–10, 10–9, and 10–8 M) for 2 h. EDN-1 suppressed dose dependently (10–11 to 10–8 M) the phagocytic activity for sperm and superoxide production of PMNs in response to capacitated sperm. Moreover, this suppression was eliminated by an ETB receptor antagonist (BQ-788). EDN-1 suppressed mRNA expression of EDN-1 and ETB but not ETA receptors in PMNs, suggesting the ETB receptor-mediated pathway. Scanning electron microscopic observation revealed that incubation of PMNs with EDN-1 (10–9 M) completely suppressed the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results provide evidence indicating that EDN-1 may be involved in the protection of sperm from phagocytosis by PMNs in the bovine oviduct, supporting sperm survival until fertilization. PMID:26781611

  6. Endothelin-1 downregulates sperm phagocytosis by neutrophils in vitro: A physiological implication in bovine oviduct immunity.

    PubMed

    Marey, Mohamed Ali; Yousef, Mohamed Samy; Liu, Jinghui; Morita, Kazuhiro; Sasaki, Motoki; Hayakawa, Hiroyuki; Shimizu, Takashi; Elshahawy, Ibrahim I; Miyamoto, Akio

    2016-04-22

    The oviduct is an active contractile tube that provides the proper environment for sperm transport, capacitation and survival. Oviductal contractions are regulated by autocrine/paracrine secretion of several factors, such as prostaglandins (PGs) and endothelin-1 (EDN-1). We have previously shown that during the preovulatory stage, sperm are exposed to polymorphonuclear neutrophils (PMNs) in the bovine oviduct, and the bovine oviduct epithelial cells (BOECs) secrete molecules including PGE2 that suppress sperm phagocytosis by PMNs in vitro. In this study, we investigated the possible effects of EDN-1 on the phagocytic activity of PMNs toward sperm. The local concentrations of EDN-1 in oviduct fluid and BOEC culture medium ranged from 10(-10) to 10(-11) M as determined by EIA. Phagocytosis and superoxide production were assayed by co-incubation of sperm pretreated to induce capacitation with PMNs exposed to EDN-1 (0, 10(-11), 10(-10), 10(-9), and 10(-8) M) for 2 h. EDN-1 suppressed dose dependently (10(-11) to 10(-8) M) the phagocytic activity for sperm and superoxide production of PMNs in response to capacitated sperm. Moreover, this suppression was eliminated by an ETB receptor antagonist (BQ-788). EDN-1 suppressed mRNA expression of EDN-1 and ETB but not ETA receptors in PMNs, suggesting the ETB receptor-mediated pathway. Scanning electron microscopic observation revealed that incubation of PMNs with EDN-1 (10(-9) M) completely suppressed the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results provide evidence indicating that EDN-1 may be involved in the protection of sperm from phagocytosis by PMNs in the bovine oviduct, supporting sperm survival until fertilization. PMID:26781611

  7. Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions.

    PubMed

    Sawant, Kirti V; Poluri, Krishna Mohan; Dutta, Amit K; Sepuru, Krishna Mohan; Troshkina, Anna; Garofalo, Roberto P; Rajarathnam, Krishna

    2016-01-01

    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. PMID:27625115

  8. The anti-inflammatory drug nimesulide rescues alpha-1-proteinase inhibitor from oxidative inactivation by phagocytosing neutrophils.

    PubMed

    Dallegri, F; Ottonello, L; Dapino, P; Bevilacqua, M

    1992-01-01

    When neutrophils are recruited to tissue sites and exposed to phagocytosable targets, they release oxidants which may be responsible for the local inactivation of alpha-1-proteinase inhibitor (A1PI). Consequently, A1PI becomes incapable of inhibiting the proteolytic activity of elastase, released at the same time by neutrophils as a result of leakage from phagocytic vacuoles. In the present paper we show that phagocytosing neutrophils inactivate A1PI via a process inhibitable by chemical agents known to interfere with the hypochlorous acid (HOCl)-generating myeloperoxidase pathway. The anti-inflammatory drug nimesulide (NMS), which is able to efficiently limit the extracellular availability of HOCl in the neutrophil surroundings, was found to prevent the inactivation of A1PI by neutrophils. The results provide evidence for a possible way to control neutrophil elastase activity by rescuing its natural inhibitor (A1PI) at inflamed tissue sites during infectious and noninfectious processes. PMID:1579712

  9. Feasibility of multiphoton microscopy-based quantification of antibiotic uptake into neutrophil granulocytes.

    PubMed

    Mahmood, Adnan; Grice, Jeffrey E; Roberts, Michael S; Prow, Tarl W

    2013-07-01

    Antibiotic levels in livestock are usually evaluated through destructive analysis. Taking advantage of the fluorescent properties of marbofloxacin (MBX) and trovafloxacin (TVX), multiphoton microscopy (MPM) was evaluated as a minimally invasive and nondestructive method to determine the penetration of TVX and MBX into sheep neutrophils. Standard curves were measured with drug-only solutions and suggested that MBX was more suited for this type of analysis. The intracellular concentration of both TVX and MBX was higher than the extracellular concentration after incubating neutrophils for 30 min at concentrations ranging from 0.1 to 100  μg/ml for both the drugs. The intracellular concentration of TVX increased with the extracellular concentration but was always greater than the extracellular concentration, suggesting active internalization. On the other hand, intracellular/extracellular ratio (I/E) peaked at 1.6-fold I/E for 1  μg/ml and then gradually decreased with increased concentration to 1.2-fold I/E at 100  μg/ml. For the first time, this study showed the use of MPM to quantify antibiotic uptake by sheep neutrophils and observed that both antibiotics were taken up by sheep neutrophils beyond extracellular levels. PMID:23824355

  10. Whole blood human neutrophil trafficking in a microfluidic model of infection and inflammation.

    PubMed

    Hamza, Bashar; Irimia, Daniel

    2015-06-21

    Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophils arriving at injury sites. Both insufficient and excessive neutrophil recruitment can be detrimental, favouring systemic spread of microbes or triggering severe tissue damage. Despite its importance in health and disease, the trafficking of neutrophils through tissues remains difficult to control and the mechanisms regulating it are insufficiently understood. These mechanisms are also complex and difficult to isolate using traditional in vivo models. Here we designed a microfluidic model of tissue infection/inflammation, in which human neutrophils emerge from a droplet-size samples of whole blood and display bi-directional traffic between this and micro-chambers containing chemoattractant and microbe-like particles. Two geometrical barriers restrict the entrance of red blood cells from the blood to the micro-chambers and simulate the mechanical function of the endothelial barrier separating the cells in blood from cells in tissues. We found that in the presence of chemoattractant, the number of neutrophils departing the chambers by retrotaxis is in dynamic equilibrium with the neutrophils recruited by chemotaxis. We also found that in the presence of microbe-like particles, the number of neutrophils trapped in the chambers is proportional to the number of particles. Together, the dynamic equilibrium between migration, reversed-migration and trapping processes determine the optimal number of neutrophils at a site. These neutrophils are continuously refreshed and responsive to the number of microbes. Further studies using this infection-inflammation-on-a-chip-model could help study the processes of inflammation resolution. The new in vitro experimental tools may also eventually help testing new therapeutic strategies to limit neutrophil accumulation in tissues during chronic inflammation, without increasing the risk for infections. PMID:25987163

  11. Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.

    PubMed

    Garratt, Luke W; Sutanto, Erika N; Ling, Kak-Ming; Looi, Kevin; Iosifidis, Thomas; Martinovich, Kelly M; Shaw, Nicole C; Kicic-Starcevich, Elizabeth; Knight, Darryl A; Ranganathan, Sarath; Stick, Stephen M; Kicic, Anthony

    2015-08-01

    Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis.Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis.A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis.Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease. PMID:25929954

  12. Trapped Surfaces

    NASA Astrophysics Data System (ADS)

    Senovilla, José M. M.

    I review the definition and types of (closed) trapped surfaces. Surprising global properties are shown, such as their "clairvoyance" and the possibility that they enter into flat portions of the spacetime. Several results on the interplay of trapped surfaces with vector fields and with spatial hypersurfaces are presented. Applications to the quasi-local definition of Black Holes are discussed, with particular emphasis set onto marginally trapped tubes, trapping horizons and the boundary of the region with closed trapped surfaces. Finally, the core of a trapped region is introduced, and its importance discussed.

  13. Trapped Surfaces

    NASA Astrophysics Data System (ADS)

    Senovilla, José M. M.

    2013-03-01

    I review the definition and types of (closed) trapped surfaces. Surprising global properties are pointed out, such as their "clairvoyance" and the possibility that they enter into flat portions of the spacetime. Several results on the interplay of trapped surfaces with vector fields and with spatial hypersurfaces are presented. Applications to the quasi-local definition of Black Holes are analyzed, with particular emphasis set onto marginally trapped tubes, trapping horizons and the boundary of the region with closed trapped surfaces. Finally, the core of a trapped region is introduced, and its importance briefly discussed.

  14. Impaired neutrophil function in 24p3 null mice contributes to enhanced susceptibility to bacterial infections

    PubMed Central

    Liu, Zhuoming; Petersen, Robert; Devireddy, L.

    2013-01-01

    Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3−/− mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3−/− mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3−/− neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3−/− neutrophils. Further, 24p3−/− neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3−/− mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3−/− mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. PMID:23543755

  15. The Novel Functions of the PLC/PKC/PKD Signaling Axis in G Protein-Coupled Receptor-Mediated Chemotaxis of Neutrophils

    PubMed Central

    Xu, Xuehua; Jin, Tian

    2015-01-01

    Chemotaxis, a directional cell migration guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. Chemotaxis is mediated by the G protein-coupled receptor (GPCR) signaling pathway. Extracellular stimuli trigger activation of the PLC/PKC/PKD signaling axis, which controls several signaling pathways. Here, we concentrate on the novel functions of PLC/PKC/PKD signaling in GPCR-mediated chemotaxis of neutrophils. PMID:26605346

  16. Extracellular guanosine regulates extracellular adenosine levels

    PubMed Central

    Cheng, Dongmei; Jackson, Travis C.; Verrier, Jonathan D.; Gillespie, Delbert G.

    2013-01-01

    The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)−6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases

  17. Disruption of a Regulatory Network Consisting of Neutrophils and Platelets Fosters Persisting Inflammation in Rheumatic Diseases

    PubMed Central

    Maugeri, Norma; Rovere-Querini, Patrizia; Manfredi, Angelo A.

    2016-01-01

    A network of cellular interactions that involve blood leukocytes and platelets maintains vessel homeostasis. It plays a critical role in the response to invading microbes by recruiting intravascular immunity and through the generation of neutrophil extracellular traps (NETs) and immunothrombosis. Moreover, it enables immune cells to respond to remote chemoattractants by crossing the endothelial barrier and reaching sites of infection. Once the network operating under physiological conditions is disrupted, the reciprocal activation of cells in the blood and the vessel walls determines the vascular remodeling via inflammatory signals delivered to stem/progenitor cells. A deregulated leukocyte/mural cell interaction is an early critical event in the natural history of systemic inflammation. Despite intense efforts, the signals that initiate and sustain the immune-mediated vessel injury, or those that enforce the often-prolonged phases of clinical quiescence in patients with vasculitis, have only been partially elucidated. Here, we discuss recent evidence that implicates the prototypic damage-associated molecular pattern/alarmin, the high mobility group box 1 (HMGB1) protein in systemic vasculitis and in the vascular inflammation associated with systemic sclerosis. HMGB1 could represent a player in the pathogenesis of rheumatic diseases and an attractive target for molecular interventions. PMID:27242789

  18. Disruption of a Regulatory Network Consisting of Neutrophils and Platelets Fosters Persisting Inflammation in Rheumatic Diseases.

    PubMed

    Maugeri, Norma; Rovere-Querini, Patrizia; Manfredi, Angelo A

    2016-01-01

    A network of cellular interactions that involve blood leukocytes and platelets maintains vessel homeostasis. It plays a critical role in the response to invading microbes by recruiting intravascular immunity and through the generation of neutrophil extracellular traps (NETs) and immunothrombosis. Moreover, it enables immune cells to respond to remote chemoattractants by crossing the endothelial barrier and reaching sites of infection. Once the network operating under physiological conditions is disrupted, the reciprocal activation of cells in the blood and the vessel walls determines the vascular remodeling via inflammatory signals delivered to stem/progenitor cells. A deregulated leukocyte/mural cell interaction is an early critical event in the natural history of systemic inflammation. Despite intense efforts, the signals that initiate and sustain the immune-mediated vessel injury, or those that enforce the often-prolonged phases of clinical quiescence in patients with vasculitis, have only been partially elucidated. Here, we discuss recent evidence that implicates the prototypic damage-associated molecular pattern/alarmin, the high mobility group box 1 (HMGB1) protein in systemic vasculitis and in the vascular inflammation associated with systemic sclerosis. HMGB1 could represent a player in the pathogenesis of rheumatic diseases and an attractive target for molecular interventions. PMID:27242789

  19. Human neutrophil surface protrusion under a point load: location independence and viscoelasticity

    PubMed Central

    Xu, Gang; Shao, Jin-Yu

    2008-01-01

    Mechanical properties of neutrophils have been recognized as key contributors to stabilizing neutrophil rolling on the endothelium during the inflammatory response. In particular, accumulating evidence suggests that surface protrusion and tether extraction from neutrophils facilitate stable rolling by relieving the disruptive forces on adhesive bonds. Using a customized optical trap setup, we applied piconewton-level pulling forces on targeted receptors that were located either on the microvillus tip (CD162) or intermicrovillus surface of neutrophils (CD18 and CD44). Under a constant force-loading rate, there always occurred an initial tent-like surface protrusion that was terminated either by rupture of the adhesion or by a “yield” or “crossover” to tether extraction. The corresponding protrusional stiffness of neutrophils was found to be between 0.06 and 0.11 pN/nm, depending on the force-loading rate and the cytoskeletal integrity, but not on the force location, the medium osmolality, nor the temperature increase from 22°C to 37°C. More importantly, we found that neutrophil surface protrusion was accompanied by force relaxation and hysteresis. In addition, the crossover force did not change much in the range of force-loading rates studied, and the protrusional stiffness of lymphocytes was similar to that of neutrophils. These results show that neutrophil surface protrusion is essentially viscoelastic, with a protrusional stiffness that stems primarily from the actin cortex, and the crossover force is independent of the receptor-cytoskeleton interaction. PMID:18815230

  20. Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion.

    PubMed

    Shao, Shuai; Cao, Tianyu; Jin, Liang; Li, Bing; Fang, Hui; Zhang, Jieyu; Zhang, Yuan; Hu, Jinhong; Wang, Gang

    2016-07-01

    Psoriasis is characterized by resistance to infections, which is regulated by antimicrobial proteins. Whether antimicrobial proteins play a pathogenic role in psoriasis remains unclear. In this study, we aimed to elucidate the role of lipocalin-2 (Lcn2), an antimicrobial protein, in the pathogenesis of psoriasis. Our results showed that Lcn2 was highly expressed in the lesional skin of psoriatic patients. The neutralization of Lcn2 alleviated epidermal hyperplasia, inflammation, and especially neutrophil infiltration in an imiquimod-induced psoriasis-like murine model. In vitro, Lcn2 stimulated human neutrophils to produce vital proinflammatory mediators, such as IL-6, IL-8, tumor necrosis factor-α, and IL-1α via a specific receptor, 24p3R, on neutrophils, which consequently activated the downstream extracellular signal-regulated kinase-1/2 and p38-mitogen-activated protein kinase signaling pathways. Moreover, Lcn2-induced neutrophil chemotaxis was concentration dependent and mediated by the extracellular signal-regulated kinase-1/2 and p38-mitogen-activated protein kinase signaling pathways in vitro. Furthermore, we demonstrated that both keratinocytes and neutrophils were the sources of Lcn2 in the lesional skin of psoriatic patients. Taken together, these results suggest that Lcn2 is involved in the pathogenesis of psoriasis by modulating neutrophil function, and that it could serve as a potential target for treating psoriasis. PMID:26979478

  1. Induction of CD18-mediated passage of neutrophils by Pasteurella haemolytica in pulmonary bronchi and bronchioles.

    PubMed

    Ackermann, M R; Brogden, K A; Florance, A F; Kehrli, M E

    1999-02-01

    demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces. PMID:9916073

  2. Hematologic comparisons of shot and live trapped cottontail rabbits.

    PubMed

    Jacobson, H A; Kirkpatrick, R L; Burkhart, H E; Davis, J W

    1978-01-01

    Comparisons were made between hematologic measurements of shot and box-trapped cottontail rabbits (Sylvilagus floridanus). Trapped rabbits had significantly (P less than 0.001) higher serum corticoid levels and segmented neutrophil percentages and significantly (P less than 0.001) lower lymphocyte percentages than did shot rabbits. Trapped rabbits also had significantly (P less than 0.05) higher packed cell volumes and blood urea nitrogen values than did shot rabbits. PMID:633520

  3. Neutrophil Functions in Periodontal Homeostasis.

    PubMed

    Cortés-Vieyra, Ricarda; Rosales, Carlos; Uribe-Querol, Eileen

    2016-01-01

    Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed. PMID:27019855

  4. Neutrophil Functions in Periodontal Homeostasis

    PubMed Central

    Cortés-Vieyra, Ricarda; Rosales, Carlos

    2016-01-01

    Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed. PMID:27019855

  5. Inhibition of cytochalasin-primed neutrophils by hyperosmolarity.

    PubMed

    Giambelluca, Miriam S; Gende, Oscar A

    2008-10-01

    Experimental and clinical investigations using hyperosmotic solutions for resuscitation of hemorrhagic shock demonstrated modulation of the inflammatory response. Decreased postinjury hyperinflammation has been attributed to a reduction in neutrophil-mediated tissue damage. This study shows that cytoskeletal disruption with cytochalasinB did not reverse or prevent the inhibitory effect of an osmolarity increase on the neutrophil cytotoxic response to a formyl peptide. In cytochalasin-primed neutrophils, the hyperosmolarity-dependent inhibition promptly reversed after returning to iso-osmotic levels. Paradoxically, an increase in osmolarity after stimulation produced an increase in the release of reactive oxygen species to the extracellular medium. The inhibitory effect of hyperosmotic NaCl can be reproduced by solutions of similar osmolarity containing N-methyl glucamine or sucrose, but solutions containing mannitol allowed an almost complete response to N-formyl methionyl leucyl phenylalanine. The effects on the release of reactive oxygen species to the extracellular media found with the OxyBURST-bovine serum albumin assay correlated with the changes of the intracellular calcium signal, indicating that the inhibition by hyperosmolarity occurs near the receptor level. PMID:18277949

  6. Feedback Amplification of Neutrophil Function.

    PubMed

    Németh, Tamás; Mócsai, Attila

    2016-06-01

    As the first line of innate immune defense, neutrophils need to mount a rapid and robust antimicrobial response. Recent studies implicate various positive feedback amplification processes in achieving that goal. Feedback amplification ensures effective migration of neutrophils in shallow chemotactic gradients, multiple waves of neutrophil recruitment to the site of inflammation, and the augmentation of various effector functions of the cells. We review here such positive feedback loops including intracellular and autocrine processes, paracrine effects mediated by lipid (LTB4), chemokine, and cytokine mediators, and bidirectional interactions with the complement system and with other immune and non-immune cells. These amplification mechanisms are not only involved in antimicrobial immunity but also contribute to neutrophil-mediated tissue damage under pathological conditions. PMID:27157638

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

    PubMed

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

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  8. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    PubMed Central

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

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  9. Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

    PubMed Central

    Kalyanaraman, B; Sohnle, P G

    1985-01-01

    A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis. PMID:2987307

  10. Optical trapping

    PubMed Central

    Neuman, Keir C.; Block, Steven M.

    2006-01-01

    Since their invention just over 20 years ago, optical traps have emerged as a powerful tool with broad-reaching applications in biology and physics. Capabilities have evolved from simple manipulation to the application of calibrated forces on—and the measurement of nanometer-level displacements of—optically trapped objects. We review progress in the development of optical trapping apparatus, including instrument design considerations, position detection schemes and calibration techniques, with an emphasis on recent advances. We conclude with a brief summary of innovative optical trapping configurations and applications. PMID:16878180

  11. COLD TRAPS

    DOEpatents

    Thompson, W.I.

    1958-09-30

    A cold trap is presented for removing a condensable component from a gas mixture by cooling. It consists of a shell, the exterior surface of which is chilled by a refrigerant, and conductive fins welded inside the shell to condense the gas, and distribute the condensate evenly throughout the length of the trap, so that the trap may function until it becomes completely filled with the condensed solid. The contents may then be removed as either a gas or as a liquid by heating the trap. This device has particuinr use as a means for removing uranium hexafluoride from the gaseous diffusion separation process during equipment breakdown and repair periods.

  12. Neutrophil Elastase Inhibitors

    PubMed Central

    Groutas, William C.; Dou, Dengfeng; Alliston, Kevin R.

    2011-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis, and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered An overview of major developments in COPD research with emphasis on low molecular weight neutrophil elastase inhibitors is described in this review. Expert opinion Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is till limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as, for instance, an HNE or MMP-12 inhibitor with an anti-inflammatory agent such as a phosphodiesterase-4 inhibitor, or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress PMID:21235378

  13. AUTOINFLAMMATORY PUSTULAR NEUTROPHILIC DISEASES

    PubMed Central

    Naik, Haley B.; Cowen, Edward W.

    2013-01-01

    SYNOPSIS The inflammatory pustular dermatoses constitute a spectrum of non-infectious conditions ranging from localized involvement to generalized disease with associated acute systemic inflammation and multi-organ involvement. Despite the variability in extent and severity of cutaneous presentation, each of these diseases is characterized by non-infectious neutrophilic intra-epidermal microabscesses. Many share systemic findings including fever, elevated inflammatory markers, inflammatory bowel disease and/or osteoarticular involvement, suggesting potential common pathogenic links (Figure 1). The recent discoveries of several genes responsible for heritable pustular diseases have revealed a distinct link between pustular skin disease and regulation of innate immunity. These genetic advances have led to a deeper exploration of common pathways in pustular skin disease and offer the potential for a new era of biologic therapy which targets these shared pathways. This chapter provides a new categorization of inflammatory pustular dermatoses in the context of recent genetic and biologic insights. We will discuss recently-described monogenic diseases with pustular phenotypes, including deficiency of IL-1 receptor antagonist (DIRA), deficiency of the IL-36 receptor antagonist (DITRA), CARD14-associated pustular psoriasis (CAMPS), and pyogenic arthritis, pyoderma gangrenosum, acne (PAPA). We will then discuss how these new genetic advancements may inform how we view previously described pustular diseases, including pustular psoriasis and its clinical variants, with a focus on historical classification by clinical phenotype. PMID:23827244

  14. Protrusive and Contractile Forces of Spreading Human Neutrophils.

    PubMed

    Henry, Steven J; Chen, Christopher S; Crocker, John C; Hammer, Daniel A

    2015-08-18

    Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil's morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell's mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (-20 ± 10 pN/post) than those residing at the geometric perimeter (-106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β2 integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of

  15. Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects.

    PubMed

    Altincicek, Boran; Stötzel, Sabine; Wygrecka, Malgorzata; Preissner, Klaus T; Vilcinskas, Andreas

    2008-08-15

    Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses. PMID:18684961

  16. A Morphological and Cytochemical Study of the Interaction between Paracoccidiodes brasiliensis and Neutrophils

    NASA Astrophysics Data System (ADS)

    Dias, Maria Fernanda R. G.; Filgueira, Absalom L.; de Souza, Wanderley

    2004-04-01

    Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils' degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.

  17. Neutrophils: game changers in glomerulonephritis?

    PubMed Central

    Mayadas, Tanya N.; Rosetti, Florencia; Ernandez, Thomas; Sethi, Sanjeev

    2010-01-01

    Glomerulonephritides represent a diverse array of diseases that have in common immune cell-mediated effector mechanisms that cause organ damage. The contribution of neutrophils to the pathogenesis of proliferative glomerulonephritis (GN) is not well recognized. Most equate neutrophils with killing pathogens and causing collateral tissue damage during acute inflammation. However, these phagocytes are endowed with additional characteristics that have been traditionally reserved for cells of the adaptive immune system. They communicate with other cells, exhibit plasticity in their responses and have the potential to coordinate and inform the subsequent immune response, thus countering the notion that they arrive, destroy and then disappear. Therefore, neutrophils, which are the first to arrive at a site of inflammation, are potential game changers in GN. PMID:20667782

  18. Fibrinogen is degraded and internalized during incubation with neutrophils, and fibrinogen products localize to electron lucent vesicles.

    PubMed Central

    Kirsch, Richard; Jaffer, Mohamed A; Woodburne, Vivienne E; Sewell, Trevor; Kelly, Sharon L; Kirsch, Ralph E; Shephard, Enid G

    2002-01-01

    A biologically relevant relationship exists between neutrophils and coagulation processes. Several studies have focused on the ability of neutrophil proteases (both intracellular and membrane-associated) to degrade fibrinogen. The present study investigates the events following the interaction of activated neutrophils with soluble fibrinogen. During incubation of PMA-stimulated neutrophils with fibrinogen at 37 degrees C, fibrinogenolysis occurred, and degraded fibrinogen became associated with the neutrophil. Immunoelectron microscopy identified these fibrinogen products to be located within electron lucent vesicles, and not on the surface of the cell, suggesting that they are internalized. Although a specific interaction between fibrinogen and the neutrophil membrane might assist uptake, in the presence of physiological concentrations of fibrinogen, internalization occurred largely via a non-specific pinocytic process. Studies at low temperature revealed that both intact and degraded forms of fibrinogen can associate with neutrophils. The fibrinogen products detected intracellularly in experiments performed at 37 degrees C might represent uptake of degraded as well as intact forms of fibrinogen, the latter being rapidly degraded intracellularly. This route of fibrinogenolysis contributes minimally to the overall extent of the degradation process, the majority occurring extracellularly. Neutrophils thus possess a proteolytic mechanism for preventing accumulation of surface ligand, perhaps allowing them to evade the immunomodulatory effects of such ligands during inflammation. PMID:12023883

  19. Effects of Docosahexaenoic Supplementation and In Vitro Vitamin C on the Oxidative and Inflammatory Neutrophil Response to Activation

    PubMed Central

    Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Tur, Josep Antoni; Pons, Antoni

    2015-01-01

    We studied the effects of diet supplementation with docosahexaenoic (DHA) and in vitro vitamin C (VitC) at physiological concentrations on oxidative and inflammatory neutrophil response to phorbol myristate acetate (PMA). Fifteen male footballers ingested a beverage enriched with DHA or a placebo for 8 weeks in a randomized double-blind study. Neutrophils were isolated from blood samples collected in basal conditions at the end of nutritional intervention. Neutrophils were cultured for 2 hours at 37°C in (a) control media, (b) media with PMA, and (c) media with PMA + VitC. PMA induces neutrophil degranulation with increased extracellular myeloperoxidase and catalase activities, nitric oxide production, expression of the inflammatory genes cyclooxygenase-2, nuclear factor κβ, interleukin 8 and tumor necrosis factor α, and interleukin 6 production. DHA diet supplementation boosts the exit of CAT from neutrophils but moderates the degranulation of myeloperoxidase granules induced by PMA. VitC facilitates azurophilic degranulation of neutrophils and increases gene expression of myeloperoxidase induced by PMA. VitC and DHA diet supplementation prevent PMA effects on inflammatory gene expression, although together they do not produce additional effects. DHA diet supplementation enhances antioxidant defences and anti-inflammatory neutrophil response to in vitro PMA activation. VitC facilitates neutrophil degranulation but prevents an inflammatory response to PMA. PMID:25960826

  20. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  1. Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus

    PubMed Central

    Swain, Dilip K.; Kushwah, Mohar Singh; Kaur, Mandheer; Dang, Ajay K.

    2015-01-01

    Aim: The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM). Materials and Methods: A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy. Results: Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows. Conclusion: The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection. PMID:27047094

  2. APPLICATION OF PROTEOMICS TO NEUTROPHIL BIOLOGY

    PubMed Central

    Luerman, Gregory C.; Uriarte, Silvia M.; Rane, Madhavi J.; McLeish, Kenneth R.

    2009-01-01

    Polymorphonuclear leukocytes or neutrophils are a primary effector cell of the innate immune system and contribute to the development of adaptive immunity. Neutrophils participate in both the initiation and resolution of inflammatory responses through a series of highly coordinated molecular and phenotypic changes. To accomplish these changes, neutrophils express numerous receptors and use multiple overlapping and redundant signal transduction pathways. Dysregulation of the activation or resolution pathways plays a role in a number of human diseases. A comprehensive understanding of the regulation of neutrophil responses can be provided by high throughput proteomic technologies and sophisticated computational analysis. The first steps in the application of proteomics to understanding neutrophil biology have been taken. Here we review the application of expression, structural, and functional proteomic studies to neutrophils. Although defining the complex molecular events associated with neutrophil activation is in the early stages, the data generated to date suggest that proteomic technologies will dramatically enhance our understanding of neutrophil biology. PMID:19580889

  3. Role of neutrophils in systemic autoimmune diseases

    PubMed Central

    2013-01-01

    Neutrophils have emerged as important regulators of innate and adaptive immune responses. Recent evidence indicates that neutrophils display marked abnormalities in phenotype and function in various systemic autoimmune diseases, and may play a central role in initiation and perpetuation of aberrant immune responses and organ damage in these conditions. This review discusses the putative roles that neutrophils and aberrant neutrophil cell death play in the pathogenesis of various systemic autoimmune diseases, including systemic lupus erythematosus, small vessel vasculitis and rheumatoid arthritis. PMID:24286137

  4. αVβ3 Integrin Regulation of Respiratory Burst in Fibrinogen Adherent Human Neutrophils

    PubMed Central

    Kim, Hye-Yeong; Skokos, Eleni A.; Myer, Deborah J.; Agaba, Perez; Gonzalez, Anjelica L.

    2015-01-01

    In response to inflammatory stimuli, microvascular endothelial cells become activated, initiating the capture and exit of neutrophils from the blood vessel and into the extravascular extracellular matrix (ECM). In the extravascular space, neutrophils bind to ECM proteins, regulating cellular functions via signaling through adhesion molecules known as integrins. The αVβ3 integrin is an important mediator of neutrophil adhesion to ECM proteins containing the Arg-Gly-Asp (RGD) peptide sequence, including fibrinogen and fibronectin. Despite the abundance of RGD sequence in the ECM, adhesion molecule-mediated neutrophil activity has been focused on the β2 (Mac-1, CD11b/CD18) and β1 integrin response to matrix proteins. Here we investigated αVβ3 integrin-mediated reactive oxidant suppression as a consequence of human neutrophil adhesion to RGD containing proteins. Using integrin ligand-modified (poly)ethylene glycol hydrogels and reactive oxygen species (ROS) sensitive fluorescent probes (dihydrotetramethylrhosamine, H2TMRos), we evaluated integrin–peptide interactions that effectively regulate ROS generation. This study demonstrates that neutrophil adhesion suppresses ROS production in an αVβ3-dependent manner. Additionally, we determine that p38 mitogen-activated protein kinase in the respiratory burst signaling pathway is interrupted by integrin-mediated adhesion. These data indicate that ECM/integrin interactions can induce αVβ3-mediated adhesion dependent downstream signaling of ROS regulation via a Mac-1 independent mechanism. PMID:25632307

  5. Three-dimensional migration of neutrophils through an electrospun nanofibrous membrane.

    PubMed

    Jin, Songwan; Park, Tae-Min; Kim, Cho-Hee; Kim, Jin-Soo; Le, Binh Duong; Jeong, Young Hun; Kwak, Jong-Young; Yoon, Sik

    2015-06-01

    The study of immune cell migration is important for understanding the immune system network, which is associated with the response to foreign cells. Neutrophils act against foreign cells before any other immune cell, and they must be able to change shape and squeeze through narrow spaces in the extracellular matrix (ECM) during migration to sites of infection. Conventional in vitro migration assays are typically performed on two-dimensional substrates that fail to reproduce the three-dimensional (3-D) nature of the ECM. Here we present an in vitro method to simulate the 3-D migration of neutrophils using an electrospun nanofibrous membrane, which is similar to the ECM in terms of morphology. We examined the properties of neutrophil movement and the effects of gravity and the presence of IL-8, which has been widely used as a chemotactic attractant for neutrophils. The number of neutrophils passing through the nanofibrous membrane were higher, and their movement was more active in the presence of IL-8. Also, we confirmed that neutrophils could migrate against gravity toward IL-8 through a nanofibrous membrane. PMID:26054764

  6. Role of gelsolin in actin depolymerization of adherent human neutrophils.

    PubMed Central

    Wang, J S; Coburn, J P; Tauber, A I; Zaner, K S

    1997-01-01

    Human neutrophils generally function adherent to an extracellular matrix. We have previously reported that upon adhesion to laminin- or fibronectin-coated, but not uncoated, plastic there is a depolymerization of actin in neutrophils. This phenomenon was not affected by inhibitors of the more well-studied components of the signal transduction pathway, specifically, pertussis toxin, an inhibitor of G-proteins, H-7 or staurosporine, inhibitors of protein kinase C, or herbimycin A, an inhibitor of nonreceptor tyrosine kinase. We therefore focused our attention on actin-binding proteins and measured the changes in the partitioning of gelsolin between the Triton X-100-soluble and -insoluble cellular fractions which occur upon neutrophil adhesion by means of quantitating anti-gelsolin antibody binding to aliquots of these fractions. It was found that approximately 90% of the total cellular gelsolin was found in the Triton X-100-soluble fraction in suspended cells, but that upon adherence to either fibronectin- or laminin-coated plastic about 40% of the soluble gelsolin could be detected in the insoluble fraction. This effect was not observed in cells adherent to uncoated plastic, wherein more than 90% of the gelsolin was found in the soluble fraction. Results of immunofluorescence microscopy of these cell preparations was consistent with this data. A gelsolin translocation to the insoluble cellular actin network may account for a part of the observed actin depolymerization. Images PMID:9017600

  7. Staphylococcal Superantigen-Like Protein 1 and 5 (SSL1 & SSL5) Limit Neutrophil Chemotaxis and Migration through MMP-Inhibition

    PubMed Central

    Koymans, Kirsten J.; Bisschop, Adinda; Vughs, Mignon M.; van Kessel, Kok P. M.; de Haas, Carla J. C.; van Strijp, Jos A. G.

    2016-01-01

    Matrix metalloproteinases (MMPs) are endopeptidases that degrade components of the extracellular matrix, but also modulate inflammation. During bacterial infections, MMPs are important in the recruitment and migration of inflammatory cells. Besides facilitating cell migration by degrading extracellular matrix components, they potentiate the action of several inflammatory molecules, including cytokines, chemokines, and antimicrobial peptides. Staphylococcus aureus secretes an arsenal of immune evasion molecules that interfere with immune cell functioning and hamper proper immune responses. An earlier study identified staphylococcal superantigen-like protein 5 (SSL5) as an MMP9 inhibitor. Since multiple MMPs are involved in neutrophil recruitment, we set up an in-depth search for additional MMP inhibitors by testing a panel of over 70 secreted staphylococcal proteins on the inhibition of the two main neutrophil MMPs: MMP8 (neutrophil collagenase) and MMP9 (neutrophil gelatinase B). We identified SSL1 and SSL5 as potent inhibitors of both neutrophil MMPs and show that they are actually broad range MMP inhibitors. SSL1 and SSL5 prevent MMP-induced cleavage and potentiation of IL-8 and inhibit the migration of neutrophils through collagen. Thus, through MMP-inhibition, SSL1 and SSL5 interfere with neutrophil activation, chemotaxis, and migration, all vital neutrophil functions in bacterial clearance. Studies on MMP-SSL interactions can have therapeutic potential and SSL based derivatives might prove useful in treatment of cancer and destructive inflammatory diseases. PMID:27399672

  8. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  9. Cathepsin G-regulated Release of Formyl Peptide Receptor Agonists Modulate Neutrophil Effector Functions*

    PubMed Central

    Woloszynek, Josh C.; Hu, Ying; Pham, Christine T. N.

    2012-01-01

    Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway. PMID:22879591

  10. Effect of deuterium oxide on neutrophil oxidative metabolism, phagocytosis, and lysosomal enzyme release

    SciTech Connect

    Tsan, M.F.; Turkall, R.M.

    1982-12-01

    We have previously shown that deuterium oxide (D/sub 2/O) enhances the oxidation of methionine, a myeloperoxidase (MPO) -mediated reaction, by human neutrophils during phagocytosis. However, D/sub 2/O has no effect on the oxidation of methionine by the purified MPO-H/sub 2/O/sub 2/-Cl- system. To explain this observation, we studied the effect of D/sub 2/O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D/sub 2/O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D/sub 2/O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D/sub 2/O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H/sub 2/O/sub 2/ production by neutrophils in the presence of latex particles or PMA were also stimulated by D/sub 2/O. In contrast, D/sub 2/O inhibited the ingestion of latex particles. D/sub 2/O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D/sub 2/O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D/sub 2/O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.

  11. Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP

    PubMed Central

    Karmakar, Mausita; Katsnelson, Michael A.; Dubyak, George R.; Pearlman, Eric

    2016-01-01

    Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K+, NLRP3 inflammasome activation and IL-1β secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca2+], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca2+]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1β secretion by neutrophils likely has a significant, wide ranging clinical impact. PMID:26877061

  12. Neutrophils in cancer: neutral no more.

    PubMed

    Coffelt, Seth B; Wellenstein, Max D; de Visser, Karin E

    2016-07-01

    Neutrophils are indispensable antagonists of microbial infection and facilitators of wound healing. In the cancer setting, a newfound appreciation for neutrophils has come into view. The traditionally held belief that neutrophils are inert bystanders is being challenged by the recent literature. Emerging evidence indicates that tumours manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumour behaviour. In this Review, we discuss the involvement of neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and therapeutic targets. PMID:27282249

  13. 7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils.

    PubMed

    Kabeya, Luciana M; Fuzissaki, Carolina N; Taleb-Contini, Silvia H; da C Ferreira, Ana Maria; Naal, Zeki; Santos, Everton O L; Figueiredo-Rinhel, Andréa S G; Azzolini, Ana Elisa C S; Vermelho, Roberta B; Malvezzi, Alberto; Amaral, Antonia T-do; Lopes, João Luis C; Lucisano-Valim, Yara Maria

    2013-10-25

    In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects. PMID:23994743

  14. Protrusive and Contractile Forces of Spreading Human Neutrophils

    PubMed Central

    Henry, Steven J.; Chen, Christopher S.; Crocker, John C.; Hammer, Daniel A.

    2015-01-01

    Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil’s morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell’s mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (−20 ± 10 pN/post) than those residing at the geometric perimeter (−106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β2 integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the

  15. Regulation of immune responses by neutrophils.

    PubMed

    Wang, Jing; Arase, Hisashi

    2014-06-01

    Neutrophils, the most abundant circulating cells in humans, are major pathogen-killing immune cells. For many years, these cells were considered to be simple killers at the "bottom" of immune responses. However, recent studies have revealed more sophisticated mechanisms associated with neutrophilic cytotoxic functions, and neutrophils have been shown to contribute to various infectious and inflammatory diseases. In this review, we discuss the key features of neutrophils during inflammatory responses, from their release from the bone marrow to their death in inflammatory loci. We also discuss the expanding roles of neutrophils that have been identified in the context of several inflammatory diseases. We further focus on the mechanisms that regulate neutrophil recruitment to inflamed tissues and neutrophil cytotoxic activities against both pathogens and host tissues. PMID:24850053

  16. Regulation of neutrophil apoptosis by modulation of PKB/Akt activation.

    PubMed

    Rane, Madhavi J; Klein, Jon B

    2009-01-01

    The serine/threonine kinase, Akt, also known as PKB (Protein Kinase B) is one important signal transduction pathway that mediates the delay of neutrophil apoptosis caused by inflammatory mediators. Proteins controlled by the PKB/Akt pathway have been reported to prevent or reverse apoptotic-signaling pathways and regulate cell survival. In this review we discuss the role of PKB/Akt activation in the regulation of neutrophil activation during inflammation, and the importance of resolving the inflammatory response by inhibiting PKB/Akt activation and neutrophil survival. Furthermore, we introduce the concept of a dynamic Akt signal complex that is altered when an extracellular signal is initiated such that changes in protein-protein interactions within the Akt signal complex regulates Akt activity and cell survival. Various substrates of PKB/Akt as well as positive and negative regulators of PKB/Akt activation are discussed which in turn inhibit or enhance cellular survival. PMID:19273208

  17. Carrageenans effect on neutrophils alone and in combination with LPS in vitro.

    PubMed

    Sokolova, E V; Karetin, Y; Davydova, V N; Byankina, A O; Kalitnik, A A; Bogdanovich, L N; Yermak, I M

    2016-07-01

    Influence of sulfated red algal polysaccharides (κ-, λ-, and κ/β-carrageenans) and degraded derivative of κ/β-carrageenan on neutrophils/monocytes activation alone and in combination with lipopolysaccharide was investigated by means of determination of reactive oxygen species production, latex microparticles engulfment, total and extracellular myeloperoxidase induction and the analysis of silhouette and contour two-dimensional images of flattened cells. Carrageenans alone can activate neutrophils with much less potency than lipopolysaccharide (LPS) and the sulfation degree of carrageenans stipulates high activity in this role. On the other hand, carrageenans especially with low contents of sulfate groups are able to interfere with LPS in vitro resulting in reducing inter- and intracellular activation of neutrophils killing mechanisms. Further research is necessary to relate these findings to actions on the whole animal or human in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1603-1609, 2016. PMID:26915063

  18. The effect of leptin on the respiratory burst of human neutrophils cultured in synovial fluid

    PubMed Central

    Rzodkiewicz, Przemysław; Gajewska, Joanna; Wojtecka-Łukasik, Elżbieta

    2015-01-01

    Objectives Leptin is a hormone responsible for nutritional status and immune competence coordination. In rheumatoid arthritis (RA) increased leptin levels were observed in both serum and synovial fluid. Its influence on development of the disease still remains unclear. So far, research on leptin's influence on the emission of reactive oxygen intermediates (ROI) measured with chemiluminescence (CL) has provided unclear and contradictory results. In this study, we evaluated the influence of leptin on oxidative activity of neutrophils isolated from blood of healthy volunteers and cultured in different amounts of synovial fluid (SF) from patients with RA. Material and methods Neutrophils’ oxidative metabolism was measured by two types of CL. The first one, luminol-dependent CL (CL-lum), allows one to determine phagocytic activity and the level of ROI generated in a myeloperoxidase-dependent manner. The second method used was lucigenin-dependent CL (CL-luc), which monitors ROI production dependent on the NADPH oxidase enzyme complex located in the cell membranes of neutrophils and enables one to determine the scope of extracellular ROI emission. Results Neutrophils stimulated by opsonized zymosan show a decrease in the level of CL-lum, proportional to the increasing concentration of both SF and serum collected from healthy donors. The observed effect of decreased CL-lum may, therefore, be dependent on the physical conditions (viscosity of fluids used). None of these experiments showed any effect of leptin on the level of CL-lum. Conclusions The present study showed that leptin does not affect the level of any of the CL types in inactive neutrophils incubated in normal serum, and it does not affect the level of oxidative activity in resting neutrophils incubated with SF. However, leptin influences extracellular ROI emission (measured by CL-luc). Leptin reduces extracellular emission of ROI, and this effect is dependent on concentration and duration of exposure to

  19. Thrombospondin-1 restrains neutrophil granule serine protease function and regulates the innate immune response during Klebsiella pneumoniae infection.

    PubMed

    Zhao, Y; Olonisakin, T F; Xiong, Z; Hulver, M; Sayeed, S; Yu, M T; Gregory, A D; Kochman, E J; Chen, B B; Mallampalli, R K; Sun, M; Silverstein, R L; Stolz, D B; Shapiro, S D; Ray, A; Ray, P; Lee, J S

    2015-07-01

    Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1(-/-)) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild-type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1(-/-) mouse lungs compared with WT controls. Lung NE activity was increased in thbs1(-/-) mice following K. pneumoniae challenge, and thbs1(-/-) neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1(-/-) neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793-801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal. PMID:25492474

  20. The P2X1 receptor is required for neutrophil extravasation during lipopolysaccharide-induced lethal endotoxemia in mice.

    PubMed

    Maître, Blandine; Magnenat, Stéphanie; Heim, Véronique; Ravanat, Catherine; Evans, Richard J; de la Salle, Henri; Gachet, Christian; Hechler, Béatrice

    2015-01-15

    Extracellular ATP is becoming increasingly recognized as an important regulator of inflammation. However, the known repertoire of P2 receptor subtypes responsible for the proinflammatory effects of ATP is sparse. We looked at whether the P2X1 receptor, an ATP-gated cation channel present on platelets, neutrophils, and macrophages, participates in the acute systemic inflammation provoked by LPS. Compared with wild-type (WT) mice, P2X1(-/-) mice displayed strongly diminished pathological responses, with dampened neutrophil accumulation in the lungs, less tissue damage, reduced activation of coagulation, and resistance to LPS-induced death. P2X1 receptor deficiency also was associated with a marked reduction in plasma levels of the main proinflammatory cytokines and chemokines induced by LPS. Interestingly, macrophages and neutrophils isolated from WT and P2X1(-/-) mice produced similar levels of proinflammatory cytokines when stimulated with LPS in vitro. Intravital microscopy revealed a defect in LPS-induced neutrophil emigration from cremaster venules into the tissues of P2X1(-/-) mice. Using adoptive transfer of immunofluorescently labeled neutrophils from WT and P2X1(-/-) mice into WT mice, we demonstrate that the absence of the P2X1 receptor on neutrophils was responsible for this defect. This study reveals a major role for the P2X1 receptor in LPS-induced lethal endotoxemia through its critical involvement in neutrophil emigration from venules. PMID:25480563

  1. Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

    PubMed Central

    Karlsson, Anna

    1999-01-01

    Wheat germ agglutinin (WGA), a lectin with specificity for N-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures. PMID:10377127

  2. Alarmins Link Neutrophils and Dendritic Cells

    PubMed Central

    Yang, De; de la Rosa, Gonzalo; Tewary, Poonam; Oppenheim, Joost J.

    2009-01-01

    Neutrophils are the first major population of leukocyte to infiltrate infected or injured tissues and are crucial for initiating host innate defense and adaptive immunity. Although the contribution of neutrophils to innate immune defense is mediated predominantly by phagocytosis and killing of microorganisms, neutrophils also participate in the induction of adaptive immune responses. At sites of infection and/or injury, neutrophils release numerous mediators upon degranulation or death, among these are alarmins which have a characteristic dual capacity to mobilize and activate antigen-presenting cells. We describe here how alarmins released by neutrophil degranulation and/or death can link neutrophils to dendritic cells by promoting their recruitment and activation, resulting in the augmentation of innate and adaptive immune responses. PMID:19699678

  3. Proteome Mapping of Adult Zebrafish Marrow Neutrophils Reveals Partial Cross Species Conservation to Human Peripheral Neutrophils

    PubMed Central

    Singh, Sachin Kumar; Sethi, Sachin; Aravamudhan, Sriram; Krüger, Marcus; Grabher, Clemens

    2013-01-01

    Neutrophil granulocytes are pivotal cells within the first line of host defense of the innate immune system. In this study, we have used a gel-based LC-MS/MS approach to explore the proteome of primary marrow neutrophils from adult zebrafish. The identified proteins originated from all major cellular compartments. Gene ontology analysis revealed significant association of proteins with different immune-related network and pathway maps. 75% of proteins identified in neutrophils were identified in neutrophils only when compared to neutrophil-free brain tissue. Moreover, cross-species comparison with human peripheral blood neutrophils showed partial conservation of immune-related proteins between human and zebrafish. This study provides the first zebrafish neutrophil proteome and may serve as a valuable resource for an understanding of neutrophil biology and innate immunity. PMID:24019943

  4. Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils.

    PubMed

    Kramer, Philip A; Prichard, Lynn; Chacko, Balu; Ravi, Saranya; Overton, E Turner; Heath, Sonya L; Darley-Usmar, Victor

    2015-09-01

    Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function. PMID:25951298

  5. Ripple Trap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    3 April 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows the margin of a lava flow on a cratered plain in the Athabasca Vallis region of Mars. Remarkably, the cratered plain in this scene is essentially free of bright, windblown ripples. Conversely, the lava flow apparently acted as a trap for windblown materials, illustrated by the presence of the light-toned, wave-like texture over much of the flow. That the lava flow surface trapped windblown sand and granules better than the cratered plain indicates that the flow surface has a rougher texture at a scale too small to resolve in this image.

    Location near: 10.7oN, 204.5oW Image width: 3 km (1.9 mi) Illumination from: lower left Season: Northern Winter

  6. Neutrophil function and dysfunction in periodontal disease.

    PubMed

    Van Dyke, T E; Vaikuntam, J

    1994-01-01

    The polymorphonuclear leukocyte or neutrophil is an integral part of the acute inflammatory response. Its function as a protective cell in the pathogenesis of periodontal disease has been studied extensively. Abnormal neutrophil function has been associated (directly or indirectly) with the pathogenesis of early onset periodontal disease. This paper reviews the recent developments in neutrophil function and dysfunction as they relate to periodontal disease progression. PMID:8032460

  7. Nicotine is Chemotactic for Neutrophils and Enhances Neutrophil Responsiveness to Chemotactic Peptides

    NASA Astrophysics Data System (ADS)

    Totti, Noel; McCusker, Kevin T.; Campbell, Edward J.; Griffin, Gail L.; Senior, Robert M.

    1984-01-01

    Neutrophils contribute to chronic bronchitis and pulmonary emphysema associated with cigarette smoking. Nicotine was found to be chemotactic for human neutrophils but not monocytes, with a peak activity at ~ 31 micromolar. In lower concentrations (comparable to those in smokers' plasma), nicotine enhanced the response of neutrophils to two chemotactic peptides. In contrast to most other chemoattractants for neutrophils, however, nicotine did not affect degranulation or superoxide production. Nicotine thus may promote inflammation and consequent lung injury in smokers.

  8. Neutrophil-Related Gene Expression And Low-Density Granulocytes Associated with Disease Activity and Response to Treatment in ANCA-Associated Vasculitis

    PubMed Central

    Grayson, Peter C.; Carmona-Rivera, Carmelo; Xu, Lijing; Lim, Noha; Gao, Zhong; Asare, Adam L.; Specks, Ulrich; Stone, John H.; Seo, Philip; Spiera, Robert F.; Langford, Carol A.; Hoffman, Gary S.; Kallenberg, Cees G.M.; St Clair, E. William; Tchao, Nadia K.; Ytterberg, Steven R.; Phippard, Deborah J.; Merkel, Peter A.; Kaplan, Mariana J.; Monach, Paul A.

    2015-01-01

    Objectives To discover biomarkers involved in the pathophysiology of ANCA-associated vasculitis (AAV) and determine if low-density granulocytes (LDGs) contribute to gene expression signatures in AAV. Methods The source of clinical data and linked biospecimens was a randomized controlled treatment trial in AAV. RNA-sequencing of whole blood from patients with AAV was performed during active disease at the baseline visit (BL) and during remission 6 months later (6M). Gene expression was compared between patients who met versus did not meet the primary trial outcome of clinical remission at 6M (responders vs. nonresponders). Measurement of neutrophil-related gene expression was confirmed in PBMCs to validate findings in whole blood. A negative selection strategy isolated LDGs from PBMC fractions. Results Differential expression between responders (n=77) and nonresponders (n=35) was detected in 2,346 transcripts at BL visit (p<0.05). Unsupervised hierarchical clustering demonstrated a cluster of granulocyte-related genes, including myeloperoxidase (MPO) and proteinase 3 (PR3). A granulocyte multi-gene composite score was significantly higher in nonresponders than responders (p<0.01) and during active disease compared to remission (p<0.01). This signature strongly overlapped an LDG signature identified previously in lupus (FDRGSEA<0.01). Transcription of PR3 measured in PBMCs was associated with active disease and treatment response (p<0.01). LDGs isolated from patients with AAV spontaneously formed neutrophil extracellular traps containing PR3 and MPO. Conclusions In AAV an increased expression of a granulocyte gene signature is associated with disease activity and decreased response to treatment. The source of this signature is likely LDGs, a potentially pathogenic cell type in AAV. PMID:25891759

  9. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

    PubMed

    Franklin, Cindy; Cesko, Elvir; Hillen, Uwe; Schilling, Bastian; Brandau, Sven

    2015-01-01

    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. PMID:26241482

  10. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease

    PubMed Central

    Franklin, Cindy; Cesko, Elvir; Hillen, Uwe

    2015-01-01

    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. PMID:26241482

  11. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with spec...

  12. Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists.

    PubMed

    Chapple, Iain Lc; Matthews, John B; Wright, Helen J; Scott, Ann E; Griffiths, Helen R; Grant, Melissa M

    2013-01-01

    Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 µM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function. PMID:22914919

  13. Trapped antihydrogen.

    PubMed

    Andresen, G B; Ashkezari, M D; Baquero-Ruiz, M; Bertsche, W; Bowe, P D; Butler, E; Cesar, C L; Chapman, S; Charlton, M; Deller, A; Eriksson, S; Fajans, J; Friesen, T; Fujiwara, M C; Gill, D R; Gutierrez, A; Hangst, J S; Hardy, W N; Hayden, M E; Humphries, A J; Hydomako, R; Jenkins, M J; Jonsell, S; Jørgensen, L V; Kurchaninov, L; Madsen, N; Menary, S; Nolan, P; Olchanski, K; Olin, A; Povilus, A; Pusa, P; Robicheaux, F; Sarid, E; el Nasr, S Seif; Silveira, D M; So, C; Storey, J W; Thompson, R I; van der Werf, D P; Wurtele, J S; Yamazaki, Y

    2010-12-01

    Antimatter was first predicted in 1931, by Dirac. Work with high-energy antiparticles is now commonplace, and anti-electrons are used regularly in the medical technique of positron emission tomography scanning. Antihydrogen, the bound state of an antiproton and a positron, has been produced at low energies at CERN (the European Organization for Nuclear Research) since 2002. Antihydrogen is of interest for use in a precision test of nature's fundamental symmetries. The charge conjugation/parity/time reversal (CPT) theorem, a crucial part of the foundation of the standard model of elementary particles and interactions, demands that hydrogen and antihydrogen have the same spectrum. Given the current experimental precision of measurements on the hydrogen atom (about two parts in 10(14) for the frequency of the 1s-to-2s transition), subjecting antihydrogen to rigorous spectroscopic examination would constitute a compelling, model-independent test of CPT. Antihydrogen could also be used to study the gravitational behaviour of antimatter. However, so far experiments have produced antihydrogen that is not confined, precluding detailed study of its structure. Here we demonstrate trapping of antihydrogen atoms. From the interaction of about 10(7) antiprotons and 7 × 10(8) positrons, we observed 38 annihilation events consistent with the controlled release of trapped antihydrogen from our magnetic trap; the measured background is 1.4 ± 1.4 events. This result opens the door to precision measurements on anti-atoms, which can soon be subjected to the same techniques as developed for hydrogen. PMID:21085118

  14. COLD TRAP

    DOEpatents

    Milleron, N.

    1963-03-12

    An improved linear-flow cold trap is designed for highvacuum applications such as mitigating back migration of diffusion pump oil moiecules. A central pot of liquid nitrogen is nested within and supported by a surrounding, vertical, helical coil of metai sheet, all enveloped by a larger, upright, cylindrical, vacuum vessel. The vertical interstices between successive turns of the coil afford lineal, axial, high-vacuum passages between open mouths at top and bottom of said vessel, while the coil, being cold by virtue of thermal contact of its innermost turn with the nitrogen pot, affords expansive proximate condensation surfaces. (AEC)

  15. VACUUM TRAP

    DOEpatents

    Gordon, H.S.

    1959-09-15

    An improved adsorption vacuum trap for use in vacuum systems was designed. The distinguishing feature is the placement of a plurality of torsionally deformed metallic fins within a vacuum jacket extending from the walls to the central axis so that substantially all gas molecules pass through the jacket will impinge upon the fin surfaces. T fins are heated by direct metallic conduction, thereby ol taining a uniform temperature at the adeorbing surfaces so that essentially all of the condensible impurities from the evacuating gas are removed from the vacuum system.

  16. Chemotactic and enzyme-releasing activity of amphipathic proteins for neutrophils. A possible role for protease in chemotaxis on substratum-bound protein gradients.

    PubMed Central

    Wilkinson, P C; Bradley, G R

    1981-01-01

    The purified amphipathic proteins, alpha s 1-casein, beta-casein, and alkali-denatured serum albumin were studied for chemotactic and enzyme-releasing effects on human neutrophil leucocytes. Evidence for chemotaxis both in fluid-phase gradients and on solid-phase gradients was obtained using visual assays. In fluid-phase gradients, neutrophils showed good orientation to gradient sources of these proteins at concentrations of 10(-4) to 10(-5) M. Solid-phase gradients of casein and of denatured albumin were prepared on glass coverslips, and the locomotion of neutrophils attached to these coverslips was filmed by time-lapse cinematography. Displacement of neutrophils towards the highest concentration of substratum-bound protein was observed, suggesting that neutrophils can show true chemotaxis on a solid-phase gradient. All three proteins induced enzyme release from neutrophils in the absence of cytochalasin B. Lysozyme release was equivalent to that released by stimulation with formyl methionyl peptide in the presence of cytochalasin B, but the proteins stimulated a smaller release of beta-glucuronidase than the peptide. The proteins stimulated release of neutrophil proteases which were able to digest both casein and denatured albumin extracellularly. It is suggested that this proteolytic activity may assist locomotion of neutrophils, especially on solid-phase protein gradients, by cleaving membrane-attached protein, thus both freeing cell-surface receptors and allowing the cell to detach itself from the substratum and continue movement. Images Figure 1 PMID:7016748

  17. A Radical Break: Restraining Neutrophil Migration.

    PubMed

    Renkawitz, Jörg; Sixt, Michael

    2016-09-12

    When neutrophils infiltrate a site of inflammation, they have to stop at the right place to exert their effector function. In this issue of Developmental Cell, Wang et al. (2016) show that neutrophils sense reactive oxygen species via the TRPM2 channel to arrest migration at their target site. PMID:27623379

  18. Chronic neutrophilic leukaemia and plasma cell-related neutrophilic leukaemoid reactions.

    PubMed

    Bain, Barbara J; Ahmad, Shahzaib

    2015-11-01

    Many cases reported as 'chronic neutrophilic leukaemia' have had an associated plasma cell neoplasm. Recent evidence suggests that the great majority of such cases represent a neutrophilic leukaemoid reaction to the underlying multiple myeloma or monoclonal gammopathy of undetermined significance. We have analysed all accessible reported cases to clarify the likely diagnosis and to ascertain whether toxic granulation, Döhle bodies and an increased neutrophil alkaline phosphatase score were useful in making a distinction between chronic neutrophilic leukaemia and a neutrophilic leukaemoid reaction. We established that all these changes occur in both conditions. Toxic granulation and Döhle bodies are more consistently present in leukaemoid reactions but also occur quite frequently in chronic neutrophilic leukaemia. The neutrophil alkaline phosphatase score is increased in both conditions and is of no value in making a distinction. PMID:26218186

  19. Evasion of Neutrophil Killing by Staphylococcus aureus

    PubMed Central

    McGuinness, Will A.; Kobayashi, Scott D.; DeLeo, Frank R.

    2016-01-01

    Staphylococcus aureus causes many types of infections, ranging from self-resolving skin infections to severe or fatal pneumonia. Human innate immune cells, called polymorphonuclear leukocytes (PMNs or neutrophils), are essential for defense against S. aureus infections. Neutrophils are the most prominent cell type of the innate immune system and are capable of producing non-specific antimicrobial molecules that are effective at eliminating bacteria. Although significant progress has been made over the past few decades, our knowledge of S. aureus-host innate immune system interactions is incomplete. Most notably, S. aureus has the capacity to produce numerous molecules that are directed to protect the bacterium from neutrophils. Here we review in brief the role played by neutrophils in defense against S. aureus infection, and correspondingly, highlight selected S. aureus molecules that target key neutrophil functions. PMID:26999220

  20. Evasion of Neutrophil Killing by Staphylococcus aureus.

    PubMed

    McGuinness, Will A; Kobayashi, Scott D; DeLeo, Frank R

    2016-01-01

    Staphylococcus aureus causes many types of infections, ranging from self-resolving skin infections to severe or fatal pneumonia. Human innate immune cells, called polymorphonuclear leukocytes (PMNs or neutrophils), are essential for defense against S. aureus infections. Neutrophils are the most prominent cell type of the innate immune system and are capable of producing non-specific antimicrobial molecules that are effective at eliminating bacteria. Although significant progress has been made over the past few decades, our knowledge of S. aureus-host innate immune system interactions is incomplete. Most notably, S. aureus has the capacity to produce numerous molecules that are directed to protect the bacterium from neutrophils. Here we review in brief the role played by neutrophils in defense against S. aureus infection, and correspondingly, highlight selected S. aureus molecules that target key neutrophil functions. PMID:26999220

  1. How Neutrophils Shape Adaptive Immune Responses

    PubMed Central

    Leliefeld, Pieter H. C.; Koenderman, Leo; Pillay, Janesh

    2015-01-01

    Neutrophils are classically considered as cells pivotal for the first line of defense against invading pathogens. In recent years, evidence has accumulated that they are also important in the orchestration of adaptive immunity. Neutrophils rapidly migrate in high numbers to sites of inflammation (e.g., infection, tissue damage, and cancer) and are subsequently able to migrate to draining lymph nodes (LNs). Both at the site of inflammation as well as in the LNs, neutrophils can engage with lymphocytes and antigen-presenting cells. This crosstalk occurs either directly via cell–cell contact or via mediators, such as proteases, cytokines, and radical oxygen species. In this review, we will discuss the current knowledge regarding locations and mechanisms of interaction between neutrophils and lymphocytes in the context of homeostasis and various pathological conditions. In addition, we will highlight the complexity of the microenvironment that is involved in the generation of suppressive or stimulatory neutrophil phenotypes. PMID:26441976

  2. Extracellular calcium sensing and extracellular calcium signaling

    NASA Technical Reports Server (NTRS)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  3. Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils.

    PubMed

    Tateno, N; Matsumoto, N; Motowaki, T; Suzuki, K; Aratani, Y

    2013-05-01

    Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H2O2 treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H2O2 due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan. PMID:23438680

  4. Very-late-antigen-4 (VLA-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke.

    PubMed

    Neumann, Jens; Riek-Burchardt, Monika; Herz, Josephine; Doeppner, Thorsten R; König, Rebecca; Hütten, Heiko; Etemire, Eloho; Männ, Linda; Klingberg, Anika; Fischer, Thomas; Görtler, Michael W; Heinze, Hans-Jochen; Reichardt, Peter; Schraven, Burkhart; Hermann, Dirk M; Reymann, Klaus G; Gunzer, Matthias

    2015-02-01

    Neuronal injury from ischemic stroke is aggravated by invading peripheral immune cells. Early infiltrates of neutrophil granulocytes and T-cells influence the outcome of stroke. So far, however, neither the timing nor the cellular dynamics of neutrophil entry, its consequences for the invaded brain area, or the relative importance of T-cells has been extensively studied in an intravital setting. Here, we have used intravital two-photon microscopy to document neutrophils and brain-resident microglia in mice after induction of experimental stroke. We demonstrated that neutrophils immediately rolled, firmly adhered, and transmigrated at sites of endothelial activation in stroke-affected brain areas. The ensuing neutrophil invasion was associated with local blood-brain barrier breakdown and infarct formation. Brain-resident microglia recognized both endothelial damage and neutrophil invasion. In a cooperative manner, they formed cytoplasmic processes to physically shield activated endothelia and trap infiltrating neutrophils. Interestingly, the systemic blockade of very-late-antigen-4 immediately and very effectively inhibited the endothelial interaction and brain entry of neutrophils. This treatment thereby strongly reduced the ischemic tissue injury and effectively protected the mice from stroke-associated behavioral impairment. Behavioral preservation was also equally well achieved with the antibody-mediated depletion of myeloid cells or specifically neutrophils. In contrast, T-cell depletion more effectively reduced the infarct volume without improving the behavioral performance. Thus, neutrophil invasion of the ischemic brain is rapid, massive, and a key mediator of functional impairment, while peripheral T-cells promote brain damage. Acutely depleting T-cells and inhibiting brain infiltration of neutrophils might, therefore, be a powerful early stroke treatment. PMID:25391494

  5. Antioxidant, antimicrobial and neutrophil-modulating activities of herb extracts.

    PubMed

    Denev, Petko; Kratchanova, Maria; Ciz, Milan; Lojek, Antonin; Vasicek, Ondrej; Blazheva, Denitsa; Nedelcheva, Plamena; Vojtek, Libor; Hyrsl, Pavel

    2014-01-01

    The present study provides a comprehensive data on the antioxidant, antimicrobial and neutrophil-modulating activities of extracts from six medicinal plants--blackberry (Rubus fruticosus) leaves, chokeberry (Aronia melanocarpa) leaves, hawthorn (Crataegus monogyna) leaves, lady's mantle (Alchemilla glabra) aerial parts, meadowsweet (Filipendula ulmaria) aerial parts and raspberry (Rubus idaeus) leaves. In order to analyze the antioxidant activity of the herbs, several methods (ORAC, TRAP, HORAC and inhibition of lipid peroxidation) were used. Blackberry leaves and meadowsweet extracts revealed the highest antioxidant activities via all methods. All extracts studied blocked almost completely the opsonized zymosan particle-activated ROS production by neutrophils from human whole blood. On the other hand, the effect of extracts on phorbol myristate acetate-activated ROS production was much milder and even nonsignificant in the case of chokeberry leaves. This latter result suggests that extracts (apart from their antioxidative activity) interfere with the signaling cascade of phagocyte activation upstream of the protein kinase C activation. The antimicrobial activity of the investigated extracts against 11 human pathogens was investigated using three different methods. Meadowsweet and blackberry leaves extracts had the highest antimicrobial effect and the lowest minimal inhibiting concentrations (MICs) against the microorganisms tested. PMID:24945135

  6. Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase

    PubMed Central

    2013-01-01

    Background Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer. Results We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis. Conclusion We conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer. PMID:24321240

  7. Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism.

    PubMed

    Antoine, Francis; Girard, Denis

    2015-01-01

    Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis. PMID:24926560

  8. Activation of bovine neutrophils by Brucella spp.

    PubMed

    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. PMID:27436438

  9. Neutrophil gene expression in rheumatoid arthritis.

    PubMed

    Cross, Andrew; Bakstad, Denise; Allen, John C; Thomas, Luke; Moots, Robert J; Edwards, Steven W

    2005-10-01

    There is now a growing awareness that infiltrating neutrophils play an important role in the molecular pathology of rheumatoid arthritis. In part, this arises from the fact that neutrophils have potent cytotoxic activity, but additionally from the fact that inflammatory neutrophils can generate a number of cytokines and chemokines that can have a direct influence on the progress of an inflammatory episode. Furthermore, the molecular properties of inflammatory neutrophils are quite different from those normally found in the circulation. For example, inflammatory neutrophils, but not blood neutrophils, can express cell surface receptors (such as MHC Class II molecules and FcgammaRI) that dramatically alter the way in which these cells can interact with ligands to modulate immune function. Cytokine/chemokine expression and surface expression of these novel cell surface receptors is dependent upon the neutrophil responding to local environmental factors to selectively up-regulate the expression of key cellular components via signalling pathways coupled to transcriptional activation. However, major changes in the expression levels of some proteins are also regulated by post-translational modifications that alter rates of proteolysis, and hence changes in the steady-state levels of these molecules. PMID:16112850

  10. Proliferating cell nuclear antigen in neutrophil fate.

    PubMed

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  11. Characterization of the Hypercitrullination Reaction in Human Neutrophils and Other Leukocytes

    PubMed Central

    Zhou, Yebin; Di Pucchio, Tiziana; Sims, Gary P.; Mittereder, Nanette; Mustelin, Tomas

    2015-01-01

    Autoantibodies against citrullinated proteins are diagnostic for rheumatoid arthritis. However, the molecular mechanisms driving protein citrullination in patients with rheumatoid arthritis remain poorly understood. Using two independent western blotting methods, we report that agents that trigger a sufficiently large influx of extracellular calcium ions induced a marked citrullination of multiple proteins in human neutrophils, monocytes, and, to a lesser extent, T lymphocytes and natural killer cells, but not B lymphocytes or dendritic cells. This response required 250–1,000 μM extracellular calcium and was prevented by EDTA. Other neutrophil activating stimuli, such as formyl-peptides, GM-CSF, IL-6, IL8, TNFα, or phorbol ester, did not induce any detectable increase in protein citrullination, suggesting that receptor-induced calcium mobilization is insufficient to trigger hypercitrullination. We conclude that loss of membrane integrity and subsequent influx of high levels of calcium, which can be triggered by perforin released from cytotoxic cells or complement mediated formation of membrane attack complexes in the joints of rheumatoid arthritis patients, are sufficient to induce extensive protein citrullination in immune cells, notably neutrophils. This mechanism may provide the citrullinated autoantigens that drive autoimmunity in this devastating disease. PMID:26078491

  12. Neutrophil dysfunction and increased susceptibility to infection.

    PubMed

    Ottonello, L; Dapino, P; Pastorino, G; Dallegri, F; Sacchetti, C

    1995-09-01

    A critical evaluation of 3 years' experience using laboratory screening to detect neutrophil dysfunction is described. Neutrophil dysfunctions in patients with recurrent bacterial infections were investigated by using the following screening tests: (1) neutrophil chemotaxis towards N-formylmethionyl peptides (FMLP) and the complement fragment C5a; (2) neutrophil production of superoxide anions (O2-) in response to phorbol myristate acetate and opsonized zymosan particles; and (3) examination of May-Grünwald and myeloperoxidase cytochemical staining of peripheral blood smears. These tests were carried out in 100 patients suffering from infections and suspected of having altered neutrophil functional competence. A minority of patients was found to have well defined neutrophil dysfunction syndromes: chronic granulomatous disease (four cases), Chediak-Higashi disease (one case) and myeloperoxidase deficiency (one case). Of the remaining 94 patients, in whom infections localized to airways and/or skin predominated, 53 cases were found to have impaired chemotaxis (41 cases) or partial defects of the O2- production. Defects of chemotaxis toward FMLP and those towards both FLMP and C5a were the most frequent abnormalities. No defect was found in the other 41 patients. Moreover, impaired neutrophil chemotaxis was found in some patients with selective IgA deficiency (five cases) or immotile cilia syndrome (seven cases). The results suggest that (a) additional screening tests are required to ameliorate the efficiency of the diagnostic work-up of the patients suspected to have neutrophil dysfunction; and (b) further evaluation, also at the molecular level, should be considered at least in selected cases of non-classified neutrophil dysfunction in order to clarify diagnosis and plan rational therapeutic strategies. PMID:7498244

  13. Neutrophil-Mediated Phagocytosis of Staphylococcus aureus

    PubMed Central

    van Kessel, Kok P. M.; Bestebroer, Jovanka; van Strijp, Jos A. G.

    2014-01-01

    Initial elimination of invading Staphylococcus aureus from the body is mediated by professional phagocytes. The neutrophil is the major phagocyte of the innate immunity and plays a key role in the host defense against staphylococcal infections. Opsonization of the bacteria with immunoglobulins and complement factors enables efficient recognition by the neutrophil that subsequently leads to intracellular compartmentalization and killing. Here, we provide a review of the key processes evolved in neutrophil-mediated phagocytosis of S. aureus and briefly describe killing. As S. aureus is not helpless against the professional phagocytes, we will also highlight its immune evasion arsenal related to phagocytosis. PMID:25309547

  14. Synergistic protection against hyperoxia-induced lung injury by neutrophils blockade and EC-SOD overexpression

    PubMed Central

    2012-01-01

    Background Oxygen may damage the lung directly via generation of reactive oxygen species (ROS) or indirectly via the recruitment of inflammatory cells, especially neutrophils. Overexpression of extracellular superoxide dismutase (EC-SOD) has been shown to protect the lung against hyperoxia in the newborn mouse model. The CXC-chemokine receptor antagonist (Antileukinate) successfully inhibits neutrophil influx into the lung following a variety of pulmonary insults. In this study, we tested the hypothesis that the combined strategy of overexpression of EC-SOD and inhibiting neutrophil influx would reduce the inflammatory response and oxidative stress in the lung after acute hyperoxic exposure more efficiently than either single intervention. Methods Neonate transgenic (Tg) (with an extra copy of hEC-SOD) and wild type (WT) were exposed to acute hyperoxia (95% FiO2 for 7 days) and compared to matched room air groups. Inflammatory markers (myeloperoxidase, albumin, number of inflammatory cells), oxidative markers (8-isoprostane, ratio of reduced/oxidized glutathione), and histopathology were examined in groups exposed to room air or hyperoxia. During the exposure, some mice received a daily intraperitoneal injection of Antileukinate. Results Antileukinate-treated Tg mice had significantly decreased pulmonary inflammation and oxidative stress compared to Antileukinate-treated WT mice (p < 0.05) or Antileukinate-non-treated Tg mice (p < 0.05). Conclusion Combined strategy of EC-SOD and neutrophil influx blockade may have a therapeutic benefit in protecting the lung against acute hyperoxic injury. PMID:22816678

  15. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  16. Neutrophil chemotactic factor release and neutrophil alveolitis in asbestos-exposed individuals

    SciTech Connect

    Hayes, A.A.; Rose, A.H.; Musk, A.W.; Robinson, B.W.

    1988-09-01

    Alveolar neutrophil accumulation occurs in asbestosis. To evaluate a possible role for release of neutrophil chemotactic factor (NCF) in the pathogenesis of asbestosis, spontaneous NCF release from alveolar macrophages obtained by bronchoalveolar lavage (BAL) in eight individuals with asbestosis, 13 asbestos-exposed individuals without asbestosis, and five control subjects has been studied. Alveolar macrophages were incubated in medium (four hours; 37 degrees C), and neutrophil responses to the supernatants were assayed in a microchemotaxis chamber. Alveolar macrophages from subjects with asbestosis released more NCF (97 +/- 19 neutrophils per high-power field (N/HPF)) than controls (3 +/- 1 N/HPF; p less than 0.01). Alveolar macrophages from individuals with asbestos exposure and increased BAL neutrophil proportions (n = 7) released more NCF (93 +/- 24 N/HPF) than individuals with asbestos exposure and normal BAL neutrophil proportions (n = 6; 11 +/- 6 N/HPF; p less than 0.02). The results show that spontaneous NCF release occurs in asbestosis and that NCF release is associated with neutrophil alveolitis in asbestos-exposed individuals without asbestosis, suggesting a pathogenic role for NCF in mediating this neutrophil alveolitis. The results of the study also suggest that the presence of crackles is a better predictor of the presence of neutrophil alveolitis than is an abnormal chest x-ray film.

  17. Moesin regulates neutrophil rolling velocity in vivo.

    PubMed

    Matsumoto, Masanori; Hirata, Takako

    2016-01-01

    During inflammation, the selectin-induced slow rolling of neutrophils on venules cooperates with chemokine signaling to mediate neutrophil recruitment into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) and CD44 as E-selectin ligands that activate integrins to induce slow rolling. We show here that in TNF-α-treated cremaster muscle venules, slow leukocyte rolling was impaired in mice deficient in moesin, a member of the ezrin-radixin-moesin (ERM) family. Accordingly, neutrophil recruitment in a peritonitis model was decreased in moesin-deficient mice when chemokine signaling was blocked with pertussis toxin. These results suggest that moesin contributes to the slow rolling and subsequent recruitment of neutrophils during inflammation. PMID:27131737

  18. [Effect of erythromycin on neutrophil adhesion molecules].

    PubMed

    Kusano, S; Mukae, H; Morikawa, T; Asai, T; Sawa, H; Morikawa, N; Oda, H; Sakito, O; Shukuwa, C; Senju, R

    1993-01-01

    The mechanisms of erythromycin (EM) in chronic lower respiratory tract diseases including diffuse panbronchiolitis (DPB) has been reported. In this study we investigated the effect of EM on peripheral neutrophil adhesion molecules such as LFA-1 and Mac-1 obtained from six healthy subjects. Pretreatment of neutrophils with each concentration (10 ng/ml approximately 100 micrograms/ml) of EM resulted in no significant reduction in the expression of LFA-1 alpha, beta and Mac-1. Moreover, EM had no capability of reducing these expressions even when neutrophils were pretreated with 1 microgram/ml of EM at time from 0 to 60 min. These findings indicate that EM does not directly reduce the expression of LFA-1 alpha, beta and Mac-1 on peripheral neutrophil obtained from healthy subjects. PMID:8450276

  19. Rosette nanotubes inhibit bovine neutrophil chemotaxis

    PubMed Central

    Le, Minh Hong Anh; Suri, Sarabjeet Singh; Rakotondradany, Felaniaina; Fenniri, Hicham; Singh, Baljit

    2010-01-01

    Migration of activated neutrophils that have prolonged lifespan into inflamed organs is an important component of host defense but also contributes to tissue damage and mortality. In this report, we used biologically-inspired RGD-tagged rosette nanotubes (RNT) to inhibit neutrophil chemotaxis. We hypothesize that RGD-RNT will block neutrophil migration through inhibition of MAPK. In this report, RNT conjugated to lysine (K–RNT) and arginine-glycine-aspartic acid-serine-lysine (RGDSK-RNT) were co-assembled in a molar ratio of 95/5. The effect of the resulting composite RNT (RGDSK/K–RNT) on neutrophil chemotaxis, cell signaling and apoptosis was then investigated. Exposure to RGDSK/K–RNT reduced bovine neutrophil migration when compared to the non-treated group (p < 0.001). Similar effect was seen following treatment with ERK1/2 or p38 MAPK inhibitors. Phosphorylation of the ERK1/2 and p38 MAPK was inhibited at 5 min by RGDSK/K–RNT (p < 0.05). The RGDSD/K-RNT did not affect the migration of neutrophils pre-treated with αvβ3 integrin antibody suggesting that both bind to the same receptor. RGDSK/K–RNT did not induce apoptosis in bovine neutrophils, which was suppressed by pre-exposing them to LPS (p < 0.001). We conclude that RGDSK/K–RNT inhibit phosphorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils. PMID:20663476

  20. Neutrophils in asthma--a review.

    PubMed

    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. PMID:25511380

  1. What really happens in the neutrophil phagosome?

    PubMed Central

    Hurst, James K.

    2015-01-01

    Current viewpoints concerning the bactericidal mechanisms of neutrophils are reviewed from a perspective that emphasizes challenges presented by the inability to duplicate ex vivo the intracellular milieu. Among the challenges considered are the influences of confinement upon substrate availability and reaction dynamics, direct and indirect synergistic interactions between individual toxins, and bacterial responses to stressors. Approaches to gauging relative contributions of various oxidative and nonoxidative toxins within neutrophils using bacteria and bacterial mimics as intrinsic probes are also discussed. PMID:22609248

  2. Human neutrophils contain and bind high molecular weight kininogen.

    PubMed Central

    Gustafson, E J; Schmaier, A H; Wachtfogel, Y T; Kaufman, N; Kucich, U; Colman, R W

    1989-01-01

    Because plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HMWK), we determined whether HMWK could mediate kallikrein's association with neutrophils. HMWK antigen (237 +/- 61 ng HMWK/10(8) neutrophils) was present in lysates of washed human neutrophils. Little if any plasma HMWK was tightly bound and nonexchangeable with the neutrophil surface. Human neutrophils were found to possess surface membrane-binding sites for HMWK but no internalization was detected at 37 degrees C. 125I-HMWK binding to neutrophils was dependent upon Zn2+. Binding of 125I-HMWK to neutrophils was specific and 90% reversible. 125I-HMWK binding to neutrophils was saturable with an apparent Kd of 9-18 nM and 40,000-70,000 sites per cell. Upon binding to neutrophils, 125I-HMWK was proteolyzed by human neutrophil elastase (HNE) into lower relative molecular mass derivatives. Furthermore, HMWK found in neutrophils also served as a cofactor for HNE secretion because neutrophils deficient in HMWK have reduced HNE secretion when stimulated in plasma deficient in HMWK or with purified kallikrein. These studies indicate that human neutrophils contain a binding site for HMWK that could serve to localize plasma or neutrophil HMWK on their surface to possibly serve as a receptor for kallikrein and to participate in HNE secretion by this enzyme. Images PMID:2738152

  3. Mechanism Underlying Levofloxacin Uptake by Human Polymorphonuclear Neutrophils

    PubMed Central

    Vazifeh, Doina; Bryskier, André; Labro, Marie-Thérèse

    1999-01-01

    The mechanism of radiolabeled levofloxacin ([3H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentration ratio of about 4 to 6. Levofloxacin accumulated in PMNs parallel to the extracellular concentration, without saturation, over the range of 2.5 to 200 mg/liter (linear regression analysis: r = 0.92; P < 0.001). The activation energy was low (36 ± 7.2 kJ/mol). Levofloxacin uptake was increased in Ca2+-depleted, EGTA-containing medium by approximately 33% (P = 0.022), while Ni2+, a Ca2+ channel inhibitor, inhibited it in a concentration-dependent manner, with the concentration that inhibited 50% of control uptake being approximately 2.65 mM. Verapamil (an l-type Ca2+ channel inhibitor) and other pharmacologic agents which modify Ca2+ homeostasis did not modify levofloxacin uptake. Interestingly, Ca2+ and Mg2+ inhibited levofloxacin uptake in a concentration-dependent manner. EGTA, Ni2+, and verapamil did not modify levofloxacin efflux; thapsigargin, a Ca2+ pool-releasing agent, modestly increased the intracellular retention of levofloxacin. In addition, contrary to other fluoroquinolones, probenecid at 1 to 10 mM did not modify either levofloxacin uptake or efflux. These data are consistent with a mechanism of passive accumulation of levofloxacin in PMNs. Extracellular Ca2+ and Mg2+ may influence the structural conformation of levofloxacin or the lipophilicity of PMN membranes, thus explaining their effect on levofloxacin uptake. PMID:9925513

  4. Interactions of human neutrophils with leukotoxic streptococci.

    PubMed Central

    Sullivan, G W; Mandell, G L

    1980-01-01

    Most strains of Streptococcus pyogenes contain a toxin which can kill neutrophils. Previous workers failed to show any correlation between leukotoxin content and virulence of animals or humans. We examined the in vitro interactions of a leukotoxic streptococcus and a nonleukotoxic variant with human neutrophils. At ratios of 200 streptococcal colony-forming units per neutrophil, the toxic strain killed 92.8 +/- 2.0% of neutrophils, and the nontoxic strain killed only 9.0 +/- 1.2%. Despite this, ingestion of the two strains was equal. Postphagocytic oxidative metabolism was equivalent with low numbers of either toxic or nontoxic streptococci but depressed with high numbers of leukotoxic streptococci. At 20 min, neutrophils were able to kill leukotoxic (99.6 +/- 0.3% killed) and nonleukotoxic streptococci (99.5 +/- 0.2% killed) equally efficiently (P = 0.42). Thus, leukotoxicity does not interfere with the ability of neutrophils to destroy streptococci. This may explain why leukotoxicity does not appear to be an important factor in streptococcal virulence. Images Fig. 1 PMID:7002789

  5. Proteomic Analysis of Neutrophil Priming by PAF.

    PubMed

    Aquino, Elaine N; Neves, Anne C D; Santos, Karina C; Uribe, Carlos E; Souza, Paulo E N; Correa, José R; Castro, Mariana S; Fontes, Wagner

    2016-01-01

    Polymorphonuclear neutrophils are the main cells of the innate immunity inflammatory response. Several factors can activate or stimulate neutrophils, including platelet-activating factor (PAF), a lipid mediator. Some authors consider the activation induced by PAF priming because it triggers limited production of reactive oxygen species (ROS) and it amplifies the response of the cell to a subsequent activator. The stimulation is reversible, which is critical for modulating the inflammatory response. Exacerbated inflammatory responses lead to serious diseases, such as systemic inflammatory response syndrome (SIRS), among others. Characterizing the stimulation of neutrophils during the possible reversion or prevention of an exaggerated inflammatory response is critical for the development of control strategies. In this study, a proteomic approach was used to identify 36 proteins that differ in abundance between quiescent neutrophils and PAFstimulated neutrophils. The identified proteins were associated with increased DNA repair processes, calcium flux, protein transcription, cytoskeleton alterations that facilitate migration and degranulation, and the release of proinflammatory cytokines and proteins that modulate the inflammatory response. Some of the identified proteins have not been previously reported in neutrophils. PMID:26631175

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

    PubMed

    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. PMID:26608308

  7. Tendon Functional Extracellular Matrix

    PubMed Central

    Screen, H.R.C.; Birk, D.E.; Kadler, K.E.; Ramirez, F; Young, M.F.

    2015-01-01

    This article is one of a series, summarising views expressed at the Orthopaedic Research Society New Frontiers in Tendon Research Conference. This particular article reviews the three workshops held under the “Functional Extracellular Matrix” stream. The workshops focused on the roles of the tendon extracellular matrix, such as performing the mechanical functions of tendon, creating the local cell environment and providing cellular cues. Tendon is a complex network of matrix and cells, and its biological functions are influenced by widely-varying extrinsic and intrinsic factors such as age, nutrition, exercise levels and biomechanics. Consequently, tendon adapts dynamically during development, ageing and injury. The workshop discussions identified research directions associated with understanding cell-matrix interactions to be of prime importance for developing novel strategies to target tendon healing or repair. PMID:25640030

  8. Neutrophils in Cancer: Two Sides of the Same Coin

    PubMed Central

    Uribe-Querol, Eileen; Rosales, Carlos

    2015-01-01

    Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions. PMID:26819959

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

    SciTech Connect

    Wu, Yang-Chang; Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu; Lan, Yu-Hsuan; Chang, Fang-Rong; Chang, Ya-Wen; Hwang, Tsong-Long

    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.

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

    PubMed

    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

  11. Concanavalin A enhances phagocytosis and killing of Candida albicans by mice peritoneal neutrophils and macrophages.

    PubMed

    Loyola, Wagner; Gaziri, Daniel Augusto; Gaziri, Luis Carlos Jabur; Felipe, Ionice

    2002-07-12

    In this study we tested the hypothesis that after administration of a single intraperitoneal dose of concanavalin A (Con-A) to mice, the proportion of neutrophils and macrophages in the peritoneal exudate and their phagocytic and candidacidal activities should change with time. The number of neutrophils in the peritoneal exudate was greatly increased 6 h after administration of Con-A, and those cells were able to kill both intracellular and extracellular yeast and germ tube forms of Candida albicans. Addition of catalase to the culture medium reduced the killing of C. albicans, suggesting that the candidacidal activity depended on the myeloperoxidase system. The survival of mice pretreated with Con-A and submitted to an inoculum of C. albicans 6 h afterwards was twice higher than that of controls, which suggests that neutrophils were able to clear the experimental infection. One day after the treatment, the population of neutrophils in the exudate was about 45%, but after 2 days it was reduced to only 5% and the candidacidal activity was also reduced. After 4 days the exudate contained over 95% of macrophages, the candidacidal activity reached a maximum, and the phagocytosis mediated by both complement receptors and mannose receptors was increased. Uptake of FITC-mannose-BSA by macrophages was maximal on about the 4th day and was inhibited by mannan, suggesting that treatment with Con-A increased the activity of mannose receptors. These results support the hypothesis that activation of cellular immunity by Con-A occurred in two phases, one dominated by neutrophils, and the other by macrophages expressing increased activity of mannose receptors. PMID:12110482

  12. Ion Trap Mass Spectrometry

    SciTech Connect

    Eiden, Greg C.

    2005-09-01

    This chapter describes research conducted in a few research groups in the 1990s in which RF quadrupole ion trap mass spectrometers were coupled to a powerful atomic ion source, the inductively coupled plasma used in conventional ICP-MS instruments. Major section titles for this chapter are: RF Quadrupole Ion Traps Features of RF Quadrupole Ion Trap Mass Spectrometers Selective Ion Trapping methods Inductively Coupled Plasma Source Ion Trap Mass Spectrometers

  13. Neutrophilic and Pauci-immune Phenotypes in Severe Asthma.

    PubMed

    Panettieri, Reynold A

    2016-08-01

    Although 2 T-helper type 2 inflammation evokes airway hyperresponsiveness and narrowing, neutrophilic or pauci-immune asthma accounts for significant asthma morbidity. Viruses, toxicants, environmental tobacco smoke exposure, and bacterial infections induce asthma exacerbations mediated by neutrophilic inflammation or by structural cell (pauci-immune) mechanisms. Therapeutic challenges exist in the management of neutrophilic and pauci-immune phenotypes because both syndromes manifest steroid insensitivity. The recognition that neutrophil subsets exist and their functions are unique poses exciting opportunities to develop precise therapies. The conventional thought to target neutrophil activation or migration globally may explain why current drug development in neutrophilic asthma remains challenging. PMID:27401627

  14. Short-Term Heat Exposure Inhibits Inflammation by Abrogating Recruitment of and Nuclear Factor-κB Activation in Neutrophils Exposed to Chemotactic Cytokines

    PubMed Central

    Choi, Mira; Salanova, Birgit; Rolle, Susanne; Wellner, Maren; Schneider, Wolfgang; Luft, Friedrich C.; Kettritz, Ralph

    2008-01-01

    Cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-8 attract neutrophils into inflammatory sites. During emigration from the blood neutrophils interact with extracellular matrix proteins such as fibronectin. Fibronectin provides β2-integrin co-stimulation, allowing GM-CSF and IL-8 to activate nuclear factor (NF)-κB, an effect that does not occur in suspension. We tested the hypothesis that exposure of mice to fever-like temperatures abrogates neutrophil recruitment and NF-κB activation in a mouse model of skin inflammation. Mice that were exposed to 40°C for 1 hour showed strongly reduced GM-CSF- and IL-8-induced neutrophilic skin inflammation. In vitro heat exposure did not interfere with neutrophil adhesion or spreading on fibronectin but strongly inhibited migration toward both cytokines. Using specific inhibitors, we found that PI3-K/Akt was pivotal for neutrophil migration and that heat down-regulated this pathway. Furthermore, neutrophils on fibronectin showed abrogated NF-κB activation in response to GM-CSF and IL-8 after heat. In vivo heat exposure of mice followed by ex vivo stimulation of isolated bone marrow neutrophils confirmed these results. Finally, less NF-κB activation was seen in the inflammatory lesions of mice exposed to fever-like temperatures as demonstrated by in situ hybridization for IκBα mRNA. These new findings suggest that heat may have anti-inflammatory effects in neutrophil-dependent inflammation. PMID:18187571

  15. Exercise, training and neutrophil microbicidal activity.

    PubMed

    Smith, J A; Telford, R D; Mason, I B; Weidemann, M J

    1990-06-01

    The concentration in human plasma of putative neutrophil-"priming" cytokines like endogenous pyrogens is known to increase significantly in response to moderate exercise (11). This is characteristic of an acute-phase response. The ability of blood neutrophils isolated from both trained and untrained human subjects (n = 11, 9) to produce microbicidal reactive oxygen species was determined using luminol-enhanced chemiluminescence both before and after one hour of aerobic exercise at 60% VO2max. Irrespective of training and stimulus concentration, exercise nearly always caused significant "priming" of the capacity of neutrophils to produce H2O2 and HOCl upon stimulation with opsonized zymosan (P less than 0.01); however, compared to their untrained counterparts, the activity of cells isolated from trained individuals was depressed about 50% at unit stimulus concentration, both before and after exercise (P less than 0.075), whilst remaining unaltered at saturating concentrations. Although neutrophil oxygenation activity is only one parameter that contributes to immunological status, regular episodes of moderate exercise may increase resistance to infection by priming the "killing capacity" of neutrophils. In contrast, prolonged periods of intensive training may lead to increased susceptibility to common infections by diminishing this activity. PMID:2115507

  16. Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia.

    PubMed

    Hillgruber, Carina; Pöppelmann, Birgit; Weishaupt, Carsten; Steingräber, Annika Kathrin; Wessel, Florian; Berdel, Wolfgang E; Gessner, J Engelbert; Ho-Tin-Noé, Benoît; Vestweber, Dietmar; Goerge, Tobias

    2015-07-27

    Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients. PMID:26169941

  17. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

    PubMed Central

    Allen, Robert C.

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (1O2*) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism. PMID:26783542

  18. [Neuro-neutrophilic Disease and Dementia].

    PubMed

    Hisanaga, Kinya

    2016-04-01

    Neuro-neutrophilic diseases are multisystem inflammatory disorders that include neuro-Behçet and neuro-Sweet disease. These disorders ectopically damage the nervous system due to the abnormal chemotaxis of neutrophils. The neutrophils' chemotaxis is induced by oral muco-cutaneous bacterial infections and the dysregulation of cytokines, including interleukins. The frequencies of human leukocyte antigen (HLA)-B51 in neuro-Behçet disease and HLA-B54 as well as Cw1 in neuro-Sweet disease significantly higher than the levels present in Japanese normal controls. Notably, their frequencies are also higher in patients exhibiting neurological complications than in patients without neurological complications. These HLA types are considered risk factors that are directly related to the etiology of these diseases. Prednisolone and colchicine, which suppress neutrophil activation, are used to treat the acute phase of both diseases. Alternatively, dapsone is prescribed to prednisolone-dependent recurrent cases of neuro-Sweet disease. Dementia is a neurological symptom of these disorders, especially in the chronic progressive subtype of neuro-Behçet disease. Other immunosuppressant drugs, including methotrexate and infliximab, are administered to patients with the chronic progressive type of neuro-Behçet disease. Neuro-neutrophilic diseases are a form of dementia considered treatable. PMID:27056853

  19. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    PubMed

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. PMID:27109773

  20. Rapid Diagnostic Device for Subclinical Mastitis Based on Electrochemical Detection of Superoxide Produced from Neutrophils in Fresh Milk

    NASA Astrophysics Data System (ADS)

    Okada, Kohei; Fukuda, Junji; Suzuki, Hiroaki

    Electrochemical microdevices were fabricated to identify mastitic cows based on the increased number of neutrophils in raw milk. Because neutrophils produce superoxide (O2·-), the amount of O2·- can be used as an early indicator for subclinical mastitis. In the microdevices, O2·- was detected on a gold electrode using superoxide dismutase immobilized via a self-assembled monolayer of cysteine. In a preliminary test using xanthine oxidase to produce O2·-, one of the devices detected the production and rapid extinction of O2·-. When neutrophils obtained from a mastitic cow were concentrated by centrifugation and introduced into the device, a current increase distinctly different from the background was observed. Furthermore, a micropillar structure was fabricated on the gold electrode to trap and collect neutrophils, thereby facilitating the concentration of these cells around the electrode. The measured current clearly depended on the number of neutrophils in raw milk samples, demonstrating the applicability of the device for rapid diagnosis of subclinical mastitis.

  1. Phylogenic analysis of adhesion related genes Mad1 revealed a positive selection for the evolution of trapping devices of nematode-trapping fungi

    PubMed Central

    Li, Juan; Liu, Yue; Zhu, Hongyan; Zhang, Ke-Qin

    2016-01-01

    Adhesions, the major components of the extracellular fibrillar polymers which accumulate on the outer surface of adhesive traps of nematode-trapping fungi, are thought to have played important roles during the evolution of trapping devices. Phylogenetic analyses based on the genes related to adhesive materials can be of great importance for understanding the evolution of trapping devices. Recently, AoMad1, one homologous gene of the entomopathogenic fungus Metarhizium anisopliae cell wall protein MAD1, has been functionally characterized as involved in the production of adhesions in the nematode-trapping fungus Arthrobotrys oligospora. In this study, we cloned Mad1 homologous genes from nematode-trapping fungi with various trapping devices. Phylogenetic analyses suggested that species which formed nonadhesive constricting ring (CR) traps more basally placed and species with adhesive traps evolved along two lineages. Likelihood ratio tests (LRT) revealed that significant positive selective pressure likely acted on the ancestral trapping devices including both adhesive and mechanical traps, indicating that the Mad1 genes likely played important roles during the evolution of nematode-trapping fungi. Our study provides new insights into the evolution of trapping devices of nematode-trapping fungi and also contributes to understanding the importance of adhesions during the evolution of nematode-trapping fungi. PMID:26941065

  2. Decreased apoptosis of beta 2- integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, Hajime; Higuchi, Hidetoshi; Teraoka, Hiroki; Takahashi, Kenji; Takahashi, Kensi; Kuwabara, Mikinori; Inanami, Osamu; Kuwabara, Mikwori

    2004-02-01

    Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+]i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37 degrees C after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+]i, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates. PMID:14984592

  3. Major neutrophil functions subverted by Porphyromonas gingivalis

    PubMed Central

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil–P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  4. Defective neutrophi