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Sample records for acetate pma-stimulated neutrophils

  1. Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions of Agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells

    PubMed Central

    Boly, Rainatou; Franck, Thierry; Kohnen, Stephan; Lompo, Marius; Guissou, Innocent Pierre; Dubois, Jacques; Serteyn, Didier; Mouithys-Mickalad, Ange

    2015-01-01

    The ethyl acetate and n-butanolic subfractions of Agelanthus dodoneifolius were investigated for their antioxidant and antimyeloperoxidase (MPO) activities. The reactive oxygen species (ROS) generation was assessed by lucigenin-enhanced chemiluminescence (CL) and dichlorofluorescein- (DCF-) induced fluorescence techniques from phorbol myristate acetate- (PMA-) stimulated equine neutrophils and human myeloid cell line HL-60, respectively. In parallel, the effects of the tested subfractions were evaluated on the total MPO release by stimulated neutrophils and on the specific MPO activity by means of immunological assays. The results showed the potent activity of the butanolic subfraction, at least in respect of the chemiluminescence test (IC50 = 0.3 ± 0.1 µg/mL) and the ELISA and SIEFED assays (IC50 = 2.8 ± 1.2 µg/mL and 1.3 ± 1.0 µg/mL), respectively. However, the ethyl acetate subfraction was found to be the most potent in the DCF assay as at the highest concentration, DCF fluorescence intensity decreases of about 50%. Moreover, we demonstrated that the ethyl acetate subfraction was rich in catechin (16.51%) while it was not easy to identify the main compounds in the butanolic subfraction using the UPLC-MS/MS technique. Nevertheless, taken together, our results provide evidence that Agelanthus dodoneifolius subfractions may represent potential sources of natural antioxidants and of antimyeloperoxidase compounds. PMID:25821497

  2. Nimbidin suppresses functions of macrophages and neutrophils: relevance to its antiinflammatory mechanisms.

    PubMed

    Kaur, Gurpreet; Sarwar Alam, M; Athar, M

    2004-05-01

    Nimbidin is a mixture of tetranortriterpenes and is the major active principle of the seed oil of Azadirachta indica A. Juss (Meliaceae) possessing potent antiinflammatory and antiarthritic activities. The present study revealed that nimbidin significantly inhibited some of the functions of macrophages and neutrophils relevant to the inflammatory response following both in vivo and in vitro exposure. Oral administration of 5-25 mg/kg nimbidin to rats for 3 consecutive days significantly inhibited the migration of macrophages to their peritoneal cavities in response to inflammatory stimuli and also inhibited phagocytosis and phorbol-12-myristate-13-acetate (PMA) stimulated respiratory burst in these cells. In vitro exposure of rat peritoneal macrophages to nimbidin also inhibited phagocytosis and PMA stimulated respiratory burst in these cells. Nimbidin also inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS) stimulated macrophages following in vitro exposure, whereas interleukin 1 (IL-1) was only weakly inhibited. Probing the mechanism of NO inhibition revealed that nimbidin ameliorated the induction of inducible NO synthase (iNOS) without any inhibition in its catalytic activity. In addition, nimbidin also attenuated degranulation in neutrophils assessed in terms of release of beta-glucuronidase, myeloperoxidase and lysozyme. The results suggest that nimbidin suppresses the functions of macrophages and neutrophils relevant to inflammation. Thus nimbidin can be valuable in treating inflammation/inflammatory diseases. PMID:15174005

  3. 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

  4. 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

  5. Effect of perfluorochemical blood substitutes on human neutrophil function.

    PubMed

    Virmani, R; Fink, L M; Gunter, K; English, D

    1984-01-01

    This investigation was undertaken to determine the influence of perfluorochemical blood substitutes (PFCs) on human neutrophil function. Neutrophils isolated from blood of healthy donors were incubated at 37 degrees C for 1 hour with 25 percent Oxypherol (perfluorotributylamine) or Fluosol-DA (perfluorodecalin and perfluorotripropylamine) in the presence of fresh autologous serum. In comparison to cells incubated with Hank's balanced salt solution (buffer), neutrophils exposed to PFCs were markedly inhibited in their chemotactic and phagocytic responses. With 25 percent PFCs, chemotaxis to zymosan-activated serum was inhibited to approximately 25 percent of control by Fluosol-DA and 11 percent by Oxypherol. Phagocytosis of polystyrene beads in the presence of fresh serum was decreased to 52 and 50 percent of control by both Oxypherol and Fluosol-DA, respectively. Neutrophils exposed to PFCs aggregated slower and with an extended activation time upon addition of phorbol myristate acetate (PMA). When activated with n-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophils exposed to PFCs aggregated at a faster rate but with a longer lag phase in comparison to control cells. Neutrophil superoxide (O-2) release stimulated by PMA also was depressed by prior exposure of cells to Oxypherol (6 nmoles O-2/1.5 X 10(6) neutrophils) compared to buffer (32 nmoles O-2/1.5 X 10(6) neutrophils). PMA-stimulated neutrophil adherence was depressed significantly by prior exposure to Fluosol-DA compared to control. In contrast, Oxypherol had insignificant influence on stimulated adherence. Since PFCs have a profound influence on several important neutrophil functions, patients receiving PFC should be monitored closely for possible infectious complications. PMID:6087518

  6. An aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung inflammation in mice.

    PubMed

    Bachoual, Rafik; Talmoudi, Wifak; Boussetta, Tarek; Braut, Françoise; El-Benna, Jamel

    2011-06-01

    Punica granatum peel aqueous extract (PGE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. In this study we investigated the effect of PGE on human neutrophil reactive oxygen species (ROS) production in vitro and on LPS-induced lung inflammation in vivo in mice. Neutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H(2)O(2) was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethyl benzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. PGE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLF)- or phorbol myristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. PGE had no effect on superoxide anion generation, suggesting that it does not directly inhibit NADPH oxidase activity or activation pathways, or scavenge superoxide anions. PGE did not scavenge H(2)O(2) but directly inhibited myeloperoxidase activity in vitro. In vivo studies showed that PGE also attenuated LPS-induced lung inflammation in mice. So this study reveals that PGE inhibits neutrophil MPO activity and attenuates LPS-induced lung inflammation in mice. Inhibition of MPO activity by PGE could explain its anti-inflammatory action. PMID:21376769

  7. Achyrocline satureioides (Lam.) D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense

    PubMed Central

    Barioni, Eric Diego; Machado, Isabel Daufenback; Rodrigues, Stephen Fernandes de Paula; Ferraz-de-Paula, Viviane; Wagner, Theodoro Marcel; Cogliati, Bruno; Corrêa dos Santos, Matheus; Machado, Marina da Silva; de Andrade, Sérgio Faloni; Niero, Rivaldo; Farsky, Sandra Helena Poliselli

    2013-01-01

    Achyrocline satureioides (Lam.) D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4) expressions and phorbol-myristate-acetate- (PMA-) stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity. PMID:23476704

  8. Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.

    PubMed Central

    Heck, L. W.; Blackburn, W. D.; Irwin, M. H.; Abrahamson, D. R.

    1990-01-01

    To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2356859

  9. Hepatocyte injury by activated neutrophils in vitro is mediated by proteases.

    PubMed Central

    Harbrecht, B G; Billiar, T R; Curran, R D; Stadler, J; Simmons, R L

    1993-01-01

    OBJECTIVE: This study determined the mechanism used by neutrophils (PMNs) to induce hepatocellular injury. SUMMARY BACKGROUND DATA: Neutrophils have been shown to be potent mediators of cell and tissue injury and have been hypothesized to contribute to the hepatic injury that occurs after trauma and infection. Oxygen radical scavengers protect the liver in vivo from inflammatory injury and it has been suggested that PMNs are the source of these toxic oxygen radicals. The specific mechanism used by PMNs to produce hepatocellular damage, however, has not been determined. METHODS: Neutrophils were cultured in vitro with hepatocytes (HCs) and stimulated with phorbol 12-myristate 13-acetate (PMA) to induce HC injury in the presence of oxygen radical scavengers and protease inhibitors. RESULTS: PMA induced a PMN-mediated HC injury that was dependent on the number of PMNs present and the concentration of PMA. Protease inhibitors reduced the extent of HC injury, while oxygen radical scavengers had no effect. Hydrogen peroxide, directly applied, was able to injure HCs, but only at concentrations greater than those that could be produced by PMA-stimulated PMNs. CONCLUSIONS: PMNs are cytotoxic to cultured HCs, predominantly due to the release of proteolytic enzymes, while HCs appear relatively resistant to oxidative injury. Involvement of neutrophil toxic oxygen radicals in hepatic damage in vivo may require impairment of HC antioxidant defenses or may involve injury to nonparenchymal liver cells with secondary effects on HCs. PMID:8342991

  10. [Perfluorocarbon emulsions and other corpuscular systems influence on neutrophil activity].

    PubMed

    Shekhtman, D G; Safronova, V G; Sklifas, A N; Alovskaia, A A; Gapeev, A B; Obraztsov, V V; Chemeris, N K

    1997-01-01

    Influence of perfluorodecalin, perfluoromethilcyclohexylpiperidine, perfluorotributylamine emulsions on active oxygen form (AOF) generation by neutrophils has been studied. All investigated emulsions stabilized both proxanol 268 and egg yolk phospholipids inhibited PMA-stimulated neutrophil activity. Castor oil emulsion also inhibited the neutrophil activity. Neutrophil response for chemotactic peptide, was unchanged in the presence of all tested emulsions. We suppose that fast hydrophobic attachment of inert submicrone emulsion particles to cell surface provokes alteration of neutrophil plasma membrane function resulting in a decrease of AOF generation. PMID:9490112

  11. Lipoic acid suppression of neutrophil respiratory burst: effect of NADPH.

    PubMed

    O'Neill, Heidi C; Rancourt, Raymond C; White, Carl W

    2008-02-01

    Lipoic acid (LA) and its reduced product dihydrolipoic acid (DHLA) are potent antioxidants with capacity to scavenge reactive oxygen species (ROS) and recycle endogenous antioxidants. LA may increase cellular glutathione (GSH), an antioxidant lacking in the lung's epithelial lining fluid in lung disorders such as idiopathic pulmonary fibrosis (IPF). Neutrophils (PMN) are key innate responders and are pivotal in clearing bacterial infection, therefore it is crucial to understand the impact LA may have on their function. Circulating neutrophils were isolated from healthy volunteers and pretreated with LA or diluent. Cells were subsequently activated with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) to induce ROS production. SOD-inhibitable reduction of acetylated cytochrome c demonstrated the PMA-dependent respiratory burst was suppressed by LA. Oxygen consumption also was diminished when PMA-stimulated cells were pretreated with LA. PMN respiratory burst was partially restored by addition of NADPH but not other pyridine nucleotides. LA did not inhibit glucose-6-phosphate dehydrogenase activity of PMN. These data together suggest that the reduction of LA to DHLA using cellular NADPH may limit the capacity of the PMN NADPH oxidase to produce superoxide. Further studies will be required to determine if LA can diminish excessive superoxide produced by PMN and/or alveolar macrophages in IPF or relevant disease models in vivo. PMID:18158760

  12. Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate

    SciTech Connect

    Gardner, J.P.; Melnick, D.A.; Malech, H.L.

    1986-02-15

    The biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA) were examined. Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu- (/sup 125/I)iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on D-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product as receptor on the surface of unstimulated cells. These observations suggest that specific granule membranes contain large amounts of formyl peptide receptor, which is biochemically identical to that found on the cell surface and can be mobilized to the cell surface with appropriate stimulation.

  13. Effect of post-exercise protein-leucine feeding on neutrophil function, immunomodulatory plasma metabolites and cortisol during a 6-day block of intense cycling.

    PubMed

    Nelson, Andre R; Jackson, Lara; Clarke, Jim; Stellingwerff, Trent; Broadbent, Suzanne; Rowlands, David S

    2013-09-01

    Whey protein and leucine ingestion following exercise increases muscle protein synthesis and could influence neutrophil function during recovery from prolonged intense exercise. We examined the effects of whey protein and leucine ingestion post-exercise on neutrophil function and immunomodulators during a period of intense cycling. In a randomized double-blind crossover, 12 male cyclists ingested protein/leucine/carbohydrate/fat (LEUPRO 20/7.5/89/22 g h(-1), respectively) or isocaloric carbohydrate/fat control (CON 119/22 g h(-1)) beverages for 1-3 h post-exercise during 6 days of high-intensity training. Blood was taken pre- and post-exercise on days 1, 2, 4 and 6 for phorbol myristate acetate (PMA)-stimulated neutrophil superoxide (O2 (-)) production, immune cell counts, amino acid and lipid metabolism via metabolomics, hormones (cortisol, testosterone) and cytokines (interleukin-6, interleukin-10). During recovery on day 1, LEUPRO ingestion increased mean concentrations of plasma amino acids (glycine, arginine, glutamine, leucine) and myristic acid metabolites (acylcarnitines C14, myristoylcarnitine; and C14:1-OH, hydroxymyristoleylcarnitine) with neutrophil priming capacity, and reduced neutrophil O2 production (15-17 mmol O2 (-) cell(-1) ± 90 % confidence limits 20 mmol O2 (-) cell(-1)). On day 2, LEUPRO increased pre-exercise plasma volume (6.6 ± 3.8 %) but haematological effects were trivial. LEUPRO supplementation did not substantially alter neutrophil elastase, testosterone, or cytokine concentrations. By day 6, however, LEUPRO reduced pre-exercise cortisol 21 % (±15 %) and acylcarnitine C16 (palmitoylcarnitine) during exercise, and increased post-exercise neutrophil O2 (-) (33 ± 20 mmol O2 (-) cell(-1)), relative to control. Altered plasma amino acid and acylcarnitine concentrations with protein-leucine feeding might partly explain the acute post-exercise reduction in neutrophil function and increased exercise-stimulated neutrophil oxidative burst on

  14. Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp.

    PubMed

    Paula, Fabiana S; Kabeya, Luciana M; Kanashiro, Alexandre; de Figueiredo, Andréa S G; Azzolini, Ana Elisa C S; Uyemura, Sérgio A; Lucisano-Valim, Yara Maria

    2009-01-01

    The tamarind (Tamarindus indica L.) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC50 (in microg/10(6)cells)=115.7+/-9.7 (LumCL) and 174.5+/-25.9 (LucCL)], than the OZ- [IC50=248.5+/-23.1 (LumCL) and 324.1+/-34.6 (LucCL)] or fMLP-stimulated cells [IC50=178.5+/-12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O2 consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 microg/10(6)cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases. PMID:19022329

  15. Impact of simultaneous stimulation of 5-lipoxygenase and myeloperoxidase in human neutrophils.

    PubMed

    Zschaler, Josefin; Arnhold, Jürgen

    2016-04-01

    Human neutrophil 5-lipoxygenase (5-LOX) oxidizes arachidonic acid (AA) to 5S-hydro(pero)xy-6E,8Z,11Z, 14Z-eicosatetraenoic acid (5-H(p)ETE) and leukotriene (LT)A4, which is further converted to the chemoattractant LTB4. These cells contain also the heme enzyme myeloperoxidase (MPO) producing several potent oxidants such as hypochlorous acid (HOCl). Previously, it was shown that MPO-metabolites influence 5-LOX product formation. Here, we addressed the question, whether a simultaneous activation of MPO and 5-LOX in neutrophils results in comparable changes of 5-LOX activity. Human neutrophils were stimulated with H2O2 or phorbol 12-myristate 13-acetate (PMA) for MPO activation and subsequently treated with calcium ionophore A23187 inducing 5-LOX product formation on endogenous AA. Special attention was drawn to neutrophil vitality, formation of MPO-derived metabolites and redox status. The pre-stimulation with H2O2 resulted in a concentration-dependent increase in the ratio of 5-HETE to the sum of LTB4+6-trans-LTB4 in consequence of MPO activation. Thereby no impairment of cell vitality and only a slightly reduction of total glutathione level was observed. An influence of MPO on 5-LOX product formation could be suggested using an MPO inhibitor. In contrast, the pre-stimulation with PMA resulted in different changes of 5-LOX product formation leading to a reduced amount of 5-HETE unaffected by MPO inhibition. Furthermore, impaired cell vitality and diminished redox status was detected after PMA stimulation. Nevertheless, a MPO-induced diminution of LTB4 was obvious. Further work is necessary to define the type of 5-LOX modification and investigate the effect of physiological MPO activators. PMID:27033421

  16. Reactive oxygen species (ROS) production in human peripheral blood neutrophils exposed in vitro to static magnetic field.

    PubMed

    Poniedziałek, Barbara; Rzymski, Piotr; Karczewski, Jacek; Jaroszyk, Feliks; Wiktorowicz, Krzysztof

    2013-12-01

    The aim of this study was to determine the effect of gradient static magnetic field (SMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. Blood samples collected from healthy individuals were incubated in an inhomogeneous SMF (in a south or north pole of the field) for 15, 30 or 45 minutes. The maximum value of induction (B max) amounted to ≈ 60 mT. To determine the strength of the ROS production, dihydrorhodamine (123DHR) as fluorophore and phorbol 12-myristate 13-acetate (PMA) as respiratory burst stimulator were used. 123DHR oxidation by ROS was measured by flow cytometry. The exposure of blood samples to SMF induced statistically significant changes in ROS production in unstimulated and PMA-stimulated neutrophils. The observed effects were highly correlated with the exposure time and depended on the orientation of the field. Although intracellular mechanisms underlying such interactions are not thoroughly understood, it could be presumed that SMF affects ROS metabolic oscillations and their formation and inactivation. This study emphasizes the importance of proper adjustment of exposure time to SMF for any potential therapeutic applications. PMID:23631724

  17. 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

  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. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.

    PubMed

    Daigneault, Marc; Preston, Julie A; Marriott, Helen M; Whyte, Moira K B; Dockrell, David H

    2010-01-01

    Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD(3)) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD(3) and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. PMID:20084270

  20. Differential effects of nylon fibre adherence on the production of superoxide anion by human polymorphonuclear neutrophilic granulocytes stimulated with chemoattractants, ionophore A23187 and phorbol myristate acetate.

    PubMed Central

    Kownatzki, E; Uhrich, S

    1987-01-01

    Human polymorphonuclear neutrophilic granulocytes were made adherent by passing them over protein-coated nylon fibre columns and compared with suspended cells for their production of superoxide anion as measured by cytochrome C reduction. The cells were stimulated with chemotactic factors, the ionophore A 23187, and the tumour promoter phorbol myristate acetate. There was no increased O2-. production by adherent cells in the absence of a stimulus. Adherent cells produced considerably higher amounts of superoxide than suspended cells when stimulated with formyl-methionyl-leucyl-phenylalanine, ionophore A 23187, C5a, C5adesArg, and the platelet activating factor 1-o-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine. In contrast, stimulation with phorbol myristate acetate did not result in higher superoxide release from adherent than from suspended cells, and leukotriene B4 and a mononuclear cell-derived chemotaxin did not stimulate either cell to release significant amounts of superoxide. It is suggested that the augmented production of oxygen radicals with certain stimuli contributes to inflammatory symptoms in situations involving adherent granulocytes. PMID:2820637

  1. 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

  2. Protein phosphorylation in human neutrophils (PMN) induced by phorbol myristate acetate (PMA) and formyl-Met-Leu-Phe (fMLP)

    SciTech Connect

    Caldwell, S.E.; McPhail, L.C.; Bass, D.A.; McCall, C.E.

    1986-03-05

    Phosphorylation may regulate PMN responses to stimulation by PMA or fMLP. They examined differences in protein phosphorylation induced by either agent using cellular subfractionation. PMN were loaded with /sup 32/P, and stimulated with 100 ..mu..g/ml PMA for 20 min, or 10/sup -6/M fMLP + 10/sup -5/M cytochalasin B for 30 sec - conditions identical to those required for optimal stimulation of the respiratory burst by each agent. PMN were sonicated, and ultracentrifuged to yield cytosolic and particulate fractions. Protein samples were analyzed by SDS-gel electrophoresis, silver staining, and autoradiography. 43 phosphoproteins were identified in cytosolic fractions, and 40 in particulate fractions. In cytosolic fractions, as many as 22 phosphoproteins were enhanced by PMA stimulation and 12 with fMLP treatment. A cluster of phosphoproteins, approx. 38-43 kd, was enhanced by PMA, but not by fMLP, stimulation. As many as 6 particulate fraction proteins were more heavily phosphorylated with PMA treatment, and 11 with fMLP stimulation. In particulate fractions, a 26 kd phosphoprotein appeared upon PMA treatment, but not with fMLP. Characterization of the phosphoproteins which differ between the two treatments should aid in understanding the differential effects of PMA and fMLP on PMN function.

  3. Monoclonal Lym-1 antibody-targeted lysis of B lymphoma cells by neutrophils. Evidence for two mechanisms of FcgammaRII-dependent cytolysis.

    PubMed

    Ottonello, L; Epstein, A L; Mancini, M; Amelotti, M; Dapino, P; Dallegri, F

    2000-11-01

    Human neutrophils incubated with the anti-HLA-DR mAb Lym-1, plus PMA, induced significant cytolysis of B lymphoma cells compared with Lym-1 and PMA alone. The effect of PMA was independent of the ability of the compound to stimulate neutrophil-respiratory burst. In fact, first, neutrophils from a patient with chronic granulomatous disease were cytolytically effective in spite of their inability to produce oxidants. Second, various kinase inhibitors exerted different effects on the PMA-stimulated cytolytic system and neutrophil-oxidative burst. Previous studies have shown the involvement of the FcgammaRII, CD11b-CD18 integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was inhibited by the anti-FcgammaRII mAb IV.3, the anti-CD18 mAb MEM 48, and the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and N-acetyl-D-glucosamine. Furthermore, the PMA- and GM-CSF-stimulated cytolysis was insensitive and sensitive to inhibition by pertussis toxin, respectively. Thus, the use of PMA and GMCSF as neutrophil stimulants uncovers the existence of distinct mechanisms of Lym-1 mAb-mediated cytolysis. PMID:11073105

  4. Suppressed neutrophil function in children with acute lymphoblastic leukemia.

    PubMed

    Tanaka, Fumiko; Goto, Hiroaki; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Naruto, Takuya; Nishimaki, Shigeru; Yokota, Shumpei

    2009-10-01

    Infection is a major obstacle in cancer chemotherapy. Neutropenia has been considered to be the most important risk factor for severe infection; however, other factors, such as impaired neutrophil function, may be involved in susceptibility to infection in patients undergoing chemotherapy. In this study, we analyzed neutrophil function in children with acute lymphoblastic leukemia (ALL). Whole blood samples were obtained from 16 children with ALL at diagnosis, after induction chemotherapy, and after consolidation chemotherapy. Oxidative burst and phagocytic activity of neutrophils were analyzed by flow cytometry. Oxidative burst of neutrophils was impaired in ALL patients. The percentage of neutrophils with normal oxidative burst after PMA stimulation was 59.0 +/- 13.2 or 70.0 +/- 21.0% at diagnosis or after induction chemotherapy, respectively, which was significantly lower compared with 93.8 +/- 6.1% in healthy control subjects (P = 0.00004, or 0.002, respectively); however, this value was normal after consolidation chemotherapy. No significant differences were noted in phagocytic activity in children with ALL compared with healthy control subjects. Impaired oxidative burst of neutrophils may be one risk factor for infections in children with ALL, especially in the initial periods of treatment. PMID:19728023

  5. 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

  6. 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

  7. 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

  8. Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C.

    PubMed

    Kessels, G C; Krause, K H; Verhoeven, A J

    1993-06-15

    Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative

  9. 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

  10. Exogenous C2 Ceramide Suppresses Matrix Metalloproteinase Gene Expression by Inhibiting ROS Production and MAPK Signaling Pathways in PMA-Stimulated Human Astroglioma Cells.

    PubMed

    Jung, Ji-Sun; Ahn, Young-Ho; Moon, Byung-In; Kim, Hee-Sun

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which play a pivotal role in invasion, migration, and angiogenesis of glioma. Therefore, controlling MMPs is potentially an important therapeutic strategy for glioma. In the present study, we found that exogenous cell-permeable short-chain C2 ceramide inhibits phorbol myristate acetate (PMA)-induced MMP-1, -3, and -9 gene expressions in U87MG and U373MG human astroglioma cells. In addition, C2 ceramide inhibited the protein secretion and enzymatic activities of MMP-1, -3, and -9. The Matrigel invasion assay and wound healing assay showed that C2 ceramide suppresses the in vitro invasion and migration of glioma cells, which appears to be involved in strong inhibition of MMPs by C2 ceramide. Subsequent mechanistic studies revealed that C2 ceramide inhibits PMA-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor (NF)-κB/activator protein (AP)-1 DNA binding activities. Furthermore, C2 ceramide significantly inhibited PMA-induced reactive oxygen species (ROS) production and NADPH oxidase 4 (NOX4) expression, and inhibition of ROS by diphenylene iodonium (DPI, NADPH oxidase inhibitor) mimicked the effects of C2 ceramide on MMP expression and NF-κB/AP-1 via inhibition of p38 MAPK. The results suggest C2 ceramide inhibits MMP expression and glioma invasion, at least partly, by modulating ROS-p38 MAPK signaling axis and other MAPK signaling pathways. PMID:27043542

  11. Exogenous C2 Ceramide Suppresses Matrix Metalloproteinase Gene Expression by Inhibiting ROS Production and MAPK Signaling Pathways in PMA-Stimulated Human Astroglioma Cells

    PubMed Central

    Jung, Ji-Sun; Ahn, Young-Ho; Moon, Byung-In; Kim, Hee-Sun

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which play a pivotal role in invasion, migration, and angiogenesis of glioma. Therefore, controlling MMPs is potentially an important therapeutic strategy for glioma. In the present study, we found that exogenous cell-permeable short-chain C2 ceramide inhibits phorbol myristate acetate (PMA)-induced MMP-1, -3, and -9 gene expressions in U87MG and U373MG human astroglioma cells. In addition, C2 ceramide inhibited the protein secretion and enzymatic activities of MMP-1, -3, and -9. The Matrigel invasion assay and wound healing assay showed that C2 ceramide suppresses the in vitro invasion and migration of glioma cells, which appears to be involved in strong inhibition of MMPs by C2 ceramide. Subsequent mechanistic studies revealed that C2 ceramide inhibits PMA-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor (NF)-κB/activator protein (AP)-1 DNA binding activities. Furthermore, C2 ceramide significantly inhibited PMA-induced reactive oxygen species (ROS) production and NADPH oxidase 4 (NOX4) expression, and inhibition of ROS by diphenylene iodonium (DPI, NADPH oxidase inhibitor) mimicked the effects of C2 ceramide on MMP expression and NF-κB/AP-1 via inhibition of p38 MAPK. The results suggest C2 ceramide inhibits MMP expression and glioma invasion, at least partly, by modulating ROS-p38 MAPK signaling axis and other MAPK signaling pathways. PMID:27043542

  12. Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

    PubMed Central

    Sudbeck, B D; Baumann, P; Ryan, G J; Breitkopf, K; Nischt, R; Krieg, T; Mauch, C

    1999-01-01

    Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. PMID:10085241

  13. 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

  14. 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

  15. 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

  16. 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

  17. Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study

    PubMed Central

    Ranguelova, Kalina; Rice, Annette B.; Khajo, Abdelahad; Triquigneaux, Mathilde; Garantziotis, Stavros; Magliozzo, Richard S.; Mason, Ronald P.

    2012-01-01

    The objective of the present study is to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In the present study sulfite (•SO3−) and sulfate (SO4•−) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, PMA-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The latter adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO4•−) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation. PMID:22326772

  18. beta. -Endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

    SciTech Connect

    Diamant, M.; Henricks, P.A.J.; Nijkamp, F.P.; de Wied, D. )

    1989-01-01

    In the present study, the immunomodulatory effect of {beta}-endorphin ({beta}-E) and shorter pro-opiomelancortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity and their non-opioid derivatives were tested. With the exception of {alpha}-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10{sup {minus}17}M and higher than 10{sup {minus}8}M were without effect. {beta}-E and dT{beta}E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations. {gamma}-E and dT{gamma}E proved to be less potent inhibitors, reaching maximal effect at higher concentrations. DE{gamma}E exerted an even less pronounced but still significant suppressive effect at the concentration of 10{sup {minus}10}M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone.

  19. NRF2 Signaling Negatively Regulates Phorbol-12-Myristate-13-Acetate (PMA)-Induced Differentiation of Human Monocytic U937 Cells into Pro-Inflammatory Macrophages

    PubMed Central

    Choi, Hye-young; Choi, Bo-hyun; Kim, Sang-Tae; Heo, Tae-Hwe; Lee, Joo Young; Park, Pil-Hoon; Kwak, Mi-Kyoung

    2015-01-01

    Blood monocytes are recruited to injured tissue sites and differentiate into macrophages, which protect against pathogens and repair damaged tissues. Reactive oxygen species (ROS) are known to be an important contributor to monocytes’ differentiation and macrophages’ function. NF-E2-related factor 2 (NRF2), a transcription factor regulating cellular redox homeostasis, is known to be a critical modulator of inflammatory responses. We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA). In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Both transcript and protein levels for pro-inflammatory cytokines, including interleukine-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNFα) were highly elevated in PMA-stimulated NRF2-silenced U937 compared to the control. In addition, PMA-inducible secretion of monocyte chemotactic protein 1 (MCP-1) was significantly high in NRF2-silenced U937. As an underlying mechanism, we showed that NRF2-knockdown U937 retained high levels of cellular ROS and endoplasmic reticulum (ER) stress markers expression; and subsequently, PMA-stimulated levels of Ca2+ and PKCα were greater in NRF2-knockdown U937 cells, which caused enhanced nuclear accumulation of nuclear factor-ҡB (NFҡB) p50 and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Whereas the treatment of NRF2-silenced U937 cells with pharmacological inhibitors of NFҡB or ERK1/2 largely blocked PMA-induced IL-1β and IL-6 expression, indicating that these pathways are associated with cell differentiation. Taken together, our results suggest that the NRF2 system functions to suppress PMA-stimulated U937 cell differentiation into pro-inflammatory macrophages and provide evidence that the ROS-PKCα-ERK-NFҡB axis is involved in PMA-facilitated differentiation of NRF2-silenced U937 cells

  20. Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs. Role of oxygen radicals and granulocyte adherence.

    PubMed

    Shasby, D M; Shasby, S S; Peach, M J

    1983-01-01

    Human granulocytes and phorbol myristate acetate (PMA) increased permeability to albumin of monolayers of cultured endothelial cells grown on micropore filters. Granulocytes from a patient with chronic granulomatous disease and PMA did not increase endothelial permeability to albumin, demonstrating that the increase in permeability is dependent on granulocyte-derived oxygen radicals. When granulocytes were separated from the endothelial cells by a micropore filter, granulocytes and PMA no longer increased endothelial permeability to albumin, demonstrating that PMA-stimulated granulocytes must be closely approximated to endothelial cells to increase endothelial permeability. The relevance of these in vitro findings to an intact microvasculature was confirmed by demonstrating that agents that reduce granulocyte adherence to endothelium reduce edema formed in isolated lungs by granulocytes and PMA, an oxygen radical dependent process. Pretreatment of granulocytes with cytochalasin B or addition of 2% dextran to isolated lung perfusates reduced granulocyte adherence and markedly reduced edema formation in isolated lungs. These studies demonstrate that PMA-stimulated granulocytes must be closely apposed to endothelial cells to increase endothelial permeability through an oxygen-radical-dependent mechanism, and they suggest that reduction of granulocyte adherence may protect against granulocyte-dependent edema. PMID:6849554

  1. 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

  2. Acetaminophen prevents oxidative burst and delays apoptosis in human neutrophils.

    PubMed

    Freitas, Marisa; Costa, Vera M; Ribeiro, Daniela; Couto, Diana; Porto, Graça; Carvalho, Félix; Fernandes, Eduarda

    2013-05-23

    Acetaminophen is a frequently prescribed over-the-counter drug to reduce fever and pain in the event of inflammatory process. As neutrophils are relevant cells in inflammatory processes, the putative interaction of acetaminophen with these cells, if present, would be of paramount importance. The present study was undertaken to evaluate the effect of acetaminophen in human neutrophils' oxidative burst and lifespan in vitro. The obtained results demonstrate that acetaminophen efficiently modulates neutrophils' oxidative burst in phorbol myristate acetate-activated neutrophils, in a concentration-dependent manner, at in vivo relevant concentrations. It was clearly demonstrated that acetaminophen is a strong scavenger of HOCl and H2O2, which probably contributed to the effect observed in neutrophils. Acetaminophen also induced the depletion of glutathione in stimulated neutrophils, suggesting its transformation into a reactive intermediate. Obtained results further revealed that acetaminophen affects programmed cell death of human neutrophils, resulting in a delay of previously stimulated neutrophils-mediated apoptosis. Overall, our data suggested that acetaminophen has considerable potential to be included in anti-inflammatory therapeutic strategies, by preventing biological damage induced by an excessive production of reactive species generated in activated neutrophils and by extending the lifespan of neutrophils, favoring the elimination of pathogens, thus contributing to tissue healing and resolution of inflammation. PMID:23518321

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Ammonium acetate

    Integrated Risk Information System (IRIS)

    Ammonium acetate ; CASRN 631 - 61 - 8 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  10. Vinyl acetate

    Integrated Risk Information System (IRIS)

    Vinyl acetate ; CASRN 108 - 05 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  11. Ethyl acetate

    Integrated Risk Information System (IRIS)

    Ethyl acetate ; CASRN 141 - 78 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  12. Phenylmercuric acetate

    Integrated Risk Information System (IRIS)

    Phenylmercuric acetate ; CASRN 62 - 38 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinog

  13. Thallium acetate

    Integrated Risk Information System (IRIS)

    Jump to main content . Integrated Risk Information System Recent Additions | Contact Us Search : All EPA IRIS • You are here : EPA Home • Research • Environmental Assessment • IRIS • IRIS Summaries Redirect Page As of September 30 , 2009 , the assessment summary for Thallium acetate is included in t

  14. 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

  15. 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

  16. Expression and role of adhesion molecule CD18 on bovine neutrophils.

    PubMed

    Nagahata, H; Nochi, H; Tamoto, K; Noda, H; Kociba, G J

    1995-01-01

    Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions. PMID:7704836

  17. Expression and role of adhesion molecule CD18 on bovine neutrophils.

    PubMed Central

    Nagahata, H; Nochi, H; Tamoto, K; Noda, H; Kociba, G J

    1995-01-01

    Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions. Images Fig. 2. Fig. 3. Fig. 4. PMID:7704836

  18. 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

  19. [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

  20. 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

  1. Effects of phorbol 12-myristate 13-acetate on triglyceride and cholesteryl ester synthesis in cultured coronary smooth muscle cells and macrophages.

    PubMed

    Moinat, M; Chevey, J M; Muzzin, P; Giacobino, J P; Kossovsky, M

    1990-02-01

    In cultured pig coronary smooth muscle cells phorbol 12-myristate 13-acetate (PMA) stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and the incorporation of [2-3H]glycerol into triglycerides 6.4- and 4.5-fold, respectively. The maximal effects occurred after 3 h of treatment and there was a return to basal values after 72 h. In the presence of 400 microM oleic acid, PMA stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and that of [2-3H]glycerol into triglycerides 5.3- and 2.3-fold, respectively. The stimulatory effects were more sustained (still significant after 72 h) and their maxima were delayed (peaks after 24 h). PMA was also found to increase 2-fold the amount of triglyceride that accumulated in the cells in the presence of oleic acid after 24 h. In macrophages IC-21, the effects of PMA were observed only in the presence of oleic acid. They consisted of a 1.9-fold stimulation in the conversion of [4-14C]cholesterol into cholesteryl esters after 72 h and of a 1.7-fold stimulation in the incorporation of [2-3H]glycerol into triglycerides after 24 h. PMA also increased the amount of triglyceride that accumulated in the cells 1.9-fold after 72 h. It is concluded that PMA, and possibly growth factors, may promote lipid storage in smooth muscle cells and that fatty acids favor long lasting effects of PMA in smooth muscle cells and are necessary for any effect of PMA in macrophages. PMID:2324651

  2. 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

  3. [Inhibition of neutrophil adhesion by pectic galacturonans].

    PubMed

    Popov, S V; Ovodova, R G; Popova, G Iu; Nikitina, I R; Ovodov, Iu S

    2007-01-01

    The inhibition of the adhesion of neutrophils to fibronectin by the fragments of the main galacturonan chain of the following pectins was demonstrated: comaruman from the marsh cinquefoil Comarum polustre, bergenan from the Siberian tea Bergenia crassifolia, lemnan from the duckweed Lemna minor, zosteran from the seagrass Zostera marina, and citrus pectin. The parent pectins, except for comaruman, did not affect the cell adhesion. Galacturonans prepared from the starting pectins by acidic hydrolysis were shown to reduce the neutrophil adhesion stimulated by phorbol 12-myristate 13-acetate (1.625 microM) and dithiothreitol (0.5 mM) at a concentration of 50-200 microg/ml. The presence of carbohydrate chains with molecular masses higher than 300, from 100 to 300, and from 50 to 100 kDa in the galacturonan fractions was proved by membrane ultrafiltration. PMID:17375675

  4. 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

  5. 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

  6. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells.

    PubMed Central

    Weiss, S J; Young, J; LoBuglio, A F; Slivka, A; Nimeh, N F

    1981-01-01

    Human neutrophils stimulated with phorbol myristate acetate were able to destroy suspensions or monolayers of cultured human endothelial cells. Neutrophil-mediated cytotoxicity was related to phorbol myristate acetate concentration, time of incubation and neutrophil number. Cytolysis was prevented by the addition of catalase, while superoxide dismutase had no effect on cytotoxicity. The addition of the heme-enzyme inhibitors, azide or cyanide, markedly stimulated neutrophil-mediated damage while exogenous myeloperoxidase failed to stimulate cytolysis. Neutrophils isolated from patients with chronic granulomatous disease did not destroy the endothelial cell targets while myeloperoxidase-deficient neutrophils successfully mediated cytotoxicity. Endothelial cell damage mediated by the myeloperoxidase deficient cells was also inhibited by catalase but not superoxide dismutase. The addition of purified myeloperoxidase to the deficient cells did not stimulate cytotoxicity. Glucose-glucose oxidase, an enzyme system capable of generating hydrogen peroxide, could replace the neutrophil as the cytotoxic mediator. The addition of myeloperoxidase at low concentrations of glucose oxidase did not increase cytolysis, but at the higher concentrations of glucose oxidase it stimulated cytotoxicity. The destruction of endothelial cells by the glucose oxidase-myeloperoxidase system was inhibited by the addition of hypochlorous acid scavengers. In contrast, neutrophil-mediated cytolysis was not effectively inhibited by the hypochlorous acid scavengers. Based on these observations, we propose that human neutrophils can destroy cultured human endothelial cells by generating cytotoxic quantities of hydrogen peroxide. PMID:6268662

  7. 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

  8. Induction of neutrophil chemotactic factor production by staurosporine in rat peritoneal neutrophils

    PubMed Central

    Edamatsu, Takeo; Xiao, Yi-Qun; Tanabe, Jun-ichi; Mue, Suetsugu; Ohuchi, Kazuo

    1997-01-01

    Incubation of rat peritoneal neutrophils in medium containing various concentrations of staurosporine (6.4–64 nM) increased the neutrophil chemotactic activity in the conditioned medium in a time- and concentration-dependent manner. Separation of the neutrophil chemotactic activity in the conditioned medium by isoelectric focusing revealed that staurosporine (64 nM) stimulated the production of basic (pH>8) neutrophil chemotactic factors, while TPA (12-O-tetradecanoylphorbol 13-acetate, 49 nM) stimulated the production of both basic (pH>8) and acidic (pH 5) neutrophil chemotactic factors. Determination by immunoassay of cytokine-induced neutrophil chemoattractant (CINC)-1, -2α, -2β and -3 in the conditioned medium at 4 h revealed that staurosporine (64 nM) and TPA (49 nM) strongly stimulated the production of CINC-3 (staurosporine, 133.0±3.8; TPA, 26.7±1.0; control, 0.32±0.01 ng ml−1, means±s.e.mean from four samples) compared to CINC-1 (staurosporine, 55.0±1.2; TPA, 12.2±0.3; control, 0.56±0.01 ng ml−1), and CINC-2α (staurosporine, 1.09±0.03; TPA, 0.90±0.02; control, <0.10 ng ml−1). CINC-2β was below the detectable amount (<0.078 ng ml−1). The level of CINC-3 mRNA in the peritoneal neutrophils was determined by reverse transcription-polymerase chain reaction. Staurosporine (64 nM) and TPA (49 nM) enhanced the level of CINC-3 mRNA time-dependently, but had no effect on GAPDH mRNA levels. Production of staurosporine-induced neutrophil chemotactic factor was inhibited by the protein kinase C inhibitors, H-7 (IC50, 12.3 μM), calphostin C (IC50, 0.77 μM) and Ro 31-8425 (24.3% inhibition at 10 μM), and by the tyrosine kinase inhibitor, genistein (IC50, 68.5 μM). Production of TPA-induced neutrophil chemotactic factor was also inhibited by both inhibitors. Both the staurosporine- and the TPA-induced increase in CINC-3 mRNA levels were suppressed by H-7 and genistein. PMID:9283699

  9. 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

  10. 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

  11. Faropenem enhances superoxide anion production by human neutrophils in vitro.

    PubMed

    Sato, K; Sato, N; Shimizu, H; Tsutiya, T; Takahashi, H; Kakizaki, S; Takayama, H; Takagi, H; Mori, M

    1999-09-01

    Neutrophils are important cellular components in the defence against infections and many studies in vitro have shown that some antibiotics affect neutrophil function. We examined the effect of faropenem, a new oral penem antibiotic on neutrophil killing function by determining the generation of superoxide anion in vitro. The production of superoxide anion was measured by chemiluminescence amplified by a Cypridina luciferin analogue in the presence of N-formyl-Met-Leu-Phe (fMLP). Faropenem significantly enhanced chemiluminescence in a dose-dependent manner. The effect of faropenem was maximal at 5 min of incubation time and continued for at least 30 min. The effect of faropenem was also observed when neutrophils were stimulated by a calcium ionophore (ionomycin), while the effect of faropenem did not change in the presence of 12-O-tetra-decanoylphorbolmyristate acetate. Cytosol Ca2+ concentration ([Ca2+]i) monitored with Fura-2 increased in response to fMLP, however, faropenem did not influence the response of [Ca2+]i to fMLP. Our results suggest that faropenem enhanced the generation of superoxide anion by neutrophils, probably at the site where cytosol Ca2+ regulates NADPH oxidase. Faropenem might be potentially advantageous in the treatment of infections because a synergic interaction of antibodies and cytocidal neutrophils is necessary for the early eradication of the pathogenic bacteria. PMID:10511400

  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. 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

  14. 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

  15. 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...

  16. [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

  17. Oxidative product formation in irradiated neutrophils. A flow cytometric analysis

    SciTech Connect

    Wolber, R.A.; Duque, R.E.; Robinson, J.P.; Oberman, H.A.

    1987-03-01

    The effect of irradiation on neutrophil oxidative function was evaluated using a flow cytometric assay of intracellular hydrogen peroxide (H/sub 2/O/sub 2/) production. This assay quantitates the H/sub 2/O/sub 2/-dependent conversion of the nonfluorescent compound, 2'-7'-dichlorofluorescein (DCFH), into fluorescent 2'-7'-dichlorofluorescein (DCF) on a single-cell basis. Intracellular H/sub 2/O/sub 2/ production in response to stimulation with phorbol myristate acetate was not affected by neutrophil irradiation at doses up to 2500 rad. In addition, irradiation of intracellular DCFH and aqueous 2'-7'-dichlorofluorescein diacetate (DCFH-DA) resulted in DCF production, which suggested that oxidative molecules produced by aqueous radiolysis were detected by this assay. This study indicates that radiation doses of 1500 to 2500 rad, which are sufficient to prevent induction of graft-versus-host disease by transfused blood components, are not deleterious to neutrophil oxidative metabolism.

  18. Analysis of the functional characteristics of L-selectin and its expression on normal and CD18-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Yamashiki, N; Yamaguchi, M

    2000-06-01

    In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18-deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly increased (P < 0.05) in four normal calves at 5-20 min after in vivo administration of epinephrine; however, no significant increase of neutrophils was found in three calves with bovine leucocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from three calves with BLAD was 61-77% of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate caused a marked decrease in the expression of L-selectin on neutrophils from both normal and BLAD calves. The sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased. Following stimulation with aggregated IgG, the transient phase of [Ca2+]i in neutrophils from normal and BLAD calves was increased; however, the sustained phase of [Ca2+]i in BLAD neutrophils was significantly lower (P < 0.05) than that of controls. Sulfatide-induced O2- production and chemiluminescent response in neutrophils from calves with BLAD were 48-51% of those of normal calves and were inhibited by genistein and wortmannin, respectively, in a dose-dependent manner. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from BLAD calves stimulated with sulfatides was 57% of that of controls. The degree of L-selectin expression on neutrophils was correlated with the intracellular signalling events and the related superoxide production. PMID:10849115

  19. Bacterial lipopolysaccharides prime human neutrophils for enhanced production of leukotriene B4.

    PubMed Central

    Doerfler, M E; Danner, R L; Shelhamer, J H; Parrillo, J E

    1989-01-01

    Neutrophils can be "primed" for an enhanced respiratory burst by lipopolysaccharide (LPS) in concentrations measurable in patients with septic shock. Leukotriene B4 (LTB4) is the primary eicosanoid product of neutrophils and is felt to be a mediator of host defense and inflammation. We investigated the in vitro effects of LPS on neutrophil production of LTB4 and the omega-oxidation metabolites of LTB4. Incubation of neutrophils with LPS in concentrations ranging from 0.01 to 100 ng/ml did not result in production of LTB4 or metabolites in the absence of a second stimulus. Priming neutrophils with LPS and then stimulating with opsonized zymosan, phorbol-myristate-acetate or a low concentration of the calcium ionophore A23187 resulted in enhanced production of LTB4. LPS priming of neutrophils occurred in a concentration dependent manner. LPS did not result in LTB4 production in response to the chemoattractant peptide FMLP. LPS priming of neutrophils had no effect on cytosolic calcium concentrations of resting or zymosan-stimulated cells. These results suggest that LPS might effect host defense and tissue injury by potentiating the effect of other stimulants on neutrophil production of LTB4. This LPS induced enhancement may represent an important pathogenetic pathway in patients with gram negative sepsis. PMID:2537852

  20. Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils.

    PubMed

    Zheng, H Y; Hassett, D J; Bean, K; Cohen, M S

    1992-09-01

    We studied the effects of oxidant stress on the catalase activity and hydrogen peroxide sensitivity of Neisseria gonorrhoeae. N. gonorrhoeae is an obligate pathogen of man that evokes a remarkable but ineffective neutrophil response. Gonococci make no superoxide dismutase but express high catalase activity. Gonococcal catalase activity increased threefold when organisms were subjected to 1.0 mM hydrogen peroxide. This increase in catalase activity was marked by a parallel increase in protein concentration recognized by a rabbit polyclonal antibody raised against the purified gonococcal enzyme. Catalase was primarily localized to the gonococcal cytoplasm in the presence or absence of stress; only a single isoenzyme of catalase could be identified. Exposure of gonococci to neutrophil-derived oxidants was accomplished by stimulating neutrophils with phorbol myristate acetate or by using gonococcal Opa variants that interacted with neutrophils with different degrees of efficiency. Gonococci exposed to neutrophils demonstrated a twofold increase in catalase activity in spite of some reduction in viability. Exposure of gonococci to 1.0 mM hydrogen peroxide made the organisms significantly more resistant to higher concentrations of hydrogen peroxide and to neutrophils than control organisms. These results suggest that catalase is an important defense for N. gonorrhoeae during attack by human neutrophils. The rapid response of this enzyme to hydrogen peroxide should be taken into consideration in studies designed to evaluate the interaction between neutrophils and gonococci. PMID:1522209

  1. Interactions between neutrophils and Leishmania braziliensis amastigotes facilitate cell activation and parasite clearance

    PubMed Central

    Carlsen, Eric D.; Jie, Zuliang; Liang, Yuejin; Henard, Calvin A.; Hay, Christie; Sun, Jiaren; de Matos Guedes, Herbert; Soong, Lynn

    2015-01-01

    Leishmania braziliensis and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in South America. However, patient prognosis and the host immune response differ considerably depending on the infecting parasite species. The mechanisms underlying these differences appear to be multifactorial, with both host and parasite components contributing to disease outcome. Because neutrophils are a prominent component of the inflammatory infiltrate in chronic cutaneous, diffuse cutaneous, and mucocutaneous lesions, we examined neutrophil activation and microbicidal activity against amastigotes of L. amazonensis and L. braziliensis. We found that murine neutrophils internalized L. braziliensis amastigotes with greater efficiency than did L. amazonensis amastigotes. Additionally, L. braziliensis infection was a potent trigger for neutrophil activation, oxidative burst, degranulation, and production of IL-22 and IL-10, while L. amazonensis amastigotes poorly induced these responses. Finally, neutrophils were able to kill L. braziliensis amastigotes, especially when cells were activated with phorbol myristate acetate (PMA). L. amazonensis amastigotes, however, were highly resistant to neutrophil microbicidal mechanisms. This study reveals, for the first time, differential neutrophil responsiveness to distinct species of Leishmania amastigotes and highlights the complexity of neutrophil-amastigote interactions during chronic leishmaniasis. PMID:25766649

  2. Platelet activating factor amplifies human neutrophil adherence to bovine endothelial cells: evidence for a lipoxygenase dependent mechanism.

    PubMed

    Damtew, B; Spagnuolo, P J

    1992-10-01

    Platelet activating factor (PAF) is a potent lipid mediator that induces the release of leukotrienes and prostaglandins from various cells and tissues. We examined the capacity of PAF alone and in combination with soluble stimuli to enhance eicosanoid synthesis and adherence of human neutrophils. Neutrophils were preincubated with PAF and washed before exposure to the soluble stimuli F-Met-Leu-Phe (FMLP), calcium ionophore A23187, and phorbol myristate acetate. Preincubation of neutrophils with 1 microM PAF enhanced the release of both LTB4 and LTC4 in response to each of the three agonists, in contrast with the unprimed neutrophils. Priming was specific for PAF since lyso-PAF was inactive. Priming concentrations of PAF also augmented the adherence of neutrophils to endothelium in the presence of the soluble agonists A23187, phorbol myristate acetate, and FMLP. The priming effect of PAF on eicosanoid release and neutrophil adherence was shown to have similar time- and dose-dependent effects. Further, the priming effects of PAF on adherence could be reversed by preincubation of neutrophils with the lipoxygenase inhibitors nordihydroguiaretic acid and 5,8,11,14-ETYA but not by preincubation with the cyclooxygenase inhibitor indomethacin. These data demonstrate that PAF amplifies neutrophil adherence to endothelium through a lipoxygenase dependent mechanism. PMID:1330924

  3. Treatment with rhDNase in patients with cystic fibrosis alters in-vitro CHIT-1 activity of isolated leucocytes.

    PubMed

    Weckmann, M; Schultheiss, C; Hollaender, A; Bobis, I; Rupp, J; Kopp, M V

    2016-09-01

    Recent data suggest a possible relationship between cystic fibrosis (CF) pharmacotherapy, Aspergillus fumigatus colonization (AC) and/or allergic bronchopulmonary aspergillosis (ABPA). The aim of this study was to determine if anti-fungal defence mechanisms are influenced by CF pharmacotherapy, i.e. if (1) neutrophils form CF and non-CF donors differ in their ability to produce chitotriosidase (CHIT-1); (2) if incubation of isolated neutrophils with azithromycin, salbutamol, prednisolone or rhDNase might influence the CHIT-1 activity; and (3) if NETosis and neutrophil killing efficiency is influenced by rhDNase. Neutrophils were isolated from the blood of CF patients (n = 19; mean age 26·8 years or healthy, non-CF donors (n = 20; 38·7 years) and stimulated with phorbol-12-myristate-13-acetate (PMA), azithromycin, salbutamol, prednisolone or rhDNase. CHIT-1 enzyme activity was measured with a fluorescent substrate. NETosis was induced by PMA and neutrophil killing efficiency was assessed by a hyphae recovery assay. Neutrophil CHIT-1 activity was comparable in the presence or absence of PMA stimulation in both CF and non-CF donors. PMA stimulation and preincubation with rhDNase increased CHIT-1 activity in culture supernatants from non-CF and CF donors. However, this increase was significant in non-CF donors but not in CF patients (P < 0·05). RhDNase reduced the number of NETs in PMA-stimulated neutrophils and decreased the killing efficiency of leucocytes in our in-vitro model. Azithromycin, salbutamol or prednisolone had no effect on CHIT-1 activity. Stimulation of isolated leucocytes with PMA and treatment with rhDNase interfered with anti-fungal defence mechanisms. However, the impact of our findings for treatment in CF patients needs to be proved in a clinical cohort. PMID:27324468

  4. 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

  5. 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

  6. 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

  7. Various Molecular Species of Diacylglycerol Hydroperoxide Activate Human Neutrophils via PKC Activation

    PubMed Central

    Kambayashi, Yasuhiro; Takekoshi, Susumu; Tanino, Yutaka; Watanabe, Keiichi; Nakano, Minoru; Hitomi, Yoshiaki; Takigawa, Tomoko; Ogino, Keiki; Yamamoto, Yorihiro

    2007-01-01

    We have proposed that diacylglycerol hydroperoxide-induced unregulated signal transduction causes oxidative stress-related diseases. In this study, we investigated which molecular species of diacylglycerol hydroperoxide activated human peripheral neutrophils. All diacylglycerol hydroperoxides, diacylglycerol hydroxides, and diacyglycerols tested in the present study induced superoxide production by neutrophils. The ability to activate neutrophils among molecular species containing the same fatty acid composition was as follows; diacylglycerol hydroperoxide>diacylglycerol hydroxide≥diacylglycerol. The diacylglycerol hydroperoxide composed of linoleate was a stronger activator for neutrophils than that composed of arachidonate. 1-Palmitoyl-2-linoleoylglycerol hydroperoxide (PLG-OOH) was the strongest stimulator for neutrophils. We reconfirmed that PLG-OOH activated protein kinase C (PKC) in neutrophils. PLG-OOH induced the phosphorylation of p47phox, a substrate of PKC and a cytosolic component of NADPH oxidase, in neutrophils, as did N-formyl-methionyl-leucyl-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. Moreover, the time course of p47phox phosphorylation was comparable to that of superoxide production. These results suggest that PLG-OOH activated intracellular protein kinase C. PLG-OOH, produced via an uncontrolled process, can act as a biological second messenger to cause inflammatory disease from oxidative stress. PMID:18392102

  8. 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

  9. 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

  10. Preparation of vinyl acetate

    DOEpatents

    Tustin, Gerald Charles; Zoeller, Joseph Robert; Depew, Leslie Sharon

    1998-01-01

    This invention pertains to the preparation of vinyl acetate by contacting a mixture of hydrogen and ketene with a heterogeneous catalyst containing a transition metal to produce acetaldehyde, which is then reacted with ketene in the presence of an acid catalyst to produce vinyl acetate.

  11. Preparation of vinyl acetate

    DOEpatents

    Tustin, G.C.; Zoeller, J.R.; Depew, L.S.

    1998-03-24

    This invention pertains to the preparation of vinyl acetate by contacting a mixture of hydrogen and ketene with a heterogeneous catalyst containing a transition metal to produce acetaldehyde, which is then reacted with ketene in the presence of an acid catalyst to produce vinyl acetate.

  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. 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

  15. 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

  16. Complement component C3b and immunoglobulin Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

    PubMed

    Worku, M; Paape, M J; Di Carlo, A; Kehrli, M E; Marquardt, W W

    1995-04-01

    Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence-related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (LAD) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from LAD, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (CL) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of CL was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from LAD-affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and LAD-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between LAD-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from LAD-affected calves.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7785817

  17. Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils.

    PubMed

    Sallenave, J M; Si Tahar, M; Cox, G; Chignard, M; Gauldie, J

    1997-06-01

    Secretory leukocyte proteinase inhibitor (SLPI) is the main neutrophil elastase (HLE) inhibitor found in the upper airways during pulmonary inflammation. It has been shown to be synthesized and secreted in vitro by epithelial cells and has been localized in tracheal glands and bronchiolar epithelial cells by immunocytochemistry. In this study, using immunodetection and immunopurification techniques with specific anti-SLPI immunoglobulin G (IgG), we show that SLPI is present as a native 14-kDa molecule in neutrophil cytosol. In addition, we demonstrate that SLPI is the major inhibitor of HLE present in neutrophil cytosol because pre-incubation with specific anti-SLPI IgG was able to inhibit completely the anti-HLE activity of the cytosol. SLPI can be secreted (probably in an inactive form) by neutrophils and its secretion is enhanced when the cells are stimulated with phorbol myristate acetate (PMA). Elafin, an elastase-specific inhibitor, is also present in minute amounts in neutrophil cytosol and its secretion can be up-regulated. The presence of SLPI in the cytosol of neutrophils may serve as a protective screen against proteinases spilling from azurophilic granules. An alternative or supplementary role may be the maintenance of a differentiated phenotype. PMID:9201260

  18. Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus

    PubMed Central

    Ridzuan, Noridzzaida; John, Cini Mathew; Sandrasaigaran, Pratheep; Maqbool, Maryam; Liew, Lee Chuen; Lim, Jonathan; Ramasamy, Rajesh

    2016-01-01

    AIM: To assess the amount and pattern of reactive oxygen species (ROS) production in diabetic patient-derived neutrophils. METHODS: Blood samples from type 2 diabetes mellitus (DM) patients and volunteers (controls) were subjected to neutrophil isolation and the assessment of neutrophil oxidative burst using chemiluminescence assay. Neutrophils were activated by using phorbol myristate acetate (PMA) and neutrophils without activation were kept as a negative control. The chemiluminescence readings were obtained by transferring cell suspension into a 1.5 mL Eppendorf tube, with PMA and luminol. Reaction mixtures were gently vortexed and placed inside luminometer for a duration of 5 min. RESULTS: Our results showed that in the resting condition, the secretion of ROS in normal non-diabetic individuals was relatively low compared to diabetic patients. However, the time scale observation revealed that the secreted ROS declined accordingly with time in non-diabetic individuals, yet such a reduction was not detected in diabetic patients where at all the time points, the secretion of ROS was maintained at similar magnitudes. This preliminary study demonstrated that ROS production was significantly higher in patients with DM compared to non-diabetic subjects in both resting and activated conditions. CONCLUSION: The respiratory burst activity of neutrophils could be affected by DM and the elevation of ROS production might be an aggravating factor in diabetic-related complications. PMID:27433296

  19. 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

  20. 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

  1. 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

  2. Phosphoproteins and the activation of the neutrophil respiratory burst oxidase

    SciTech Connect

    Okamura, N.; Curnutte, J.T.; Babior, B.M.

    1987-05-01

    The respiratory burst oxidase is a neutrophil enzyme that converts oxygen to O/sub 2//sup -/. It is dormant in resting cells but is activated when the cells are exposed to phorbol myristate acetate (PMA). PMA also induces the incorporation of /sup 32/P into certain neutrophil proteins. To determine whether phosphorylation of these proteins is related to oxidase activation, protein phosphorylation was studied in patients with chronic granulomatous disease (GCD), a group of inherited conditions in which oxidase activity is missing. In normals, neutrophil activation by PMA is associated with the phosphorylation inter alia of 48K proteins at pI 7.3 and 7.8. There is also inconstant phosphorylation of a 48K protein at pI 6.8. In 4 patients with X-linked chronic granulomatous disease (CGD), phosphorylation of pp48/6.8 and pp48/7.3 was absent, while in autosomal recessive CGD, phosphorylation of all 3 of these proteins was absent in 3 patients and significantly diminished in a fourth. These results suggest that the phosphorylation of these proteins is related to the activation of the respiratory burst oxidase. By peptide mapping, these 3 proteins appear to consist of a single peptide species whose pI variability may be due to post-translational modification. The only phosphoamino acid found in pp48/7.3 was phosphoserine.

  3. 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

  4. 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

  5. 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

  6. Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition.

    PubMed

    Jancinová, Viera; Perecko, Tomás; Nosál, Radomír; Kostálová, Daniela; Bauerová, Katarína; Drábiková, Katarína

    2009-06-10

    Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1-100 microM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane. PMID:19371737

  7. 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.

  8. 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

  9. Interferon-γ enhances phorbol myristate acetate-induced cell attachment and tumor necrosis factor production via the NF-κB pathway in THP-1 human monocytic cells.

    PubMed

    Kurihara, Yuichi; Furue, Masutaka

    2013-06-01

    During inflammation, activated macrophages express adhesion molecules and produce cytokines that interact with other hematopoietic and stromal cells. THP-1 non-adherent human monocytic cells differentiate into plastic-adherent macrophages via αVβ3 integrin, by ERK activation in the presence of phorbol myristate acetate (PMA). This has proven to be a valuable model for investigating functional monocyte/macrophage diversity. Interferon-γ (IFN-γ) is a Th1-cytokine that is crucial in macrophage activation. In this study, we investigated the effects of IFN-γ on adhesion and the secretion of tumor necrosis factor (TNF) by PMA-stimulated THP-1 cells. IFN-γ is incapable of inducing cell attachment and TNF production; however, it cumulatively upregulated PMA-induced basal adhesion and TNF production. IFN-γ increased αV integrin, ICAM-1 and VCAM-1 expression and among these PMA-induced cell surface adhesion molecules, the blocking antibody for αV integrin suppressed adhesion and TNF production. Furthermore, IFN-γ enhanced PMA-induced NF-κB phosphorylation and not ERK phosphorylation. Accordingly, the NF-κB pathway inhibitor (BAY 11-7082) inhibited the enhancing effect of IFN-γ on adhesion and TNF production. By contrast, the MEK inhibitor (U0126) almost completely eliminated PMA-induced basal adhesion and TNF production. In conclusion, IFN-γ regulates macrophage activation by mediating the NF-κB signaling pathway. PMID:23589028

  10. 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

  11. 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

  12. Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Nochi, H; Tamoto, K; Araiso, T; Noda, H; Kociba, G J

    1996-09-01

    1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated. PMID:8880976

  13. Human neutrophil adhesion to bovine aortic endothelium. Evidence for endothelial lipoxygenase activity.

    PubMed

    Damtew, B; Goldsmith, G; Tserng, K Y; Spagnuolo, P J

    1993-06-01

    We examined the effect of phorbol myristate acetate on cultured bovine aortic endothelial cells to determine the role of endothelial cells in neutrophil-endothelial cell adhesive interactions. Confluent endothelial cells were preincubated with phorbol myristate acetate and other inflammatory signals including N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe), the ionophore A23187, and thrombin; washed extensively; and incubated with 51Cr-labeled neutrophils. Preincubation of endothelium with A23187, phorbol ester, or thrombin increased adherence of neutrophils by 3.1-, 5.7-, and 3.7-fold over baseline. In contrast, f-Met-Leu-Phe preincubation failed to increase adhesion over baseline. Supernatants from endothelium preincubated with phorbol failed to augment adherence of untreated endothelial cells. Preincubation of endothelium with lipoxygenase inhibitors nordihydroguaiaretic acid (50 microM), 5,8,11,14-eicosatetraenoic acid (50 microM), and BW755C (50 microM) inhibited the effect of phorbol preincubation of endothelium significantly by 55, 27, and 22%, respectively. In contrast, inhibitors of cyclooxygenase and thromboxane synthase or thromboxane receptor antagonists had no effect on phorbol-induced adhesion. Specific desensitization of neutrophil adhesion to phorbol-treated endothelium could be demonstrated by prior exposure of neutrophils to low concentrations of leukotriene B4 (3.8 x 10(-10) M). Endothelium preincubated with phorbol but not f-Met-Leu-Phe or thrombin produced several fatty acid peaks at 280 nm, one of which comigrated with authentic leukotriene B4 (LTB4). This peak, isolated and purified, increased endothelial cell adherence in a temporal fashion in the same way as LTB4 and was demonstrated to be LTB4 by ultraviolet spectroscopy, high-performance liquid chromatography, and mass spectroscopy. These data demonstrate that endothelial cell-derived lipoxygenase metabolites, in particular LTB4, are involved, in part, in the acute regulation of

  14. 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

  15. 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

  16. 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

  17. Activation and regulation of arachidonic acid release in rabbit peritoneal neutrophils

    SciTech Connect

    Tao, W.

    1988-01-01

    Arachidonic acid release in rabbit neutrophils can be enhanced by the addition of chemotactic fMet-Leu-Phe, platelet-activating factor, PAF, or the calcium ionophore A23187. Over 80% of the release ({sup 3}H)arachidonic acid comes from phosphatidylcholine and phosphatidylinositol. The release is dose-dependent and increases with increasing concentration of the stimulus. The A23187-induced release increases with increasing time of the stimulation. ({sup 3}H)arachidonic acid release, but not the rise in the concentration of intracellular calcium, is inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The ({sup 3}H)arachidonic acid released by A23187 is potentiated while that release by fMET-Leu-Phe or PAF is inhibited in phorbol 12-myristate 13-acetate, PMA, treated rabbit neutrophils. The protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine, H-7, has no effect on the potentiation by PMA of the A23187-induced release, it prevents the inhibition by PMA of the release produced by PAF or fMet-Leu-Phe. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. The diacylglycerol kinase inhibitor R59022 increases the level of diacylglycerol in neutrophils stimulated with fMet-Leu-Phe. Furthermore, R59022 potentiates ({sup 3}H) arachidonic acid release produced by fMet-Leu-Phe. This potentiation is not inhibited by H-7, in fact, it is increased in H-7-treated neutrophils.

  18. 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

  19. 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

  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. 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

  2. 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

  3. Acetate Dependence of Tumors

    PubMed Central

    Comerford, Sarah A.; Huang, Zhiguang; Du, Xinlin; Wang, Yun; Cai, Ling; Witkiewicz, Agnes; Walters, Holly; Tantawy, Mohammed N.; Fu, Allie; Manning, H. Charles; Horton, Jay D.; Hammer, Robert E.; McKnight, Steven L.; Tu, Benjamin P.

    2014-01-01

    SUMMARY Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation and the regulation of gene expression. How highly glycolytic or hypoxic tumors are able to produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions remains poorly understood. Here we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors. PMID:25525877

  4. 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

  5. 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.

  6. Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1.

    PubMed Central

    Tözeren, A; Mackie, L H; Lawrence, M B; Chan, P Y; Dustin, M L; Springer, T A

    1992-01-01

    This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton. Images FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 8 FIGURE 9 PMID:1358239

  7. Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

    PubMed Central

    Mykoniatis, Andreas; Shen, Le; Fedor-Chaiken, Mary; Tang, Jun; Tang, Xu; Worrell, Roger T.; Delpire, Eric; Turner, Jerrold R.; Matlin, Karl S.

    2010-01-01

    In secretory epithelial cells, the basolateral Na+-K+-2Cl− cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl− secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle “pinchase” dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, ∼80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and ∼40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway. PMID:19864322

  8. A New Chemiluminescent Method for Evaluation of the Functional Activity of Neutrophils in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Proskurnina, E V; Sozarukova, M M; Polimova, A M; Prudnikova, M A; Ametov, A S; Vladimirov, Yu A

    2016-06-01

    Functional activity of neutrophils was evaluated by the chemiluminescent method with successive double stimulation by soluble stimuli with different mechanisms of action: phorbol-12-myristate-13-acetate (PMA) and phormyl-methionyl-leucyl-phenilalanine (fMLP). The study was carried out in 26 patients receiving oral sugar-reducing therapy. In addition to the functional activity of neutrophils, the levels of TBA reactive products, inflammation markers, blood clotting values, and biochemical parameters were measured. The results showed mainly reduction of the granulocytic component of the immune system in the patients. PMID:27388632

  9. 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...

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Inflammatory cells’ role in acetic acid-induced colitis

    PubMed Central

    Sanei, Mohammad H.; Hadizadeh, Fatemeh; Adibi, Peyman; Alavi, Sayyed Ali

    2014-01-01

    Background: Free radicals are the known mechanisms responsible for inducing colitis with two origins: Inflammatory cells and tissues. Only the inflammatory cells can be controlled by corticosteroids. Our aim was to assess the importance of neutrophils as one of the inflammatory cells in inducing colitis and to evaluate the efficacy of corticosteroids in the treatment of inflammatory bowel disease (IBD). Materials and Methods: Thirty-six mice were divided into six groups of six mice each. Colitis was induced in three groups by exposing them to acetic acid through enema (group 1), ex vivo (group 3), and enema after immune suppression (group 5). Each group had one control group that was exposed to water injection instead of acetic acid. Tissue samples were evaluated and compared based on macroscopic damages and biochemical and pathological results. Results: Considering neutrophilic infiltration, there were significant differences between groups 1, 3, 5, and the control of group 1. Groups 3, 5, and their controls, and group 1 and the control of group 3 had significant differences in terms of goblet depletion. Based on tissue originated H2O2, we found significant differences between group 1 and its control and group 3, and also between groups 5 and the control of group 3. All the three groups were significantly different from their controls based on Ferric Reducing Ability of Plasma (FRAP) and such differences were also seen between group 1 with two other groups. Conclusion: Neutrophils may not be the only cause of oxidation process in colitis, and also makes the effectiveness of corticosteroids in the treatment of this disease doubtful. PMID:25337523

  20. Neutrophil function and cortisol:DHEAS ratio in bereaved older adults.

    PubMed

    Khanfer, Riyad; Lord, Janet M; Phillips, Anna C

    2011-08-01

    Bereavement is a common life event for older adults and is associated with increased risk of morbidity and mortality, though the underlying reasons for this link are poorly understood. Although physical and emotional stressors and ageing are known to suppress immunity, few studies have explored the impact of bereavement upon immunity in the older population. We therefore hypothesised that the emotional stress of bereavement would suppress immune function, specifically neutrophil bactericidal activity, in older adults. A between-subjects design was used to examine the effect of recent bereavement (<2 months) on neutrophil function in elders. Participants were 24 bereaved and 24 age- and sex-matched non-bereaved controls all aged 65+ years. Neutrophil phagocytosis of Escherichia coli (E. coli) and stimulated superoxide production were assessed. Cortisol and dehydroepiandrosterone-sulphate (DHEAS) levels were determined in serum to assess potential mechanisms. Depressive and anxiety symptoms were measured by questionnaire. Neutrophil superoxide production was significantly reduced among the bereaved when challenged with E. coli (p=0.05), or phorbol 12-myristate 13-acetate (p=0.009). Further, the bereaved group had a significantly higher cortisol:DHEAS ratio compared to controls (p=0.03). There was no difference in neutrophil phagocytosis between the two groups. The psychological questionnaire results showed that the bereaved had significantly greater depressive and anxiety symptoms than the non-bereaved. The emotional stress of bereavement is associated with suppressed neutrophil superoxide production and with a raised cortisol:DHEAS ratio. The stress of bereavement exaggerates the age-related decline in HPA axis and combines with immune ageing to further suppress immune function, which may help to the explain increased risk of infection in bereaved older adults. PMID:21420485

  1. 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.

  2. 4-Methylcoumarin Derivatives Inhibit Human Neutrophil Oxidative Metabolism and Elastase Activity

    PubMed Central

    Fuzissaki, Carolina N.; Andrade, Micássio F.; Azzolini, Ana Elisa C.S.; Taleb-Contini, Silvia H.; Vermelho, Roberta B.; Lopes, João Luis C.; Lucisano-Valim, Yara Maria

    2013-01-01

    Abstract Increased neutrophil activation significantly contributes to the tissue damage in inflammatory illnesses; this phenomenon has motivated the search for new compounds to modulate their effector functions. Coumarins are natural products that are widely consumed in the human diet. We have evaluated the antioxidant and immunomodulator potential of five 4-methylcoumarin derivatives. We found that the 4-methylcoumarin derivatives inhibited the generation of reactive oxygen species by human neutrophils triggered by serum-opsonized zymosan or phorbol-12-myristate-13-acetate; this inhibition occurred in a concentration-dependent manner, as revealed by lucigenin- and luminol-enhanced chemiluminescence assays. Cytotoxicity did not mediate this inhibitory effect. The 7,8-dihydroxy-4-methylcoumarin suppressed the neutrophil oxidative metabolism more effectively than the 6,7- and 5,7-dihydroxy-4-methylcoumarins, but the 5,7- and 7,8-diacetoxy-4-methylcoumarins were less effective than their hydroxylated counterparts. An analysis of the biochemical pathways suggested that the 6,7- and 7,8-dihydroxy-4-methylcoumarins inhibit the protein kinase C-mediated signaling pathway, but 5,7-dihydroxy-4-methylcoumarin, as well as 5,7- and 7,8-diacetoxy-4-methylcoumarins do not significantly interfere in this pathway of the activation of the human neutrophil oxidative metabolism. The 4-methylcoumarin derivatives bearing the catechol group suppressed the elastase and myeloperoxidase activity and reduced the 1,1-diphenyl-2-picrylhydrazyl free radical the most strongly. Interestingly, the 5,7-dihydroxy-4-methylcoumarin scavenged hypochlorous acid more effectively than the o-dihydroxy-substituted 4-methylcoumarin derivatives, and the diacetoxylated 4-methylcoumarin derivatives scavenged hypochlorous acid as effectively as the 7,8-dihydroxy-4-methylcoumarin. The significant influence of small structural modifications in the inhibitory potential of 4-methylcoumarin derivatives on the

  3. 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

  4. Reactive oxygen species in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbation of chronic obstructive pulmonary disease.

    PubMed

    Vaitkus, Mindaugas; Lavinskiene, Simona; Barkauskiene, Diana; Bieksiene, Kristina; Jeroch, Jolanta; Sakalauskas, Raimundas

    2013-12-01

    Chronic airway inflammation can be mediated by an enhanced neutrophil oxidative burst. However, the role of bacteria in the pathogenesis of chronic obstructive pulmonary disease (COPD) exacerbations is highly controversial. The aim of this study was to evaluate the production of reactive oxygen species (ROS) in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbations of COPD (AECOPD). A total of 40 patients with AECOPD, 10 healthy nonsmokers, and 10 "healthy" smokers were enrolled into the study. Peripheral blood and sputum samples were obtained during exacerbation and after recovery. Neutrophils were isolated by high-density gradient centrifugation and magnetic separation. ROS production by neutrophils was investigated after stimulation with phorbol-myristate-acetate and Staphylococcus aureus bacteria. ROS production by neutrophils was assessed as the mean fluorescent intensity using a flow cytometer. IL-8 levels in serum and induced sputum were determinant by ELISA. Spontaneous ROS production was significantly higher in neutrophils from the patients with bacterial AECOPD as compared with nonbacterial AECOPD and stable COPD (P <0.05). ROS production stimulated with PMA and with Staphylococcus aureus was significantly higher in neutrophils isolated from the patients with bacterial AECOPD as compared with nonbacterial and stable COPD (P <0.05). The serum and induced sputum IL-8 levels were significantly increased in the patients with bacterial AECOPD than nonbacterial AECOPD, stable COPS, and "healthy" smokers and nonsmokers (P <0.05) and higher in the induced sputum as the compared with serum in all studied groups (P <0.05). Enlarge CRP level was documented during AECOPD than in all other groups (P <0.05). A markedly increased ROS production in sputum neutrophils during bacterial AECOPD shows an inflammatory response reflecting enhanced local inflammation, which can be mediated by bacterial colonization. PMID:23872721

  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 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

  7. 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

  8. 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

  9. [Nomegestrol acetate: clinical pharmacology].

    PubMed

    Lello, S

    2009-10-01

    Progestogens are used in clinical practice in some conditions. Their effects depend on their chemical structure, pharmacokinetics, pharmacodynamics, with important differences among various progestogens. Generally, progestins are classified according to their parent molecule, of which often they keep some features. Derivatives of 19-nor-progesterone are characterized by high selectivity of action on progestin receptor. In particular, nomegestrol acetate (NomAc) shows an important progestational potency, neutral gluco-lipid profile, and antigonadotropic activity. It is used for treating menstrual cycle disorders and for hormone replacement therapy in menopause in association with an estrogen. In future, thanks to its antigonadotropic activity, NomAc will be used in estroprogestin combinations in fertile women, thus taking advantage of its tolerability profile and obtaining numerous non-contraceptive benefits as well. PMID:19749678

  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. 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

  12. 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

  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. Anti-inflammatory effects of secondary metabolites of marine Pseudomonas sp. in human neutrophils are through inhibiting P38 MAPK, JNK, and calcium pathways.

    PubMed

    Yang, Shun-Chin; Sung, Ping-Jyun; Lin, Chwan-Fwu; Kuo, Jimmy; Chen, Chun-Yu; Hwang, Tsong-Long

    2014-01-01

    Activated neutrophils play a significant role in the pathogenesis of many inflammatory diseases. The metabolites of marine microorganisms are increasingly employed as sources for developing new drugs; however, very few marine drugs have been studied in human neutrophils. Herein, we showed that secondary metabolites of marine Pseudomonas sp. (N11) significantly inhibited superoxide anion generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils, with IC50 values of 0.67±0.38 µg/ml and 0.84±0.12 µg/ml, respectively. In cell-free systems, neither superoxide anion-scavenging effect nor inhibition of elastase activity was associated with the suppressive effects of N11. N11 inhibited the phosphorylation of p38 MAP kinase and JNK, but not Erk and Akt, in FMLP-induced human neutrophils. Also, N11 dose-dependently attenuated the transient elevation of intracellular calcium concentration in activated neutrophils. In contrast, N11 failed to alter phorbol myristate acetate-induced superoxide anion generation, and the inhibitory effects of N11 were not reversed by protein kinase A inhibitor. In conclusion, the anti-inflammatory effects of N11 on superoxide anion generation and elastase release in activated human neutrophils are through inhibiting p38 MAP kinase, JNK, and calcium pathways. Our results suggest that N11 has the potential to be developed to treat neutrophil-mediated inflammatory diseases. PMID:25474595

  15. Effects of Alchornea cordifolia on elastase and superoxide anion produced by human neutrophils.

    PubMed

    Kouakou-Siransy, Gisèle; Sahpaz, Sevser; Nguessan, G Irié; Datté, Jacques Yao; Brou, Jérome Kablan; Gressier, Bernard; Bailleul, François

    2010-02-01

    The ability of Alchornea cordifolia (Schum. and Thonn.) Müll. Arg. (Euphorbiaceae) leaves to inhibit human neutrophil elastase (HNE) and superoxide anion (O(2)(*-)) activities was evaluated on aqueous and ethyl acetate extracts as they allow for a targeted extraction of polyphenols. The direct effect of A. cordifolia extracts on HNE and O(2)(*-) was assessed in an acellular system. Results showed that extracts scavenge HNE and O(2)(*-) in a dose-dependent manner. Better activity was exhibited by the ethyl acetate extract with lower IC(50) (2.2 and 4. 1 mg/L for HNE and O(2)(*-), respectively) than for the aqueous extract. Cellular systems including isolated human polymorphonuclear neutrophils (PMN) were investigated to assess the effect of extracts on PMN metabolism. PMN were stimulated with 4beta-phorbol-12-myristate-13-acetate (PMA), calcium ionophore (CaI), or N-formyl-methionyl-leucine-phenylalanine (fMLP), each stimulant having its own stimulation pathway. From the IC(50) obtained, it can be concluded that A. cordifolia reduces HNE and O(2)(*-) liberation. Furthermore it was demonstrated that A. cordifolia extracts have no cytotoxic activity on PMN by measuring release of the cytosolic enzyme lactate dehydrogenase. As the ethyl acetate extract offers a higher rate of total phenols than the aqueous extract as well as better scavenging activity, it can be supposed that polyphenols, which are well known for their potent antioxidant and antielastase activity, are implicated in the activity of the plant. Phenolic substances such as quercetin, myricetin-3-glucopyranoside, myricetin-3-rhamnopyranoside, and proanthocyanidin A2 were identified in the ethyl acetate extract. In conclusion, the study provides proof of ethnomedical claims and partly explains the mechanisms of the anti-inflammatory action of A. cordifolia leaves. PMID:20645828

  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. 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

  2. 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

  3. 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

  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. 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

  6. 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

  7. 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

  8. 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

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

    SciTech Connect

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

    1991-02-01

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

  10. Phospholipase A{sub 2} is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls

    SciTech Connect

    Tithof, P.K.; Schiamberg, E.; Ganey, P.E.; Peters-Golden, M.

    1996-01-01

    Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs), activates neutrophils to produce superoxide anion (O{sub 2}{sup {minus}}) by a mechanism that involves phospholipase C-dependent hydrolysis of membrane phosphoinositides; however, subsequent signal transduction mechanisms are unknown. This study determines whether phospholipase A{sub 2}-dependent release of arachidonic acid is involved in PCB-induced O{sub 2}{sup {minus}} production. O{sub 2}{sup {minus}} production was measured in vitro in glycogen-elicited, rat neutrophils in the presence and absence of the inhibitors of phospholipase A{sub 2}: quinacrine, 4-bromophenacyl bromide (BPB), and manoalide. All three agents significantly decreased the amount of O{sub 2}{sup {minus}} detected during stimulation of neutrophils with Aroclor 1242. Similar inhibition occurred when neutrophils were activated with the classical stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate. The effects of BPB and manoalide were not a result of cytotoxicity or other nonspecific effects. Significant release of {sup 3}H-arachidonic acid preceded O{sub 2}{sup {minus}} production in neutrophils stimulated with Aroclor 1242 or fMLP. Manoalide, at a concentration that abolished O{sub 2}{sup {minus}} production, also inhibited the release of {sup 3}H-arachidonate. Aspirin, zileuton, or WEB 2086 did not affect Aroclor 1242-induced O{sub 2}{sup {minus}} production, suggesting that eicosanoids and platelet-activating factor are not needed for neutrophil activation by PCBs. Activation of phos-pholipase A{sub 2} and O{sub 2}{sup {minus}} production do not appear to involve the Ah receptor. These data suggest that Aroclor 1242 stimulates neutrophils to produce O{sub 2}{sup {minus}} by a mechanism that involves phospholipase A{sub 2}-dependent release of arachiodonic acid. 49 refs., 6 figs., 2 tabs.

  11. 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.

  12. 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.

  13. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may be produced by the calcium hydroxide neutralization of acetic acid. (b) The ingredient meets...

  14. 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

  15. 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

  16. 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

  17. Antibiofilm Properties of Acetic Acid

    PubMed Central

    Bjarnsholt, Thomas; Alhede, Morten; Jensen, Peter Østrup; Nielsen, Anne K.; Johansen, Helle Krogh; Homøe, Preben; Høiby, Niels; Givskov, Michael; Kirketerp-Møller, Klaus

    2015-01-01

    Bacterial biofilms are known to be extremely tolerant toward antibiotics and other antimicrobial agents. These biofilms cause the persistence of chronic infections. Since antibiotics rarely resolve these infections, the only effective treatment of chronic infections is surgical removal of the infected implant, tissue, or organ and thereby the biofilm. Acetic acid is known for its antimicrobial effect on bacteria in general, but has never been thoroughly tested for its efficacy against bacterial biofilms. In this article, we describe complete eradication of both Gram-positive and Gram-negative biofilms using acetic acid both as a liquid and as a dry salt. In addition, we present our clinical experience of acetic acid treatment of chronic wounds. In conclusion, we here present the first comprehensive in vitro and in vivo testing of acetic acid against bacterial biofilms. PMID:26155378

  18. 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

  19. 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

  20. 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

  1. [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

  2. 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

  3. 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

  4. High levels of fish oil enhance neutrophil development and activation and influence colon mucus barrier function in a genetically susceptible mouse model.

    PubMed

    Duriancik, David M; Comstock, Sarah S; Langohr, Ingeborg M; Fenton, Jenifer I

    2015-11-01

    Dietary fatty acids influence immunologic homeostasis, but their effect on initiation of colitis, an immune-mediated disease, is not well established. Previously, our laboratory demonstrated that high doses of dietary fish oil (FO) increased colon inflammation and dysplasia in a model of infection-induced colitis. In the current study, we assessed the effects of high-dose dietary FO, 6% by weight, on colon inflammation, neutrophil recruitment and function, and mucus layer integrity in a genetically susceptible, colitis-prone mouse model in the absence of infection. FO-fed SMAD3(-/-) mice had increased colon inflammation evidenced by increased numbers of systemic and local neutrophils and increased neutrophil chemoattractant and inflammatory cytokine gene expression in the colon. Mucus layer thickness in the cecum and goblet cell numbers in the cecum and colon in FO-fed mice were reduced compared to control. FO consumption affected colitis in male and female mice differently. Compared to female control mice, neutrophils from FO-fed female mice had reduced reactive oxygen species (ROS) upon ex vivo stimulation with phorbol myristate acetate while FO-fed male mice produced increased ROS compared to control-fed male mice. In summary, dietary FO impaired mucus layer integrity and was associated with colon inflammation characterized by increased neutrophil numbers and altered neutrophil function. High-dose FO may have detrimental effects in populations genetically susceptible for inflammatory bowel disease and these effects may differ between males and females. PMID:26297475

  5. The structure of neutrophil defensin genes.

    PubMed

    Linzmeier, R; Michaelson, D; Liu, L; Ganz, T

    1993-04-26

    Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil-specific defensins. Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related. The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and -2) or Paneth cells (HD-5) are organized differently: HD-5 had only two exons, and MCP-1 and -2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell-specific expression. PMID:8477861

  6. 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

  7. 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

  8. Defective neutrophil chemotaxis in juvenile periodontitis.

    PubMed Central

    Clark, R A; Page, R C; Wilde, G

    1977-01-01

    Neutrophil chemotaxis was evaluated in nine patients with juvenile periodontitis, with normal subjects and patients with the adult form of periodontitis as controls. Defective chemotactic responses were observed in neutrophils from seven of nine juvenile patients, and a reduced level of complement-derived chemotactic activity was demonstrated in serum from four patients. These determinations were normal in all the patients with adult periodontitis. Serum from five of the juvenile patients contained a heat-stable, non-dialyzable factor that markedly inhibited the chemotaxis of normal neutrophils. Thus the characteristic tissue destruction seen in juvenile periodontitis may be, at least in part, a consequence of a failure of host defense mechanisms. PMID:591063

  9. Clinical Microfluidics for Neutrophil Genomics and Proteomics

    PubMed Central

    Kotz, Kenneth T.; Xiao, Wenzong; Miller-Graziano, Carol; Qian, Wei-Jun; Russom, Aman; Warner, Elizabeth A.; Moldawer, Lyle L.; De, Asit; Bankey, Paul E.; Petritis, Brianne O.; Camp, David G.; Rosenbach, Alan E.; Goverman, Jeremy; Fagan, Shawn P.; Brownstein, Bernard H.; Irimia, Daniel; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N.; Smith, Richard D.; Davis, Ronald W.; Tompkins, Ronald G.; Toner, Mehmet

    2010-01-01

    Neutrophils play critical roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood and develop ‘on-chip’ processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Lastly, we implement this tool as part of a near patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of patients in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting. PMID:20802500

  10. The role of neutrophils in inflammation resolution.

    PubMed

    Jones, Hefin R; Robb, Calum T; Perretti, Mauro; Rossi, Adriano G

    2016-04-01

    The fundamental role played by neutrophils for an efficient, acute inflammatory response has long been appreciated, with the underlying molecular and cellular mechanisms largely elucidated over the past decades. However, more recent work suggests that the biological functions exerted by this fascinating leucocyte are somewhat more extensive than previously acknowledged. Here we discuss how extravasated neutrophils govern the initiation of the resolution phase of inflammation by enabling activation of pro-resolving circuits to ensure the safe conclusion of the inflammatory response. The neutrophil 'alarm bell' on resolution is effected through release of soluble mediators as well as apoptotic bodies and other vesicles, which, in turn, can inform and modify the microenvironment ultimately leading to termination of the inflammatory response coinciding with re-establishment of tissue homeostasis and functionality. PMID:27021499

  11. 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

  12. A variable immunoreceptor in a subpopulation of human neutrophils

    PubMed Central

    Puellmann, Kerstin; Kaminski, Wolfgang E.; Vogel, Mandy; Nebe, C. Thomas; Schroeder, Josef; Wolf, Hans; Beham, Alexander W.

    2006-01-01

    Neutrophils are thought to rely solely on nonspecific immune mechanisms. Here we provide molecular biological, immunological, ultrastructural, and functional evidence for the presence of a T cell receptor (TCR)-based variable immunoreceptor in a 5–8% subpopulation of human neutrophils. We demonstrate that these peripheral blood neutrophils express variable and individual-specific TCRαβ repertoires and the RAG1/RAG2 recombinase complex. The proinflammatory cytokine granulocyte colony-stimulating factor regulates expression of the neutrophil immunoreceptor and RAG1/RAG2 in vivo. Specific engagement of the neutrophil TCR complex protects from apoptosis and stimulates secretion of the neutrophil-activating chemokine IL-8. Our results, which also demonstrate the presence of the TCR in murine neutrophils, suggest the coexistence of a variable and an innate host defense system in mammalian neutrophils. PMID:16983085

  13. Dynamics of neutrophil migration in lymph nodes during infection

    PubMed Central

    Chtanova, Tatyana; Schaeffer, Marie; Han, Seong-Ji; van Dooren, Giel G.; Nollmann, Marcelo; Herzmark, Paul; Chan, Shiao Wei; Satija, Harshita; Camfield, Kristin; Aaron, Holly; Striepen, Boris; Robey, Ellen A.

    2008-01-01

    Summary While the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes, or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning laser microscopy (TPSLM) to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We find that neutrophils form both small, transient or large, persistent swarms via a strikingly coordinated migration pattern. We provide evidence that cooperative action of neutrophils and parasite egress from host cells can trigger swarm formation. Neutrophil swarm formation coincides in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses. PMID:18718768

  14. Nucleotide chloramines and neutrophil-mediated cytotoxicity.

    PubMed

    Bernofsky, C

    1991-03-01

    Hypochlorite is a reactive oxidant formed as an end product of the respiratory burst in activated neutrophils. It is responsible for killing bacteria and has been implicated in neutrophil-mediated tissue injury associated with the inflammatory process. Although hypochlorite is a potent cytotoxic agent, the primary mechanism by which it exerts its effect is unclear. This review examines evidence that the primary event in hypochlorite cytotoxicity is the loss of adenine nucleotides from the target cell. This loss appears to be mediated by the formation of adenine nucleotide chloramines which are reactive intermediates with a free radical character and are capable of forming stable ligands with proteins and nucleic acids. PMID:1848195

  15. Neutrophils come of age in chronic inflammation

    PubMed Central

    Caielli, Simone; Banchereau, Jacques; Pascual, Virginia

    2013-01-01

    Neutrophils have long been known to participate in acute inflammation, but a role in chronic inflammatory and autoimmune diseases is now emerging. These cells are key players in the recognition and elimination of pathogens, but they also sense self components, including nucleic acids and products of sterile tissue damage. While this normally contributes to tissue repair, it can also lead to the release of highly immunogenic products that can trigger and/or amplify autoimmune pathogenic loops. Understanding the mechanisms that underlie neutrophil activation, migration, survival and their various forms of death in health and disease might provide us with new approaches to treat chronic inflammatory conditions. PMID:23127555

  16. Human Neutrophils Convert the Sebum-derived Polyunsaturated Fatty Acid Sebaleic Acid to a Potent Granulocyte Chemoattractant*

    PubMed Central

    Cossette, Chantal; Patel, Pranav; Anumolu, Jaganmohan R.; Sivendran, Sashikala; Lee, Gue Jae; Gravel, Sylvie; Graham, François D.; Lesimple, Alain; Mamer, Orval A.; Rokach, Joshua; Powell, William S.

    2008-01-01

    Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP+, the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B4, indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of

  17. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium acetate. 184.1185 Section 184.1185 Food and... Substances Affirmed as GRAS § 184.1185 Calcium acetate. (a) Calcium acetate (Ca (C2H3O2)2, CAS Reg. No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may...

  18. Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils

    PubMed Central

    Sandilands, Gavin P; McCrae, Jame; Hill, Kathryn; Perry, Martin; Baxter, Derek

    2006-01-01

    We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic ‘stores’ of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac −1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G–Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy. PMID:17034427

  19. 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

  20. 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

  1. 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

  2. Oxidation of methionine residues in proteins of activated human neutrophils.

    PubMed Central

    Fliss, H; Weissbach, H; Brot, N

    1983-01-01

    A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins. PMID:6580633

  3. 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

  4. Migration of canine neutrophils to chitin and chitosan.

    PubMed

    Usami, Y; Okamoto, Y; Minami, S; Matsuhashi, A; Kumazawa, N H; Tanioka, S; Shigemasa, Y

    1994-12-01

    Suspension of chitin and chitosan particles (mean size of 1 micron) were found to attract canine neutrophils chemotactically as determined by a checkerboard assay through polycarbonate filter with 5 microns pore size in Blind well chamber. Suspension of chitin induced chemokinetic migrations of the neutrophils. These evidences might reflect accumulation of neutrophils to chitin- and chitosan-implanted regions in dogs. PMID:7696425

  5. Effects of the Ethyl Acetate Fraction of Alchornea triplinervia on Healing Gastric Ulcer in Rats

    PubMed Central

    Lima, Zeila P.; Bonamin, Flavia; Calvo, Tamara R.; Vilegas, Wagner; Santos, Lourdes C.; Rozza, Ariane L.; Pellizzon, Claudia H.; Rocha, Lucia R. M.; Hiruma-Lima, Clélia A.

    2011-01-01

    Alchornea triplinervia (Spreng.) Muell. Arg (Euphorbiaceae) is a medicinal plant commonly used by people living in the Cerrado region of Brazil to treat gastrointestinal ulcers. We previously described the gastroprotective action of methanolic extract (ME) of Alchornea triplinervia and the ethyl acetate fraction (EAF) in increasing of prostaglandin E2 (PGE2) gastric levels in the mucosa. In this work we evaluated the effect of EAF in promoting the healing process in rats with acetic acid-induced gastric ulcers. In addition, toxicity was investigated during treatment with EAF. After 14 days of treatment with EAF, the potent stimulator of gastric cell proliferation contributed to the acceleration of gastric ulcer healing. Upon immunohistochemical analysis, we observed a pronounced expression of COX-2, mainly in the submucosal layer. The 14-day EAF treatment also significantly increased the number of neutrophils in the gastric mucosa regeneration area. The EAF induced angiogenesis on gastric mucosa, observed as an increase of the number of blood vessels supplying the stomach in rats treated with EAF. Oral administration for 14 days of the ethyl acetate fraction from Alchornea triplinervia accelerated the healing of gastric ulcers in rats by promoting epithelial cell proliferation, increasing the number of neutrophils and stimulation of mucus production. This fraction, which contained mainly phenolic compounds, contributed to gastric mucosa healing. PMID:26791640

  6. 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

  7. 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

  8. Myeloperoxidase in human neutrophil host defence.

    PubMed

    Nauseef, William M

    2014-08-01

    Human neutrophils represent the predominant leucocyte in circulation and the first responder to infection. Concurrent with ingestion of microorganisms, neutrophils activate and assemble the NADPH oxidase at the phagosome, thereby generating superoxide anion and hydrogen peroxide. Concomitantly, granules release their contents into the phagosome, where the antimicrobial proteins and enzymes synergize with oxidants to create an environment toxic to the captured microbe. The most rapid and complete antimicrobial action by human neutrophils against many organisms relies on the combined efforts of the azurophilic granule protein myeloperoxidase and hydrogen peroxide from the NADPH oxidase to oxidize chloride, thereby generating hypochlorous acid and a host of downstream reaction products. Although individual components of the neutrophil antimicrobial response exhibit specific activities in isolation, the situation in the environment of the phagosome is far more complicated, a consequence of multiple and complex interactions among oxidants, proteins and their by-products. In most cases, the cooperative interactions among the phagosomal contents, both from the host and the microbe, culminate in loss of viability of the ingested organism. PMID:24844117

  9. Neutrophilic dermatoses and inflammatory bowel diseases.

    PubMed

    Marzano, A V; Menicanti, C; Crosti, C; Trevisan, V

    2013-04-01

    Pyoderma gangrenosum (PG) and Sweet's Syndrome (SS) are inflammatory skin diseases caused by the accumulation of neutrophils in the skin and, rarely, in internal organs, which led to coining the term of neutrophilic dermatoses (ND) to define these conditions. Recently, ND have been included among the autoinflammatory diseases, which are forms due to mutations of genes regulating the innate immune responses. Both PG and SS are frequently associated with inflammatory bowel diseases (IBD), a group of chronic intestinal disorders which comprises ulcerative colitis and Crohn's disease and whose pathogenesis involves both the innate and adaptive immunity in genetically prone individuals. Patients with IBD develop PG in 1-3% of cases, while SS is rarer. PG presents with deep erythematous-to-violaceous painful ulcers with undermined borders, but bullous, pustular, and vegetative variants can also occur. SS, also known as acute febrile neutrophilic dermatosis, is characterized by the abrupt onset of fever, peripheral neutrophilia, tender erythematous skin lesions and a diffuse neutrophilic dermal infiltrate. In this review that will be focused on PG and SS, we will describe also the aseptic abscesses syndrome, a new entity within the spectrum of ND which frequently occurs in association with IBD and is characterized by deep abscesses mainly involving the spleen and skin and by polymorphic cutaneous manifestations including PG- and SS-like lesions. PMID:23588144

  10. Interaction of neutrophils with vascular smooth muscle: identification of a neutrophil-derived relaxing factor.

    PubMed

    Rimele, T J; Sturm, R J; Adams, L M; Henry, D E; Heaslip, R J; Weichman, B M; Grimes, D

    1988-04-01

    Experiments were designed to study the interaction of rat peritoneal neutrophils with the vascular smooth muscle of the rat aorta. Rings of aorta, suspended in 10-ml organ chambers containing a physiologic salt solution, were precontracted with phenylephrine. Neutrophils (1 X 10(5) -4 X 10(7) cells/organ chamber) caused a cell number-dependent relaxation of the rat aorta that was augmented by superoxide dismutase (100 U/ml) or changing the oxygen content from 95 to 21%. The neutrophil-induced smooth muscle relaxation occurred in rings with and without endothelium and in rings precontracted with increasing concentrations of phenylephrine, prostaglandin F2 alpha or KCI. Catalase (1000 U/ml) and mannitol (1 X 10(-3) M) did not block the neutrophil-induced relaxation, whereas phenazine methosulfate (1 X 10(-5) M), hydroquinone (3 X 10(-5) M) and methylene blue (1 X 10(-5) M) reversed the neutrophil-induced relaxation. Pre-exposure of endothelium-rubbed rings to neutrophils (2 X 10(7) cells/organ chamber; 15 min) depressed the subsequent concentration-response curve to phenylephrine but augmented the relaxation induced by the phosphodiesterase inhibitor zaprinast (1 X 10(-5) M). The effluent from a column restraining the neutrophils induced a relaxation of endothelium-rubbed aortic rings that was prevented by methylene blue (1 X 10(-5) M). These results demonstrate that rat neutrophils release a factor that has a pharmacologic profile similar to that previously reported for the relaxing factor released from the vascular endothelium. PMID:3129547

  11. Inhibition of Human Neutrophil Responses by Essential Oil of Artemisia kotuchovii and Its Constituents

    PubMed Central

    Schepetkin, Igor A.; Kushnarenko, Svetlana V.; Özek, Gulmira; Kirpotina, Liliya N.; Utegenova, Gulzhakhan A.; Kotukhov, Yuriy A.; Danilova, Alevtina N.; Özek, Temel; Başer, K. Hüsnü Can; Quinn, Mark T.

    2015-01-01

    Essential oils were obtained by hydrodistillation of the flowers+leaves and stems of Artemisia kotuchovii Kupr. (AKEOf+l and AKEOstm, respectively) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The primary components of the oils were estragole, (E)- and (Z)-β-ocimenes, methyl eugenol, limonene, spathulenol, β-pinene, myrcene, and (E)-methyl cinnamate. Seventy four constituents were present at concentrations from 0.1 to 1.0%, and 34 compounds were identified in trace (<0.1%) amounts in one or both plant components. Screening of the essential oils for biological activity showed that AKEOstm, but not AKEOf+l, inhibited N-formyl-Met-Leu-Phe (fMLF)-stimulated Ca2+ flux and chemotaxis and phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production in human neutrophils. Selected pure constituents, representing >96% of the AKEOstm composition, were also tested in human neutrophils and HL-60 cells transfected with N-formyl peptide receptor 1 (FPR1). We found that one component, 6-methyl-3,5-heptadien-2-one (MHDO), inhibited fMLF- and interleukin 8 (IL-8)-stimulated Ca2+ flux, fMLF-induced chemotaxis, and PMA-induced ROS production in human neutrophils. MHDO also inhibited fMLF-induced Ca2+ flux in FPR1-HL60 cells. These results suggest that MHDO may be effective in modulating some innate immune responses, possibly by an inhibition of neutrophil migration and ROS production. PMID:25959257

  12. Effects of phosphodiester and phosphorothioate ODN2216 on leukotriene synthesis in human neutrophils and neutrophil apoptosis.

    PubMed

    Viryasova, Galina M; Golenkina, Ekaterina A; Galkina, Svetlana I; Gaponova, Tatjana V; Romanova, Yulia M; Sud'ina, Galina F

    2016-06-01

    Polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the initiation and resolution of the inflammatory response, and neutrophil apoptosis is a critical step in resolving inflammation. We examined the effects of oligodeoxynucleotide (ODN) species with different numbers of phosphodiester and phosphorothioate bonds on leukotriene synthesis in PMNLs and on neutrophil apoptosis. Our modifications were based on the well-known ODN2216 molecule (Krug et al., 2001). Treatment of cultured human neutrophils with ODN2216 accelerated apoptosis except in the case of a species with only phosphodiester bonds. The ODNs with poly(g) (phosphorothioate) sequences at both ends and a phosphodiester inner core had maximal effects on leukotriene synthesis in neutrophils and inhibited formation of 5-lipoxygenase metabolites. Addition of phosphodiester and phosphorothioate ODNs to PMNLs produced distinct effects on superoxide and nitric oxide formation: phosphorothioate-containing ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. Altogether, our results describe strong activation of neutrophil's cellular responses by phosphorothioate ODN2216. We propose that phosphorothioate modification of ODNs represents a potential mechanism of PMNL activation. PMID:27036535

  13. Changes in Neutrophil Functions in Astronauts

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Simons, Elizabeth R.; Castro, Victoria; Pierson, Duane L.

    2002-01-01

    Neutrophil functions (phagocytosis, oxidative burst, degranulation) and expression of surface markers involved in these functions were studied in 25 astronauts before and after 4 space shuttle missions. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch (preflight or L-10), immediately after landing (landing or R+0), and again at 3 days after landing (postflight or R+3). Blood samples were also collected from 9 healthy low-stressed subjects at 3 time points simulating a 10-day shuttle mission. The number of neutrophils increased at landing by 85 percent when compared to the preflight numbers. Neutrophil functions were studied in whole blood using flow cytometric methods. Phagocytosis of E.coli-FITC and oxidative burst capacity of the neutrophils following the 9 to 11 day missions were lower at all three sampling points than the mean values for control subjects. Phagocytosis and oxidative burst capacity of the astronauts was decreased even 10-days before space flight. Mission duration appears to be a factor in phagocytic and oxidative functions. In contrast, following the short-duration (5-days) mission, these functions were unchanged from control values. No consistent changes in degranulation were observed following either short or medium length space missions. The expression of CD16, CD32, CD11a, CD11b, CD11c, L-selectin and CD36 was measured and found to be variable. Specifically, CD16 and CD32 did not correlate with the changes in oxidative burst and phagocytosis. We can conclude from this study that the stresses associated with space flight can alter the important functions of neutrophils.

  14. 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

  15. Desmopressin Acetate in Intracranial Haemorrhage

    PubMed Central

    Kapapa, Thomas; Röhrer, Stefan; Struve, Sabine; Petscher, Matthias; König, Ralph; Wirtz, Christian Rainer; Woischneck, Dieter

    2014-01-01

    Introduction. The secondary increase in the size of intracranial haematomas as a result of spontaneous haemorrhage or trauma is of particular relevance in the event of prior intake of platelet aggregation inhibitors. We describe the effect of desmopressin acetate as a means of temporarily stabilising the platelet function. Patients and Methods. The platelet function was analysed in 10 patients who had received single (N = 4) or multiple (N = 6) doses of acetylsalicylic acid and 3 patients (control group) who had not taken acetylsalicylic acid. All subjects had suffered intracranial haemorrhage. Analysis was performed before, half an hour and three hours after administration of desmopressin acetate. Statistical analysis was performed by applying a level of significance of P ≤ 0.05. Results. (1) Platelet function returned to normal 30 minutes after administration of desmopressin acetate. (2) The platelet function worsened again after three hours. (3) There were no complications related to electrolytes or fluid balance. Conclusion. Desmopressin acetate can stabilise the platelet function in neurosurgical patients who have received acetylsalicylic acid prior to surgery without causing transfusion-related side effects or a loss of time. The effect is, however, limited and influenced by the frequency of drug intake. Further controls are needed in neurosurgical patients. PMID:25610644

  16. Inhibition of Neutrophil Exocytosis Ameliorates Acute Lung Injury in Rats

    PubMed Central

    Uriarte, Silvia M.; Rane, Madhavi J.; Merchant, Michael L.; Jin, Shunying; Lentsch, Alex B.; Ward, Richard A.; McLeish, Kenneth R.

    2013-01-01

    Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALf). Administration of TAT-SNAP-23, but not TAT-Control, significantly reduced albumin leakage, total protein levels in the BALf, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALf neutrophils and a decrease in neutrophil granule proteins in BALf. Similar degree of neutrophil accumulation in the lungs and/or BALf suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALf revealed that components of the complement and coagulation pathways were significantly reduced in BALf from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI. PMID:23364427

  17. Reductive opening of carbohydrate phenylsulfonylethylidene (PSE) acetals.

    PubMed

    Chéry, Florence; Cabianca, Elena; Tatibouët, Arnaud; De Lucchi, Ottorino; Lindhorst, Thisbe K; Rollin, Patrick

    2015-11-19

    The phenylsulfonylethylidene (PSE) acetal is a relatively new protecting group in carbohydrate chemistry. However, carbohydrate-derived phenylsulfonylethylidene (PSE) acetals show a different behavior in reductive desulfonylation than simple symmetrical acetals. Here we have investigated various SET-type reaction conditions in order to open PSE acetals regioselectively and to produce chiral ω-hydroxyethenyl ethers. Whereas sodium amalgam leads to a mixture of regioisomeric vinyl ethers besides the ethylidene acetal, samarium iodide is suited for regioselective ring opening. This is shown with seven different carbohydrate PSE acetals, both of the 1,3-dioxane and the 1,3-dioxolane type. PMID:26469209

  18. 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

  19. 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

  20. Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

    PubMed

    Fine, N; Hassanpour, S; Borenstein, A; Sima, C; Oveisi, M; Scholey, J; Cherney, D; Glogauer, M

    2016-07-01

    Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state. PMID:27270666

  1. 21 CFR 522.533 - Deslorelin acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Deslorelin acetate. (a) Specifications. Each implant contains 2.1 milligrams deslorelin acetate. (b) Sponsor.... One implant per mare. (ii) Indications for use. For inducing ovulation within 48 hours in...

  2. Carbon-isotopic analysis of dissolved acetate

    NASA Technical Reports Server (NTRS)

    Gelwicks, J. T.; Hayes, J. M.

    1990-01-01

    Heating of dried, acetate-containing solids together with oxalic acid dihydrate conveniently releases acetic acid for purification by gas chromatography. For determination of the carbon-isotopic composition of total acetate, the acetate-containing zone of the chromatographic effluent can be routed directly to a combustion furnace coupled to a vacuum system allowing recovery, purification, and packaging of CO2 for mass-spectrometric analysis. For analysis of methyl carbon, acetic acid can be cryogenically trapped from the chromatographic effluent, then transferred to a tube containing excess NaOH. The tube is evacuated, sealed, and heated to 500 degrees C to produce methane by pyrolysis of sodium acetate. Subsequent combustion of the methane allows determination of the 13C content at the methyl position in the parent acetate. With typical blanks, the standard deviation of single analyses is less than 0.4% for acetate samples larger than 5 micromoles. A full treatment of uncertainties is outlined.

  3. Ozone decomposition in aqueous acetate solutions

    SciTech Connect

    Sehested, K.; Holcman, J.; Bjergbakke, E.; Hart, E.J.

    1987-01-01

    The acetate radical ion reacts with ozone with a rate constant of k = (1.5 +/- 0.5) x 10Z dmT mol s . The products from this reaction are CO2, HCHO, and O2 . By subsequent reaction of the peroxy radical with ozone the acetate radical ion is regenerated through the OH radical. A chain decomposition of ozone takes place. It terminates when the acetate radical ion reacts with oxygen forming the unreactive peroxy acetate radical. The chain is rather short as oxygen is developed, as a result of the ozone consumption. The inhibiting effect of acetate on the ozone decay is rationalized by OH scavenging by acetate and successive reaction of the acetate radical ion with oxygen. Some products from the bimolecular disappearance of the peroxy acetate radicals, however, react further with ozone, reducing the effectiveness of the stabilization.

  4. 21 CFR 184.1721 - Sodium acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and....1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and animal tissues....

  5. 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

  6. Neutrophil myeloperoxidase destruction by ultraviolet irradiation

    SciTech Connect

    Hanker, J.; Giammara, B.; Strauss, G.

    1988-01-01

    The peroxidase activity of enriched leukocyte preparations on coverslips was determined cytochemically with a newly developed method. The techniques utilizes diaminobenzidine medium and cupric nitrate intensification and is suitable for analysis with light microscopy, SEM, and TEM. Blood specimens from control individuals were studied with and without in vitro UV irradiation and compared with those from psoriasis patients exposed therapeutically to various types of UV in phototherapy. All UV irradiated samples showed diminished neutrophil myeloperoxidase (MP) activity although that of the principal eosinophil peroxidase was unaffected. The SEMs supported the contention that decreased neutrophil MP activity might be related to UV induced degranulation. It is believed to be possible, eventually, to equate the observed MP degranulation effect after UV irradiation with diminished ability to fight bacterial infections.

  7. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

    NASA Astrophysics Data System (ADS)

    Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B.

    2014-03-01

    Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks `outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

  8. 21 CFR 582.6185 - Calcium acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium acetate. 582.6185 Section 582.6185 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium acetate. (a) Product. Calcium acetate. (b) Conditions of use. This substance is...

  9. Kinetics of the Methanogenic Fermentation of Acetate

    PubMed Central

    Fukuzaki, Satoshi; Nishio, Naomichi; Nagai, Shiro

    1990-01-01

    Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions. A second-order substrate inhibition model, qch4 = qmS/[Ks + S + (S2/Ki)], where S was the concentration of undissociated acetic acid, not ionized acetic acid, could be applicable in both cases. The analysis resulted in substrate saturation constants, Ks, of 4.0 μM for the acclimatized sludge and 104 μM for M. barkeri. The threshold concentrations of undissociated acetic acid when no further acetate utilization was observed were 0.078 μM (pH 7.50) for the acclimatized sludge and 4.43 μM (pH 7.45) for M. barkeri. These kinetic results suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization and that the acclimatized sludge in which Methanothrix spp. appeared dominant could utilize acetate better and survive at a lower concentration of undissociated acetic acid than could M. barkeri. Images PMID:16348323

  10. 21 CFR 522.2476 - Trenbolone acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... days. (A) 140 milligrams (mg) trenbolone acetate (one implant consisting of 7 pellets, each pellet containing 20 mg trenbolone acetate) per implant dose. (B) 140 mg trenbolone acetate (one implant consisting... 29 mg tylosin tartrate) per implant dose. (ii) Indications for use. For improved feed...

  11. 21 CFR 522.2476 - Trenbolone acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... days. (A) 140 milligrams (mg) trenbolone acetate (one implant consisting of 7 pellets, each pellet containing 20 mg trenbolone acetate) per implant dose. (B) 140 mg trenbolone acetate (one implant consisting... 29 mg tylosin tartrate) per implant dose. (ii) Indications for use. For improved feed...

  12. 21 CFR 522.2476 - Trenbolone acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... days. (A) 140 milligrams (mg) trenbolone acetate (one implant consisting of 7 pellets, each pellet containing 20 mg trenbolone acetate) per implant dose. (B) 140 mg trenbolone acetate (one implant consisting... 29 mg tylosin tartrate) per implant dose. (ii) Indications for use. For improved feed...

  13. 21 CFR 522.2476 - Trenbolone acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... days. (A) 140 milligrams (mg) trenbolone acetate (one implant consisting of 7 pellets, each pellet containing 20 mg trenbolone acetate) per implant dose. (B) 140 mg trenbolone acetate (one implant consisting... 29 mg tylosin tartrate) per implant dose. (ii) Indications for use. For improved feed...

  14. 21 CFR 582.1721 - Sodium acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium acetate. 582.1721 Section 582.1721 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1721 Sodium acetate. (a) Product. Sodium acetate. (b) Conditions of use. This substance is...

  15. 21 CFR 582.1721 - Sodium acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium acetate. 582.1721 Section 582.1721 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1721 Sodium acetate. (a) Product. Sodium acetate. (b) Conditions of use. This substance is...

  16. 21 CFR 582.1721 - Sodium acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium acetate. 582.1721 Section 582.1721 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1721 Sodium acetate. (a) Product. Sodium acetate. (b) Conditions of use. This substance is...

  17. 21 CFR 582.1721 - Sodium acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium acetate. 582.1721 Section 582.1721 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1721 Sodium acetate. (a) Product. Sodium acetate. (b) Conditions of use. This substance is...

  18. 21 CFR 582.1721 - Sodium acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium acetate. 582.1721 Section 582.1721 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1721 Sodium acetate. (a) Product. Sodium acetate. (b) Conditions of use. This substance is...

  19. 21 CFR 582.1005 - Acetic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Acetic acid. 582.1005 Section 582.1005 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1005 Acetic acid. (a) Product. Acetic acid. (b) Conditions of use. This substance is...

  20. 21 CFR 184.1005 - Acetic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Acetic acid. 184.1005 Section 184.1005 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD....1005 Acetic acid. (a) Acetic acid (C2H4O2, CAS Reg. No. 64-19-7) is known as ethanoic acid. It...

  1. 21 CFR 582.1005 - Acetic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Acetic acid. 582.1005 Section 582.1005 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1005 Acetic acid. (a) Product. Acetic acid. (b) Conditions of use. This substance is...

  2. 21 CFR 582.1005 - Acetic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Acetic acid. 582.1005 Section 582.1005 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1005 Acetic acid. (a) Product. Acetic acid. (b) Conditions of use. This substance is...

  3. 21 CFR 582.1005 - Acetic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Acetic acid. 582.1005 Section 582.1005 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1005 Acetic acid. (a) Product. Acetic acid. (b) Conditions of use. This substance is...

  4. 21 CFR 582.1005 - Acetic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Acetic acid. 582.1005 Section 582.1005 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS....1005 Acetic acid. (a) Product. Acetic acid. (b) Conditions of use. This substance is...

  5. Acet-oxy-γ-valerolactone.

    PubMed

    Tristram, Cameron; Gainsford, Graeme J; Hinkley, Simon

    2013-06-01

    Levulinyl cellulose esters have been produced as an effective renewable binder for architectural coatings. The title compound, C7H10O4 (systematic name: 2-methyl-5-oxo-tetra-hydro-furan-2-yl acetate), assigned as the esterifying species, was isolated and crystallized to confirm the structure. In the crystal, the mol-ecules pack in layers parallel to (102) utilizing weak C-H⋯O inter-actions. PMID:23795112

  6. Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

    PubMed

    Colom, Bartomeu; Bodkin, Jennifer V; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A; Nourshargh, Sussan

    2015-06-16

    Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic. PMID:26047922

  7. 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

  8. Leptin as a uremic toxin interferes with neutrophil chemotaxis.

    PubMed

    Ottonello, Luciano; Gnerre, Paola; Bertolotto, Maria; Mancini, Marina; Dapino, Patrizia; Russo, Rodolfo; Garibotto, Giacomo; Barreca, Tommaso; Dallegri, Franco

    2004-09-01

    Leptin is a pleiotropic molecule involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Hypoleptinemia characterizing starvation has been strictly related to increased susceptibility to infection secondary to malnutrition. Nevertheless, ESRD is characterized by high susceptibility to bacterial infection despite hyperleptinemia. Defects in neutrophils play a crucial role in the infectious morbidity, and several uremic toxins that are capable of depressing neutrophil functions have been identified. Only a few and contrasting reports about leptin and neutrophils are available. This study provides evidence that leptin inhibits neutrophil migration in response to classical chemoattractants. Moreover, serum from patients with ESRD inhibits migration of normal neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. Finally, the serum inhibitory activity can be effectively prevented by immune depletion of leptin. The results also show, however, that leptin by itself is endowed with chemotactic activity toward neutrophils. The two activities-inhibition of the cell response to chemokines and stimulation of neutrophil migration-could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable [Ca(2+)](i) mobilization, oxidant production, or beta(2)-integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties that are capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. Taking into account the crucial role of neutrophils in host defense, the leptin-mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears as a potential mechanism that contributes to the establishment of infections in ERSD. PMID:15339985

  9. Neutrophils from patients with SAPHO syndrome show no signs of aberrant NADPH oxidase-dependent production of intracellular reactive oxygen species

    PubMed Central

    Wekell, Per; Björnsdottir, Halla; Björkman, Lena; Sundqvist, Martina; Christenson, Karin; Osla, Veronica; Berg, Stefan; Fasth, Anders; Welin, Amanda; Bylund, Johan

    2016-01-01

    Objective. We aimed to investigate if aberrant intracellular production of NADPH oxidase-derived reactive oxygen species (ROS) in neutrophils is a disease mechanism in the autoinflammatory disease SAPHO syndrome, characterized by synovitis, acne, pustulosis, hyperostosis and osteitis, as has previously been suggested based on a family with SAPHO syndrome-like disease. Methods. Neutrophil function was explored in a cohort of four patients with SAPHO syndrome, two of whom were sampled during both inflammatory and non-inflammatory phase. Intracellular neutrophil ROS production was determined by luminol-amplified chemiluminescence in response to phorbol myristate acetate. Results. Cells from all patients produced normal amounts of ROS, both intra- and extracellularly, when compared with internal controls as well as with a large collection of healthy controls assayed in the laboratory over time (showing an extensive inter-personal variability in a normal population). Further, intracellular production of ROS increased during the inflammatory phase. Neutrophil activation markers were comparable between patients and controls. Conclusion. Dysfunctional generation of intracellular ROS in neutrophils is not a generalizable feature in SAPHO syndrome. Secondly, serum amyloid A appears to be a more sensitive inflammatory marker than CRP during improvement and relapses in SAPHO syndrome. PMID:27121779

  10. Characterization of prostanoid receptors on rat neutrophils.

    PubMed Central

    Wise, H; Jones, R L

    1994-01-01

    1. The effects of various prostanoid agonists have been compared on the increase in intracellular free calcium ([Ca2+]i) and the aggregation reaction of rat peritoneal neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). 2. Prostaglandin E2 (PGE2) and the specific IP-receptor agonist, cicaprost, both inhibited the FMLP-induced increase in [Ca2+]i (IC50 33 nM and 18 nM respectively) and the FMLP-induced aggregation reaction (IC50 5.6 nM and 7.9 nM respectively). PGD2, PGF2 alpha, and the TP-receptor agonist, U 46619, were inactive at the highest concentration tested (1 microM). 3. The EP1-receptor agonist, 17-phenyl-omega-trinor PGE2, and the EP3-receptor agonists, GR 63799X and sulprostone, had no inhibitory effect on FMLP-stimulated rat neutrophils. 4. PGE1 (EP/IP-receptor agonist) and iloprost (IP-receptor agonist) inhibited the FMLP-induced increase in [Ca2+]i with IC50 values of 34 nM and 38 nM respectively. The EP2-receptor agonists, butaprost and misoprostol (1 microM), inhibited both FMLP-stimulated [Ca2+]i and aggregation. However another EP2-receptor agonist, AH 13205, was inactive in both assays. 5. Prostanoid receptors present on rat neutrophils were further characterized by measuring [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) accumulation. Only those agonists capable of stimulating [3H]-cyclic AMP accumulation were able to inhibit both FMLP-stimulated [Ca2+]i and aggregation. 6. These results indicate that rat neutrophils possess inhibitory IP and EP-receptors; the relative potencies of PGE2, misoprostol and butaprost are those expected for the EP2-receptor subtype. No evidence for DP, FP, TP or EP1 and EP3-receptors was obtained. PMID:7834211