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Sample records for acetate pma-stimulated neutrophils

  1. Effect of cellulose acetate materials on the oxidative burst of human neutrophils.

    PubMed

    Moore, M A; Kaplan, D S; Picciolo, G L; Wallis, R R; Kowolik, M J

    2001-06-05

    Following adverse clinical events involving seven patients undergoing renal dialysis using 12-year-old cellulose acetate hemodialyzers, this in vitro study was proposed in an effort to characterize the inflammatory response to the constituent cellulose acetate (CA) fiber materials. Chemiluminescence (CL) and apoptosis assays were used to determine whether human neutrophils were activated by CA fiber materials and/or are sensitive to degradation/alteration of these fibers over time. Furthermore, the study examined in vitro assays with human neutrophils using a CA film, the solvents used in the film preparation and CA resin. The film could be cut to identical sized pieces in an effort to compare hemodialysis material effects in standardized amounts. For the CL assays, 60-min exposure was followed by secondary stimulation with n-formyl-met-leu-phe (fMLP) or phorbol-12-myristate-13-acetate (PMA). Short-term exposure (60-min postintroduction to CA materials) increased the inflammatory response as measured by the respiratory burst of neutrophils (p < or =.05), with CA fiber exposure significantly compared with cells alone. There was a trend toward an increased response with exposure to older fibers with secondary PMA stimulation. Apoptosis was increased 12% with exposure to the more aged fibers versus 2% with the new fibers. The fiber storage component, glycerol, significantly inhibited the oxidative response (p < or =.001; > or =80% suppression with concentrations of 5-20%). The solvents used in film preparation, N,N-dimethylacetamide and tetrahydrofuran, produced greater than a 70% and 60% suppression, respectively, of CL activity for all concentrations > or =1%. More work is needed to determine the specific nature of the interaction of inflammatory cells with CA materials, but early evidence suggests that neutrophils are activated by CA and display an altered response to more aged fibers.

  2. 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

  3. [Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils].

    PubMed

    Mikhal'chik, E V; Smolina, N V; Astamirova, T C; Gorudko, I V; Grigor'eva, D V; Ivanov, V A; Sokolov, A V; Kostevich, V A; Cherenkevich, S N; Panasenko, O M

    2013-01-01

    It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase--4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC50 = 20 microM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a "vicious circle" of neutrophil activation at the inflammatory site.

  4. Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

    PubMed Central

    Gorgojo, Juan; Scharrig, Emilia; Gómez, Ricardo M.; Harvill, Eric T.; Rodríguez, Maria Eugenia

    2017-01-01

    B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts. PMID:28095485

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

    PubMed

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

    2011-08-01

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

  6. Omega-3 fatty acids modulate Weibel-Palade body degranulation and actin cytoskeleton rearrangement in PMA-stimulated human umbilical vein endothelial cells.

    PubMed

    Bürgin-Maunder, Corinna S; Brooks, Peter R; Russell, Fraser D

    2013-11-08

    Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  7. Stimulus specific effect of ibuprofen on chemiluminescence of sheep neutrophils

    SciTech Connect

    Tahamont, M.V.; Margiotta, M.; Gee, M.H.

    1986-03-05

    The authors have shown that pretreatment with ibuprofen inhibits free radical release from complement stimulated neutrophils. To further examine the effect of ibuprofen on neutrophil free radical release, they stimulated neutrophils with the synthetic peptide, FMLP, phorbol myristate acetate (PMA), or zymosan-activated plasma (ZAP). Pure (>95%), viable (>95%) sheep neutrophils (2 x 10/sup 6/) were placed in HEPES buffer, luminol, drug or vehicle and stimulated in the luminometer with one of the stimuli. The chemiluminescence (CL) response was recorded and the drug treated samples were compared to vehicle treated controls. Ibuprofen had a dose dependent effect on CL in ZAP stimulated neutrophils. At the highest dose (10/sup -2/M) these cells produced only 37 +/- 7% of the CL response observed in the control cells. In contrast, at the same dose, ibuprofen did not significantly attenuate CL seen in FMLP stimulated cells, with these cells producing 79 +/- 7% of the control cells; nor did ibuprofen effect PMA stimulated CL, as these cells produced a CL response that was 85 +/- 8% of the control cells. Ibuprofen appears to have a stimulus specific effect on free radical release in activated neutrophils. It is also apparent that ibuprofen inhibits complement stimulated free radical release by some mechanism independent of its cyclooxygenase inhibitory effect.

  8. 12-tetradecanoyl-phorbol-13-acetate (PMA) produces injury to isolated rat lungs in the presence and absence of perfused neutrophils

    SciTech Connect

    Carpenter, L.J.; Roth, R.A.

    1986-03-01

    PMA produced injury to isolated, perfused rat lungs when eutrophils were added to or omitted from the buffer/albumin perfusion medium. When a high dose of PMA (57 ng/ml) was added to medium devoid of added neutrophils, perfusion pressure and lung weight increased. Together, superoxide dismutase (500 U/ml) and catalase (400 U/ml) had no effect on the increases in lung weight or perfusion pressure. However, papaverine (0.5 mM) prevented both the increase in perfusion pressure and fluid accumulation. When a concentration of PMA (14 ng/ml) that did not by itself cause lungs to accumulate fluid was added to perfusion medium containing neutrophils (1 x 10/sup 8/), perfusion pressures increased and lungs accumulated fluid. This concentration of PMA stimulated neutrophils (1 x 10/sup 8/) to release superoxide. Addition of superoxide dismutase (500 U/ml) and catalase (400 U/ml) to this medium prevented the increase in lung weight, but not the increase in perfusion pressure. Papaverine (0.5 mM) attenuated the increase in perfusion pressure and prevented fluid accumulation in these lungs. In summary, high concentrations of PMA produce lung injury which is independent of oxygen radicals; at lower concentrations it produces injury which is neutrophil-dependent and mediated by oxygen radicals.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Dietary fiber and the short-chain fatty acid acetate promote resolution of neutrophilic inflammation in a model of gout in mice.

    PubMed

    Vieira, Angélica T; Galvão, Izabela; Macia, Laurence M; Sernaglia, Érica M; Vinolo, Marco Aurélio R; Garcia, Cristiana C; Tavares, Luciana P; Amaral, Flávio A; Sousa, Lirlândia P; Martins, Flaviano S; Mackay, Charles R; Teixeira, Mauro M

    2017-01-01

    Gout is a disease characterized by the deposition of monosodium urate (MSU) crystals in the joints. Continuous gout episodes may lead to unresolved inflammatory responses and tissue damage. We investigated the effects of a high-fiber diet and acetate, a short-chain fatty acid (SCFA) resulting from the metabolism of fiber by gut microbiota, on the inflammatory response in an experimental model of gout in mice. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. The onset of inflammatory response induced by MSU crystals was not altered in animals given a high-fiber diet, but the high-fiber diet induced faster resolution of the inflammatory response. Similar results were obtained in animals given the SCFA acetate. Acetate was effective, even when given after injection of MSU crystals at the peak of the inflammatory response and induced caspase-dependent apoptosis of neutrophils that accounted for the resolution of inflammation. Resolution of neutrophilic inflammation was associated with decreased NF-κB activity and enhanced production of anti-inflammatory mediators, including IL-10, TGF-β, and annexin A1. Acetate treatment or intake of a high-fiber diet enhanced efferocytosis, an effect also observed in vitro with neutrophils treated with acetate. In conclusion, a high-fiber diet or one of its metabolic products, acetate, controls the inflammatory response to MSU crystals by favoring the resolution of the inflammatory response. Our studies suggest that what we eat plays a determinant role in our capacity to fine tune the inflammatory response.

  14. Differentiation of cellular processes involved in the induction and maintenance of stimulated neutrophil adherence.

    PubMed

    English, D; Gabig, T G

    1986-05-01

    Neutrophil adherence stimulated by phorbol myristate acetate (PMA) was investigated by quantitating the attachment of 51Cr-labeled neutrophils to plastic surfaces and to the endothelium of umbilical veins mounted in compartmentalized Lucite chambers. PMA-induced adherence could be functionally separated into an induction phase requiring cellular metabolism and a Mg++ dependent maintenance phase that was independent of cellular metabolism. Thus, metabolic inhibitors (N-ethylmaleimide, 2-deoxyglucose) blocked adherence when added to neutrophils prior to PMA, but did not cause detachment of cells adhering as a consequence of prior exposure to PMA. PMA failed to induce adherence of neutrophils incubated at low (0.4 degree C) temperature, but temperature reduction, even for prolonged periods, did not cause detachment of adherent cells. Thus, the attractive forces that mediate stimulated adherence persist independently of any sustained metabolic response to the inducing stimulus. However, removal of Mg++ from the media above adherent cells resulted in immediate detachment, indicating that the cation was required for the persistent expression or maintenance of the attractive forces involved. The extent of stimulated adherence correlated well with the extent of degranulation when rates were varied by limiting the incubation time or stimulus concentration. This correlation was not absolute; in the absence of Mg++, PMA induced degranulation normally but failed to enhance adherence. To explain these findings, we investigated the possibility that PMA-stimulated adherence was maintained by Mg++-dependent cellular adherence molecules released during exocytosis. Supernatants of stimulated neutrophils were devoid of adherence-promoting activity, and only weak activity was recovered in supernatants of mechanically disrupted neutrophils. PMA effectively stimulated the tight adherence of degranulated neutrophil cytoplasts to plastic surfaces and did so in the absence of stimulated

  15. 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

  16. Endogenous glucocorticoids modulate neutrophil function in a murine model of haemolytic uraemic syndrome

    PubMed Central

    Gómez, S A; Fernández, G C; Camerano, G; Dran, G; Rosa, F A; Barrionuevo, P; Isturiz, M A; Palermo, M S

    2005-01-01

    Haemolytic uraemic syndrome (HUS) is caused by Shiga-toxin-producing Escherichia coli (STEC). Although, Shiga toxin type 2 (Stx2) is responsible for the renal pathogenesis observed in patients, the inflammatory response, including cytokines and polymorphonuclear neutrophils (PMN), plays a key role in the development of HUS. Previously, we demonstrated that Stx2 injection generates an anti-inflammatory reaction characterized by endogenous glucocorticoid (GC) secretion, which attenuates HUS severity in mice. Here, we analysed the effects of Stx2 on the pathogenic function of PMN and the potential role of endogenous GC to limit PMN activation during HUS development in a murine model. For this purpose we assessed the functional activity of isolated PMN after in vivo treatment with Stx2 alone or in simultaneous treatment with Ru486 (GC receptor antagonist). We found that Stx2 increased the generation of reactive oxygen intermediates (ROI) under phobol-myristate-acetate (PMA) stimulation and that the simultaneous treatment with Ru486 strengthened this effect. Conversely, both treatments significantly inhibited in vitro phagocytosis. Furthermore, Stx2 augmented in vitro PMN adhesion to fibrinogen (FGN) and bovine serum albumin (BSA) but not to collagen type I (CTI). Stx2 + Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2. Whereas Stx2 significantly increased migration towards N-formyl-methionyl-leucyl-phenylalanine (fMLP), Stx2 + Ru486 treatment enhanced and accelerated this process. The percentage of apoptotic PMN from Stx2-treated mice was higher compared with controls, but equal to Stx2 + Ru486 treated mice. We conclude that Stx2 activates PMN and that the absence of endogenous GC enhances this activation suggesting that endogenous GC can, at least partially, counteract PMN inflammatory functions. PMID:15606615

  17. Autophagy Is Impaired in Neutrophils from Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Kuwabara, Wilson Mitsuo Tatagiba; Curi, Rui; Alba-Loureiro, Tatiana Carolina

    2017-01-01

    We tested the hypothesis that changes reported on functions of neutrophils from streptozotocin-induced diabetic rats involve autophagy impairment. Wistar rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.), and the measurements were carried out 2 weeks afterward. Neutrophils were collected through intraperitoneal cavity lavage after 4 h of i.p. oyster glycogen type 2 injection. Neutrophils cultured with PMA (20 nM) for 1 h were used for analysis of plasma membrane integrity, DNA fragmentation, and mitochondrial depolarization by flow cytometry; expression of Atg5, Atg14, Beclin1, LC3BII, and Rab9 by RT-PCR; the contents of caspase 3, LC3BII/LC3BI, and pS6 by western blotting; ATP content by fluorescence essay; reactive oxygen species production by chemiluminescence (Luminol), and autophagy by immunofluorescence tracking LC3B cleavage. Herein, neutrophils from diabetic rats had high DNA fragmentation, depolarization of mitochondrial membrane, low content of ATP, and high content of cleaved caspase 3 after PMA stimulation. Neutrophils from diabetic rats also had low expression of LC3B, failed to increase the expression of Rab9 and Atg14 induced by PMA stimulation. Neutrophils from diabetic animals also had low cleavage of LC3BI to LC3BII and do not present punctate structures that label autophagosomal membranes after stimulus. The changes of neutrophil function reported in diabetic rats do involve impaired autophagy. The suppression of autophagy in neutrophils from diabetic rats may be associated with the activation of the mTOR signaling as indicated by the high content of pS6. PMID:28163707

  18. Endomorphins delay constitutive apoptosis and alter the innate host defense functions of neutrophils.

    PubMed

    Azuma, Yasutaka; Ohura, Kiyoshi; Wang, Pao-Li; Shinohara, Mitsuko

    2002-04-01

    Recent studies have shown that opioid peptides are released from cells of the immune system during inflammation and stress, and are associated with altered immune responses. Moreover, concentrations of opioid peptides are increased in peripheral blood and at the sites of inflammatory reactions. The aim of this study was to evaluate immunological effects of opioid peptides endomorphins 1 and 2 on constitutive apoptosis, superoxide anion production, hydrogen peroxide production, adhesion, phagocytosis, and chemotaxis of neutrophils. Neutrophils were isolated by peritoneal lavage from rats. Endomorphins 1 and 2 significantly delayed constitutive neutrophil apoptosis. The delay of neutrophil apoptosis was markedly attenuated by LY294002, a phosphoinositide 3-kinase inhibitor. Moreover, endomorphins 1 and 2 activated the phosphoinositide 3-kinase pathway as determined by phosphorylation of BAD. In contrast, endomorphins 1 and 2 blocked the production of superoxide anion and hydrogen peroxide by PMA-stimulated neutrophils. In addition, endomorphins 1 and 2 inhibited neutrophil adhesion to fibronectin. Moreover, endomorphins 1 and 2 potentiated neutrophil chemotaxis toward zymosan-activated serum and IL-8, respectively. However, endomorphins 1 and 2 did not alter phagocytosis of Escherichia coli by neutrophils. These results suggest that endomorphins 1 and 2 may act to delay neutrophil apoptosis and alter the natural immune functions of neutrophils.

  19. 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.

  20. 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

  1. Immunomodulation of the neutrophil respiratory burst by endomorphins 1 and 2.

    PubMed

    Azuma, Y; Wang, P L; Shinohara, M; Ohura, K

    2000-12-01

    Opioid peptides were found to be released from cells of the immune system during inflammation and stress, and were associated with altered immune responses. Production of superoxide anions by PMA-stimulated neutrophils was markedly inhibited in a concentration-dependent manner by preincubation for 15 min with 10(-18) - 10(-6) M of the endogenous opioid peptides endomorphin 1 or 2. Inhibition was prevented by prior treatment with the micro-opioid receptor-selective antagonist beta-funaltrexamine at 10(-12) - 10(-8) M, but not the delta-opioid receptor-selective antagonist naltrindole. In contrast, endomorphins 1 and 2 caused significant potentiation of superoxide anion production in unstimulated neutrophils. These results suggest that the endogenous opioid peptides endomorphins 1 and 2 may modulate the production of superoxide anions in neutrophils via mu-opioid receptors.

  2. Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs.

    PubMed

    Bochsler, P N; Slauson, D O; Neilsen, N R

    1990-10-01

    The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 microgram/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5-45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 micrograms/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10(-5) M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant

  3. 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.

  4. 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

  5. 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

  6. Flecainide acetate acetic acid solvates.

    PubMed

    Veldre, Kaspars; Actiņs, Andris; Eglite, Zane

    2011-02-01

    Flecainide acetate forms acetic acid solvates with 0.5 and 2 acetic acid molecules. Powder X-ray diffraction, differential thermal analysis/thermogravimetric, infrared, and potentiometric titration were used to determine the composition of solvates. Flecainide acetate hemisolvate with acetic acid decomposes to form a new crystalline form of flecainide acetate. This form is less stable than the already known polymorphic form at all temperatures, and it is formed due to kinetic reasons. Both flecainide acetate nonsolvated and flecainide acetate hemisolvate forms crystallize in monoclinic crystals, but flecainide triacetate forms triclinic crystals. Solvate formation was not observed when flecainide base was treated with formic acid, propanoic acid, and butanoic acid. Only nonsolvated flecainide salts were obtained in these experiments.

  7. In vivo and in vitro assessment of porcine neutrophil activation responses to chemoattractants: flow cytometric evidence for the selective absence of formyl peptide receptors.

    PubMed

    Fletcher, M P; Stahl, G L; Longhurst, J C

    1990-04-01

    Interest in the role that activated granulocytes play in C5a-induced myocardial ischemia prompted us to investigate and compare activation responses of pig and human neutrophils. The responses of Hypaque-Ficoll purified porcine (P-PMN) and human neutrophils (H-PMN) to stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, phorbol myristate acetate (PMA), and calcium ionophore A23187 (A23187) were compared by flow cytometrically measured changes in the cells' forward (FWD-SC) (a measure of shape/volume change) and right angle (90 degrees-SC) light scatter (a measure of secretion), and in the distribution of the membrane potential sensitive fluorescent probe di-O-C (3). FMLP, C5a, and Zymosan-activated serum (ZAS stimulated chemotaxis and FMLP vs. PMA-stimulated adherence to plastic were also compared. Unstimulated P-PMN had lower FWD-SC and 90 degrees-SC than H-PMN (39.4 +/- 1.4 vs. 48.4 +/- 2.0 P less than 0.05, and 32.7 +/- 2.7 vs. 52.4 +/- 1.5 units, P less than 0.005, for FWD-SC and 90 degrees-SC of P-PMN vs. H-PMN, respectively). P-PMN selectively failed to increase their FWD-SC upon stimulation with FMLP (0.0 +/- 0.5% vs. 26.1 +/- 6.8%, P-PMN vs. H-PMN), or decrease their 90 degrees-SC when treated with cytochalasin B + FMLP (secretion) (2.4 +/- 0.1% vs. -35.8 +/- 4.6% change in 90 degrees-SC, P-PMN vs. H-PMN), while responding comparably to C5a, PMA, and A23187. P-PMN failed to depolarize in response to FMLP but responded similarly to H-PMN when activated by C5a, A23187, and PMA. P-PMN's chemotactic response to FMLP was selectively absent since the cells responded well to purified pig C5a. FMLP stimulated significant increases in H-PMN adherence to bovine serum albumin-coated plastic (44.1 +/- 6.7% vs. 12.6 +/- 3.7%, FMLP vs. buffer, P less than 0.025), but failed to increase adherence of P-PMN above baseline 0.68 +/- 0.20% vs. 2.12 +/- 1.90%, FMLP vs. buffer, P greater than 0.05. PMA (100 ng/ml) stimulated comparable increases in adherence in

  8. Mesoxalaldehyde acetals

    SciTech Connect

    Gordeeva, G.N.; Kalashnikov, S.M.; Popov, Yu.N.; Kruglov, E.A.; Imashev, U.B.

    1987-11-10

    The treatment of methylglyoxal acetals by alkyl nitrites in the presence of the corresponding aliphatic alcohols and hydrochloric acid leads to the formation of linear mesoxalaldehyde acetals, whose structure was established by NMR spectroscopy and mass spectrometry. The major pathways for the decomposition of these molecules upon electron impact were established.

  9. Management of neutrophilic dermatoses.

    PubMed

    Schadt, Courtney R; Callen, Jeffrey P

    2012-01-01

    Neutrophilic dermatoses, including Sweet's syndrome, pyoderma gangrenosum, and rheumatoid neutrophilic dermatitis, are inflammatory conditions of the skin often associated with underlying systemic disease. These are characterized by the accumulation of neutrophils in the skin. The associated conditions, potential for systemic neutrophilic infiltration, and therapeutic management of these disorders can be similar. Sweet's syndrome can often be effectively treated with a brief course of systemic corticosteroids. Pyoderma gangrenosum, however, can be recurrent, and early initiation of a steroid-sparing agent is prudent. Second-line treatment for both of these conditions includes medications affecting neutrophil function, in addition to immunosuppressant medications.

  10. Neutrophil Dysfunction in Sepsis

    PubMed Central

    Zhang, Fang; Liu, An-Lei; Gao, Shuang; Ma, Shui; Guo, Shu-Bin

    2016-01-01

    Objective: Sepsis is defined as life-threatening organ dysfunction due to a dysregulated host response to infection. In this article, we reviewed the correlation between neutrophil dysfunction and sepsis. Data Sources: Articles published up to May 31, 2016, were selected from the PubMed databases, with the keywords of “neutrophil function”, “neutrophil dysfunction”, and “sepsis”. Study Selection: Articles were obtained and reviewed to analyze the neutrophil function in infection and neutrophil dysfunction in sepsis. Results: We emphasized the diagnosis of sepsis and its limitations. Pathophysiological mechanisms involve a generalized circulatory, immune, coagulopathic, and/or neuroendocrine response to infection. Many studies focused on neutrophil burst or cytokines. Complement activation, impairment of neutrophil migration, and endothelial lesions are involved in this progress. Alterations of cytokines, chemokines, and other mediators contribute to neutrophil dysfunction in sepsis. Conclusions: Sepsis represents a severe derangement of the immune response to infection, resulting in neutrophil dysfunction. Neutrophil dysfunction promotes sepsis and even leads to organ failure. Mechanism studies, clinical practice, and strategies to interrupt dysregulated neutrophil function in sepsis are desperately needed. PMID:27824008

  11. Stimulation of neutrophils by tumor necrosis factor

    SciTech Connect

    Klebanoff, S.J.; Vadas, M.A.; Harlan, J.M.; Sparks, L.H.; Gamble, J.R.; Agosti, J.M.; Waltersdorph, A.M.

    1986-06-01

    Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H/sub 2/O/sub 2/ production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.

  12. 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).

  13. Neutrophils in cancer.

    PubMed

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

    2016-09-01

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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Effects of dietary supplementation with eicosapentaenoic acid or gamma-linolenic acid on neutrophil phospholipid fatty acid composition and activation responses.

    PubMed

    Fletcher, M P; Ziboh, V A

    1990-10-01

    Previous data that alimentation with fish oil rich in eicosapentaenoic acid (EPA; 20:n-3) or vegetable oil rich in gamma-linolenic acid (GLA; 18:3n-6) can reduce symptoms of inflammatory skin disorders lead us to determine the effects of dietary supplements of oils rich in EPA or GLA on guinea pig (GP) neutrophil (PMN) membrane potential (delta gamma), secretion, and superoxide (O2-) responses. Weanling GPs were initially fed diets supplemented with olive oil (less than 0.1% EPA; less than 0.1% GLA) for 2 weeks, followed by a crossover by two sets of animals to diets supplemented with fish oil (19% EPA) or borage oil (25% GLA). At 4-week intervals, 12% sterile casein-elicited peritoneal neutrophils (PMN) were assessed for membrane polyunsaturated fatty acid (PUFA) profiles and FMLP-, LTB4-, and PMA-stimulated delta gamma changes, changes in flow cytometrically measured forward scatter (FWD-SC) (shape change), 90 degrees scatter (90 degrees -SC) in cytochalasin B-pretreated-PMN (secretion response), and superoxide responses, GP incorporated EPA and GLA (as the elongation product, dihomo-GLA or DGLA) into their PMN phospholipids by 4 weeks. The peritoneal PMN of all groups demonstrated broad resting FWD-SC and poor activation-related FWD-SC increases, suggesting in vivo activation. While secretion was comparable in the three groups in response to FMLP, there was a trend toward inhibition of LTB4-stimulated 90 degrees -SC loss in both fish and borage oil groups. This was significant only with borage oil (21.7 +/- 2.1 vs 15.3 +/- 1.2% loss of baseline 90 degrees -SC, olive vs borage: P = 0.03). PMN from borage- and fish oil-fed GPs showed a progressively lower O2- response to FMLP than the olive oil group (73.9 +/- 3.9 and 42.9 +/- 6.8% of olive oil response for borage and fish oils, respectively; P less than 0.005 and P less than 0.01, respectively, at 12 weeks), while PMA-stimulated O2- was inhibited only in the fish oil-fed group and only at 12 weeks (62.0 +/- 2

  20. Extracellular proton release by stimulated neutrophils

    SciTech Connect

    van Zwieten, R.; Wever, R.; Hamers, M.N.; Weening, R.S.; Roos, D.

    1981-07-01

    We have tried to elucidate the mechanism of phagosome acidification in human neutrophils. Assuming that phenomena occurring at the plasma membrane reflect reactions in the phagocytic vacuoles, we have stimulated human neutrophils with agents that induce a ''respiratory burst,'' and we have measured the release of protons into the extracellular medium. Phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles each caused a rapid release of protons, concomitant with the increase in oxygen consumption. The stimulated release of protons was strictly coupled to the increase respiration of the cells, because inhibition of the respiration of either anaerobiosis, chlorpromazine, or glycolytic inhibitors also inhibited the release of protons. Also, in the presence of the above-mentioned stimulating agents, neutrophils from three patients with chronic granulomatous disease enhanced neither respiration not proton release. In normal cells, the ratio of deltaH+/-deltaO2 was 1.04 +/- 0.19 (mean +/ SD, n . 13). The mechanism of this proton release is not clear. The amount of lactic and carbonic acid produced by stimulated neutrophils was inadequate to explain the amount of protons released. Perhydroxyl radicals were also ruled out as the source of the protons. Because the cells did not release measurable amounts of phosphate ions, a phosphate-hydroxyl-ion antiport was also excluded. Finally, the lack of any effect of uncouplers renders it unlikely that a respiration-driven proton gradient is built up across the plasma membrane.

  1. Neutrophil paralysis in sepsis.

    PubMed

    Alves-Filho, José C; Spiller, Fernando; Cunha, Fernando Q

    2010-09-01

    Sepsis develops when the initial host response is unable to contain the primary infection, resulting in widespread inflammation and multiple organ dysfunction. The impairment of neutrophil migration into the infection site, also termed neutrophil paralysis, is a critical hallmark of sepsis, which is directly related to the severity of the disease. Although the precise mechanism of this phenomenon is not fully understood, there has been much advancement in the understanding of this field. In this review, we highlight the recent insights into the molecular mechanisms of neutrophil paralysis during sepsis.

  2. Constitutive apoptosis in equine peripheral blood neutrophils in vitro

    PubMed Central

    Brazil, Timothy J.; Dixon, Padraic M.; Haslett, Christopher; Murray, Joanna; McGorum, Bruce C.

    2014-01-01

    The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone. PMID:25239298

  3. Constitutive apoptosis in equine peripheral blood neutrophils in vitro.

    PubMed

    Brazil, Timothy J; Dixon, Padraic M; Haslett, Christopher; Murray, Joanna; McGorum, Bruce C

    2014-12-01

    The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone.

  4. The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

    PubMed Central

    Femling, Jon K.; Cherny, Vladimir V.; Morgan, Deri; Rada, Balázs; Davis, A. Paige; Czirják, Gabor; Enyedi, Peter; England, Sarah K.; Moreland, Jessica G.; Ligeti, Erzsébet; Nauseef, William M.; DeCoursey, Thomas E.

    2006-01-01

    Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and

  5. Synthesis of chlorinated flavonoids with anti-inflammatory and pro-apoptotic activities in human neutrophils.

    PubMed

    Freitas, Marisa; Ribeiro, Daniela; Tomé, Sara M; Silva, Artur M S; Fernandes, Eduarda

    2014-10-30

    Neutrophils are considered the central cells of acute inflammation. Flavonoids have been suggested as therapeutic agents to avoid damages induced by inflammatory processes. It is well known the reactivity of flavonoids with hypochlorous acid produced by neutrophils, to form stable mono and dichlorinated products. In this study, we synthesized novel chlorinated flavonoids and investigated their effect in neutrophils' oxidative burst and in its lifespan, in comparison with the parent non-chlorinated flavonoids. The obtained results demonstrate that chlorinated flavonoids were more efficient than their parent compounds in modulating neutrophils' oxidative burst in phorbol myristate acetate-activated neutrophils. Some of the tested flavonoids drive neutrophil apoptosis in a caspase 3-dependent fashion. The present data showed that 8-chloro-3',4',5,7-tetrahydroxyflavone (4a) constitute an alternative anti-inflammatory therapy, due to the proven ability to suppress mechanisms engaged at the onset and progression of inflammation.

  6. Ischemia Induced Neutrophil Activation and Diapedesis is Lipoxygenase Dependent.

    DTIC Science & Technology

    2007-11-02

    activity in mediating PMN activation and diapedesis . Anesthetized rabbits (n = 8) underwent 3 h of bilateral hindlimb ischemia. At 10 min of reperfusion...enhanced response of 337% to PMA stimulation. To study diapedesis , plasma collected at 10 min of reperfusion was introduced into plastic chambers taped...abolished PMN activation (51 +/- 12 fM DCF/cell) and ischemic plasma induced diapedesis into the plastic chamber (38 +/- 18 PMN/mm(exp 3)).

  7. 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...

  8. 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

  9. Neutrophil disorders and their management

    PubMed Central

    Lakshman, R; Finn, A

    2001-01-01

    Neutrophil disorders are an uncommon yet important cause of morbidity and mortality in infants and children. This article is an overview of these conditions, with emphasis on clinical recognition, rational investigation, and treatment. A comprehensive list of references is provided for further reading. Key Words: neutrophil disorders • chronic granulomatous disease • neutrophil chemotaxis • phagocytosis PMID:11271792

  10. The Neutrophil Nucleus and Its Role in Neutrophilic Function.

    PubMed

    Carvalho, Leonardo Olivieri; Aquino, Elaine Nascimento; Neves, Anne Caroline Dias; Fontes, Wagner

    2015-09-01

    The cell nucleus plays a key role in differentiation processes in eukaryotic cells. It is not the nucleus in particular, but the organization of the genes and their remodeling that provides the data for the adjustments to be made according to the medium. The neutrophil nucleus has a different morphology. It is a multi-lobed nucleus where some researchers argue no longer function. However, studies indicate that it is very probable the occurrence of chromatin remodeling during activation steps. It may be that the human neutrophil nucleus also contributes to the mobility of neutrophils through thin tissue spaces. Questions like these will be discussed in this small review. The topics include morphology of human neutrophil nucleus, maturation process and modifications of the neutrophil nucleus, neutrophil activation and chromatin modifications, causes and consequences of multi-lobulated segmented morphology, and importance of the nucleus in the formation of neutrophil extracellular traps (NETs).

  11. 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.

  12. Recent advances in understanding neutrophils

    PubMed Central

    Deniset, Justin F.; Kubes, Paul

    2016-01-01

    Neutrophils have long been regarded as key effectors of the innate immune response during acute inflammation. Recent evidence has revealed a greater functional diversity for these cells than previously appreciated, expanding roles for neutrophils in adaptive immunity and chronic pathologies. In this review, we summarize some of the evolving paradigms in the neutrophil field and highlight key advances that have contributed to our understanding of neutrophil behavior and function in vivo. We examine the concept of neutrophil subsets and polarization, we discuss novel immunomodulatory roles for neutrophils in shaping the immune response, and, finally, we identify technical advances that will further enhance our ability to track the function and fate of neutrophils. PMID:28105328

  13. High concentrations of glucose reduce the oxidative metabolism of dog neutrophils in vitro

    PubMed Central

    2013-01-01

    Background Dogs are commonly affected by hyperglycemic conditions. Hyperglycemia compromises the immune response and favors bacterial infections; however, reports on the effects of glucose on neutrophil oxidative metabolism and apoptosis are conflicting in humans and rare in dogs. Considering the many complex factors that affect neutrophil oxidative metabolism in vivo, we investigated in vitro the specific effect of high concentrations of glucose on superoxide production and apoptosis rate in neutrophils from healthy dogs. Results The capacity of the neutrophils to reduce tetrazolium nitroblue decreased significantly in the higher concentration of glucose (15.13 ± 9.73% (8 mmol/L) versus 8.93 ± 5.71% (16 mmol/L)). However, there were no changes in tetrazolium nitroblue reduction at different glucose concentrations when the neutrophils were first activated with phorbol myristate acetate. High concentrations of glucose did not affect the viability and apoptosis rate of canine neutrophils either with or without prior camptothecin stimulation. This study provides the first evidence that high concentrations of glucose inhibit the oxidative metabolism of canine neutrophils in vitro in a manner similar to that which occurs in humans, and that the decrease in superoxide production did not increase the apoptosis rate. Conclusions A high concentration of glucose reduces the oxidative metabolism of canine neutrophils in vitro. It is likely that glucose at high concentrations rapidly affects membrane receptors responsible for the activation of NADPH oxidase in neutrophils; therefore, the nonspecific immune response can be compromised in dogs with acute and chronic hyperglycemic conditions. PMID:23388121

  14. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    PubMed

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

  15. A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.

    PubMed

    Jeffery, Unity; Gray, Robert D; LeVine, Dana N

    2016-11-21

    Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

  16. Occupational triphenyltin acetate poisoning: a case report.

    PubMed Central

    Colosio, C; Tomasini, M; Cairoli, S; Foà, V; Minoia, C; Marinovich, M; Galli, C L

    1991-01-01

    A case of triphenyltin acetate (TPTA) poisoning is described. The patient, who had been exposed mainly to cutaneous absorption, showed acute stages of an urticarial eruption, signs of hepatic injury, slight glucose intolerance, and electroencephalographic abnormalities. Concomitant with the highest concentrations of tin in plasma and the peak of tin excretion in urine, neutrophils did not show the normal increase in actin polymerisation after stimulation with a chemotactic peptide (100 nM fMLP). The peak of urinary excretion of tin occurred between the fifth and the sixth day after poisoning; subsequently, the rate of excretion became slow, suggesting biphasic kinetics with the possibility of a cumulative trend. Images PMID:1825604

  17. The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Kostrzewa, Artur; Łuczak, Michał; Jagodziński, Paweł P; Baer-Dubowska, Wanda

    2012-06-01

    The aim of this study was to evaluate the effect of betanin, one of the beetroot major components, on ROS production, DNA damage and apoptosis in human resting and stimulated with phorbol 12-myristate13-acetate polymorphonuclear neutrophils, one of the key elements of the inflammatory response. Incubation of neutrophils with betanin in the concentration range 2-500 µM resulted in significant inhibition of ROS production (by 15-46%, depending on the ROS detection assay). The antioxidant capacity of betanin was most prominently expressed in the chemiluminescence measurements. This compound decreased also the percentage of DNA in comet tails in stimulated neutrophils, but only at the 24 h time point. In resting neutrophils an increased level of DNA in comet tails was observed. Betanin did not affect the activity of caspase-3, in resting neutrophils, but significantly enhanced the enzyme activity in stimulated neutrophils. The western blot analysis showed, however, an increased level of caspase-3 cleavage products as a result of betanin treatment both in resting and stimulated neutrophils. The results indicate that betanin may be responsible for the effect of beetroot products on neutrophil oxidative metabolism and its consequences, DNA damage and apoptosis. The dose and time dependent effects on these processes require further studies.

  18. The uremic toxin methylguanidine increases the oxidative metabolism and accelerates the apoptosis of canine neutrophils.

    PubMed

    Bosco, A M; Almeida, B F M; Pereira, P P; Dos Santos, D B; Neto, Á J S; Ferreira, W L; Ciarlini, P C

    2017-03-01

    We investigated the hypothesis that the increased concentration of plasma methylguanidine (MG) increases oxidative metabolism and accelerates apoptosis of neutrophils from dogs with chronic kidney disease (CKD). To achieve this, the levels of MG were quantified in healthy (n=16) and uremic dogs with CKD stage 4 of according to the guidelines of the International Renal Interest Society (IRIS, 2015) (n=16) using high performance liquid chromatography (HPLC). To evaluate the isolated effect of MG on neutrophil oxidative metabolism and apoptosis, neutrophils isolated from 12 healthy dogs were incubated with the highest concentration of plasma MG (0.005g/L) observed in dogs with CKD. Neutrophil oxidative metabolism was assessed by flow cytometry, using the probes hydroethidine for superoxide production and 2',7'-dichlorofluorescein diacetate for hydrogen peroxide production, with or without phorbol myristate acetate (PMA) stimulus. Neutrophil apoptosis and viability were also evaluated in flow cytometer using the Annexin V-PE system, with or without the apoptosis-inducing effect of camptothecin. Uremic dogs presented higher concentrations of MG (p<0.0001), increased oxidative stress and primed neutrophils with higher apoptosis rate. The neutrophil abnormalities observed in vivo were also reproduced in vitro, using cells isolated from healthy dogs and incubated with MG. We obtained strong evidence that in dogs with CKD, increased MG levels contributed to oxidative stress and potentially compromised the non-specific immune response by altering the oxidative metabolism and viability of canine neutrophils.

  19. Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

    PubMed

    Feyen, J H; Petersen, D N; Kream, B E

    1988-04-01

    We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

  20. Formation of Neutrophil Extracellular Traps under Low Oxygen Level

    PubMed Central

    Branitzki-Heinemann, Katja; Möllerherm, Helene; Völlger, Lena; Husein, Diab M.; de Buhr, Nicole; Blodkamp, Stefanie; Reuner, Friederike; Brogden, Graham; Naim, Hassan Y.; von Köckritz-Blickwede, Maren

    2016-01-01

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

  1. 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.

  2. 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

  3. Effect of exhaustive exercise on human neutrophils in athletes.

    PubMed

    Yamada, M; Suzuki, K; Kudo, S; Totsuka, M; Simoyama, T; Nakaji, S; Sugawara, K

    2000-01-01

    In order to investigate the effect of exercise on the capacity of neutrophils to produce reactive oxygen species (ROS), eight male cross-country skiers underwent maximal exercise. Peripheral blood samples were taken pre-exercise, 0 h, 1 h, and 2 h after finishing maximal exercise. Leukocyte counts significantly increased (p < 0. 05), particularly lymphocytes (p < 0.05), just after the exercise period (0 h) and significantly increased again (p < 0.05), particularly neutrophils (p < 0.05), 2 h after the exercise compared with pre-exercise values. The capacity of isolated neutrophils to produce ROS was assessed by lucigenin (Lg)-dependent chemiluminescence (CL) and luminol (Lm)-dependent CL on stimulation with opsonized zymosan (OZ) and phorbol myristate acetate (PMA). Just after exercise, the LgCL response was not affected, while the response of LmCL mixed with sodium azide, which inhibits catalase and myeloperoxidase (MPO) activity, was significantly enhanced (p < 0.05). In addition, just after exercise, the level of serum growth hormone increased significantly (p < 0.05). The serum cortisol level also increased significantly just after and 1 h after exercise (p < 0.05). These data indicated that maximal exercise not only mobilized neutrophils from marginated pools into the circulation, but also caused increased ROS generation.

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

    PubMed

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

    2006-01-01

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

  5. Hypochlorous acid regulates neutrophil extracellular trap release in humans.

    PubMed

    Palmer, L J; Cooper, P R; Ling, M R; Wright, H J; Huissoon, A; Chapple, I L C

    2012-02-01

    Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

  6. G Protein-Coupled Receptor 43 Modulates Neutrophil Recruitment during Acute Inflammation

    PubMed Central

    Nicholls, Alyce J.; Oliveira, Ana Carolina; Mason, Linda J.; Binge, Lauren; Mackay, Charles R.; Wong, Connie H. Y.

    2016-01-01

    Fermentation of dietary fibre in the gut yields large amounts of short chain fatty acids (SCFAs). SCFAs can impart biological responses in cells through their engagement of ‘metabolite-sensing’ G protein-coupled receptors (GPCRs). One of the main SCFA receptors, GPR43, is highly expressed by neutrophils, which suggests that the actions of GPR43 and dietary fibre intake may affect neutrophil recruitment during inflammatory responses in vivo. Using intravital imaging of the small intestine, we found greater intravascular neutrophil rolling and adhesion in Gpr43−/−mice in response to LPS at 1 h. After 4 h of LPS challenge, the intravascular rolling velocity of GPR43-deficient neutrophils was reduced significantly and increased numbers of neutrophils were found in the lamina propria of Gpr43−/−mice. Additionally, GPR43-deficient leukocytes demonstrated exacerbated migration into the peritoneal cavity following fMLP challenge. The fMLP-induced neutrophil migration was significantly suppressed in wildtype mice that were treated with acetate, but not in Gpr43−/−mice, strongly suggesting a role for SCFAs in modulating neutrophil migration via GPR43. Indeed, neutrophils of no fibre-fed wildtype mice exhibited elevated migratory behaviour compared to normal chow-fed wildtype mice. Interestingly, this elevated migration could also be reproduced through simple transfer of a no fibre microbiota into germ-free mice, suggesting that the composition and function of microbiota stemming from a no fibre diet mediated the changes in neutrophil migration. Therefore, GPR43 and a microbiota composition that allows for SCFA production function to modulate neutrophil recruitment during inflammatory responses. PMID:27658303

  7. Inhibition of neutrophil activation by alpha1-acid glycoprotein.

    PubMed Central

    Costello, M J; Gewurz, H; Siegel, J N

    1984-01-01

    We report that alpha1-acid glycoprotein (AAG), a naturally occurring human plasma protein and acute phase reactant of uncertain biological function, inhibits human neutrophil aggregation and superoxide anion generation induced by a variety of stimuli including zymosan treated serum, formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. Inhibition was transient, directly proportional to the glycoprotein concentration and inversely proportional to the concentration of the stimulus added. Desialyzation, resulting in the removal of a substantial portion of the molecule's negative charge, did not alter the effectiveness of AAG. Removal of the penultimate galactose residues from desialyzed AAG resulted in a slight but significant reversal of inhibition, suggesting that the heteropolysaccharide units of AAG may be important for inhibition of cellular function. We therefore suggest that the acute phase glycoprotein AAG may be a significant modulator of neutrophil as well as platelet and lymphocyte function during inflammation. PMID:6321072

  8. Inhibition of neutrophil priming and tyrosyl phosphorylation by cepharanthine, a nonsteroidal antiinflammatory drug.

    PubMed

    Kobuchi, H; Li, M J; Matsuno, T; Yasuda, T; Utsumi, K

    1992-12-01

    Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.

  9. Neutrophil in Viral Infections, Friend or Foe?

    PubMed Central

    Drescher, Brandon; Bai, Fengwei

    2012-01-01

    Polymorphonuclear leukocytes or neutrophils are the first immune cells to the site of injury and microbial infection. Neutrophils are crucial players in controlling bacterial and fungal infections, and in particular secondary infections, by phagocytosis, degranulation and neutrophil extracellular traps (NETs). While neutrophils have been shown to play important roles in viral pathogenesis, there is a lack of detailed investigation. In this article, we will review recent progresses toward understanding the role of neutrophils in viral pathogenesis. PMID:23178588

  10. Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

    SciTech Connect

    Suematsu, M.; Oshio, C.; Miura, S.; Suzuki, M.; Houzawa, S.; Tsuchiya, M.

    1988-08-30

    Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release in luminol-dependent photoemission from stimulated neutrophils.

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

    PubMed

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

    1986-07-01

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

  12. 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

  13. 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.

  14. 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.

  15. Functional relationship of the cytochrome b to the superoxide-generating oxidase of human neutrophils.

    PubMed

    Gabig, T G; Schervish, E W; Santinga, J T

    1982-04-25

    A subcellular particulate fraction containing the NADPH-dependent O2.--generating oxidase from stimulated human neutrophils was prepared. This fraction was depleted of certain enzyme markers of primary and secondary granules and was devoid of measurable myeloperoxidase, both enzymatically and spectrally. When prepared from neutrophils which had been previously stimulated with phorbal myristate acetate, this fraction contained cyanide-insensitive, pyridine nucleotide-dependent O2.--generating activity with a specific activity of 260 nmol min-1 mg-1. O2.--generating activity is completely ablated by p-chloromercuribenzoate exposure. Preparations from normal unstimulated neutrophils or stimulated neutrophils from a male patient with chronic granulomatous disease had negligible amounts of this O2.--generating enzymatic activity. The dominant chromophore in this preparation was a b-type cytochrome, the spectral and functional characteristics of which are further described herein. Pyridine nucleotide-dependent reduction of the intrinsic cytochrome b closely parallels O2.- generation in this preparation. Specifically, reduction occurs in preparations from phorbal myristate acetate-stimulated neutrophils and is absent in unstimulated or stimulated p-chloromercuribenzoate-inactivated preparations.

  16. Neutrophil Extracellular Traps Go Viral

    PubMed Central

    Schönrich, Günther; Raftery, Martin J.

    2016-01-01

    Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs. PMID:27698656

  17. Characterization of canine neutrophil granules.

    PubMed Central

    O'Donnell, R T; Andersen, B R

    1982-01-01

    The purpose of this study was to isolate distinct populations of canine neutrophil granules and to compare them with neutrophil granules from other species. Size, shape, density, and content of canine neutrophil granules were determined. Neutrophils obtained by Ficoll-Hypaque sedimentation were homogenized, and granule populations were separated by isopycnic centrifugation on a linear sucrose gradient (rho, 1.14 to 1.22 g/ml). The most dense granule population (rho, 1.197 g/ml) contained all of the myeloperoxidase, beta-glucuronidase, and elastase, more than half of the acid beta-glycerophosphatase, and most of the lysozyme. The population with intermediate density (rho, 1.179 g/ml) contained lactoferrin, vitamin B12-binding protein, and the remainder of the acid beta-glycerophosphatase and lysozyme. The least dense granule population did not contain a major peak of any of the enzymes or binding proteins tested but was distinguished by density and morphology. The size and shape of the granules were determined from scanning electron micrographs and assessment of shape was aided by transmission electron micrographs. By these methods three populations of canine neutrophil granules were characterized and named: myeloperoxidase granules, vitamin B12-binding protein granules, and low-density granules. Images PMID:6292095

  18. Nanofabrication in cellulose acetate.

    PubMed

    Zeng, Hongjun; Lajos, Robert; Metlushko, Vitali; Elzy, Ed; An, Se Young; Sautner, Joshua

    2009-03-07

    We have demonstrated nanofabrication with commercialized cellulose acetate. Cellulose acetate is used for bulk nanofabrication and surface nanofabrication. In bulk nanofabrication, cellulose acetate reacts with an e-beam and permanent patterns are formed in it instead of being transferred to other substrates. We have studied the nano relief modulation performance of cellulose acetate before and after development. The depth of the nanopatterns is magnified after development, and is varied by exposing dosage and line width of the pattern. The thinnest 65 nm wide line is achieved in the bulk fabrication. We also demonstrate a binary phase Fresnel lens array which is directly patterned in a cellulose acetate sheet. Because of its unique mechanical and optical properties, cellulose is a good candidate for a template material for soft imprinting lithography. In the surface nanofabrication, cellulose acetate thin film spin-coated on silicon wafers is employed as a new resist for e-beam lithography. We achieved 50 nm lines with 100 nm pitches, dots 50 nm in diameter, and single lines with the smallest width of 20 nm. As a new resist of e-beam lithography, cellulose acetate has high resolution comparable with conventional resists, while having several advantages such as low cost, long stock time and less harmfulness to human health.

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

    PubMed Central

    Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

    2016-01-01

    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

  20. Priming by grepafloxacin on respiratory burst of human neutrophils: its possible mechanism.

    PubMed

    Niwa, Masayuki; Kanamori, Yutaka; Hotta, Koichi; Matsuno, Hiroyuki; Kozawa, Osamu; Fujimoto, Sadaki; Uematsu, Toshihiko

    2002-10-01

    Grepafloxacin is a broad-spectrum fluoroquinolone derivative that has good tissue penetration. We demonstrated that grepafloxacin showed a priming effect on neutrophil respiratory burst, triggered by either a chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4), but not by the phorbol ester phorbol 12-myristate 13-acetate (PMA). The priming effect of grepafloxacin on fMLP-stimulated superoxide generation by human neutrophils correlated with the penetration of grepafloxacin into cells. Removal of extracellular grepafloxacin did not inhibit the priming effect on fMLP-stimulated superoxide generation. Furthermore, grepafloxacin induced the translocation of p47-phox and p67-phox to the membrane fraction of neutrophils, whereas tyrosine phosphorylation was hardly observed in neutrophils exposed to grepafloxacin. The priming effect of grepafloxacin on superoxide generation from neutrophils was not inhibited by treatment with pertussis toxin, a protein-tyrosine kinase inhibitor (ST-638) or a protein kinase C inhibitor (calphostin C), or chelation of extracellular calcium. Grepafloxacin did not change the fMLP receptor-binding properties. Taken together, these findings suggest that grepafloxacin evokes a priming effect on neutrophil superoxide generation intracellularly through the translocation of p47-phox and even p67-phox protein to the membrane fractions. GTP binding protein, protein-tyrosine phosphorylation and protein kinase C activation are not involved in the priming effect.

  1. Neutrophil adhesion and activation under flow

    PubMed Central

    Zarbock, Alexander; Ley, Klaus

    2009-01-01

    Neutrophil recruitment into inflamed tissue in response to injury or infection is tightly regulated. Reduced neutrophil recruitment can result in a reduced ability to fight invading microorganisms. During inflammation, neutrophils roll along the endothelial wall of postcapillary venules and integrate inflammatory signals. Neutrophil activation by selectins and chemokines regulates integrin adhesiveness. Binding of activated integrins to their counter-receptors on endothelial cells induces neutrophil arrest and firm adhesion. Adherent neutrophils can be further activated to undergo cytoskeletal rearrangement, crawling, transmigration, superoxide production and respiratory burst. Signaling through G-protein coupled receptors, selectin ligands, Fc receptors and outside-in signaling of integrins are all involved in neutrophil activation, but their interplay in the multistep process of recruitment are only beginning to emerge. This review provides an overview of signaling in rolling and adherent neutrophils. PMID:19037827

  2. APPLICATION OF PROTEOMICS TO NEUTROPHIL BIOLOGY

    PubMed Central

    Luerman, Gregory C.; Uriarte, Silvia M.; Rane, Madhavi J.; McLeish, Kenneth R.

    2009-01-01

    Polymorphonuclear leukocytes or neutrophils are a primary effector cell of the innate immune system and contribute to the development of adaptive immunity. Neutrophils participate in both the initiation and resolution of inflammatory responses through a series of highly coordinated molecular and phenotypic changes. To accomplish these changes, neutrophils express numerous receptors and use multiple overlapping and redundant signal transduction pathways. Dysregulation of the activation or resolution pathways plays a role in a number of human diseases. A comprehensive understanding of the regulation of neutrophil responses can be provided by high throughput proteomic technologies and sophisticated computational analysis. The first steps in the application of proteomics to understanding neutrophil biology have been taken. Here we review the application of expression, structural, and functional proteomic studies to neutrophils. Although defining the complex molecular events associated with neutrophil activation is in the early stages, the data generated to date suggest that proteomic technologies will dramatically enhance our understanding of neutrophil biology. PMID:19580889

  3. Isolation and Functional Analysis of Human Neutrophils.

    PubMed

    Kuhns, Douglas B; Long Priel, Debra A; Chu, Jessica; Zarember, Kol A

    2015-11-02

    This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described.

  4. Isolation and Functional Analysis of Human Neutrophils

    PubMed Central

    Kuhns, Douglas B.; Long Priel, Debra A.; Chu, Jessica; Zarember, Kol A.

    2015-01-01

    This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described. PMID:26528633

  5. 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.

  6. 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.

  7. Alpha(1)-acid glycoprotein is contained in bovine neutrophil granules and released after activation.

    PubMed

    Rahman, Mizanur M D; Miranda-Ribera, Alba; Lecchi, Cristina; Bronzo, Valerio; Sartorelli, Paola; Franciosi, Federica; Ceciliani, Fabrizio

    2008-09-15

    The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein alpha(1)-acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50-60kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42-45kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15min. AGP's mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.

  8. 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.

  9. Neutrophils: Cinderella of innate immune system.

    PubMed

    Kumar, V; Sharma, A

    2010-11-01

    Neutrophils are the first line of innate immune defense against infectious diseases. However, since their discovery by Elie Metchnikoff, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during acute infections. Now, 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. Along with releasing various antimicrobial molecules, neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation. 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 neutrophils in the regulation of both innate and adaptive immunity during acute infectious or inflammatory conditions.

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

    PubMed

    Sørensen, Ole E; Borregaard, Niels

    2016-05-02

    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.

  11. Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

    PubMed Central

    Carlsen, Eric D.; Hay, Christie; Henard, Calvin A.; Popov, Vsevolod; Garg, Nisha Jain

    2013-01-01

    Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis. PMID:23918780

  12. Redox state and O2*- production in neutrophils of Crohn's disease patients.

    PubMed

    Biagioni, Chiara; Favilli, Fabio; Catarzi, Serena; Marcucci, Tommaso; Fazi, Marilena; Tonelli, Francesco; Vincenzini, Maria T; Iantomasi, Teresa

    2006-02-01

    The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohn's disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2*-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-alpha)- or phorbol 12-myristate 13-acetate (PMA)-induced O2*- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and beta-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-alpha cannot be caused by excessive reactive oxygen species (ROS) production induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony-stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2*- production and adhesion, caused, in part, by an anomalous response to TNF-alpha, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the

  13. Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases

    PubMed Central

    Nikfarjam, Bahareh Abd; Adineh, Mohtaram; Hajiali, Farid

    2017-01-01

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

  14. Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells.

    PubMed

    Parker, Amber; Cuddihy, Sarah L; Son, Tae G; Vissers, Margreet C M; Winterbourn, Christine C

    2011-10-01

    Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H(2)O(2) to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H(2)O(2) was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.

  15. The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability.

    PubMed Central

    Parsons, P. E.; Sugahara, K.; Cott, G. R.; Mason, R. J.; Henson, P. M.

    1987-01-01

    The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability was examined. Although neutrophil migration was not associated with a change in epithelial permeability, prolonged neutrophil-epithelial contact following migration resulted in an increase in epithelial permeability. These results were not altered by catalase, a specific neutrophil elastase inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone or cyclohexamide. This suggests that neutrophil migration does not occur via an H2O2-induced reversible mechanism of junctional opening, which we describe herein. PMID:3314530

  16. The effects and comparative differences of neutrophil specific chemokines on neutrophil chemotaxis of the neonate.

    PubMed

    Fox, Samuel E; Lu, Wenge; Maheshwari, Akhil; Christensen, Robert D; Calhoun, Darlene A

    2005-02-07

    Neutrophil specific chemokines are potent chemoattractants for neutrophils. IL-8/CXCL8 is the most extensively studied member of this group, and its concentrations increase during inflammatory conditions of the newborn infant including sepsis and chronic lung disease. A significant amount of information exists on the effects of IL-8/CXCL8 on neutrophil chemotaxis of neonates, but little is known about the other neutrophil specific chemokines. The aim of this study was to determine the relative potency of the neutrophil specific chemokines on chemotaxis of neonatal neutrophils and to compare this effect with the effect on adult neutrophils. Neutrophils were isolated from cord blood or healthy adult donors and incubated in a Neuroprobe chemotaxis chamber. Chemokine concentrations ranging from 1-1000 ng/mL were used. Differences in chemotactic potency existed among the seven neutrophil specific chemokines. Specifically, at 100 ng/mL, the order was IL-8/CXCL8>GRO-alpha/CXCL1>GCP-2/CXCL6>NAP-2/CXCL7>ENA-78/CXCL5>GRO-gamma/CXCL2>GRO-beta/CXCL3. This pattern was observed for adult and neonatal neutrophils. We conclude that (1) neutrophils from cord blood exhibit the same pattern of potency for each ELR chemokine as neutrophils from adults, and (2) migration of neonatal neutrophils is significantly less than that of adults at every concentration examined except the lowest (1 ng/mL).

  17. Effect of tannic acid, resveratrol and its derivatives, on oxidative damage and apoptosis in human neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Ignatowicz, Ewa; Krajka-Kuźniak, Violetta; Baer-Dubowska, Wanda

    2015-10-01

    In this study we compared the antioxidant and DNA protective activity of tannic acid and stilbene derivatives, resveratrol, 3,5,4(')-trimethoxystilbene (TMS) and pterostilbene in human neutrophils stimulated to oxidative burst by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in relation to apoptosis induction. All polyphenols within the concentration range 1-100 μM reduced the intracellular ROS and H2O2 production in the TPA-stimulated cells. Tannic acid was the most effective polyphenol in protection against DNA damage induced by TPA. In the resting neutrophils resveratrol and to lesser extent other polyphenols increased DNA damage and increased the level of p53. Pretreatment of the TPA-stimulated cells with tannic acid or stilbenes led to the induction of apoptosis. The most significant effect was observed as a result of treatment with TMS and resveratrol. These compounds appeared the most effective inducers of p53 in the TPA-challenged neutrophils, what may suggest that pro-apoptotic activity of these stilbenes might be related to p53 activation. Overall, the results of our present study demonstrate that tannic acid and stilbenes modulate the ROS production, ultimately leading to cell apoptosis in human neutrophils stimulated to oxidative burst. In resting neutrophils they exhibit pro-oxidant activity, which is accompanied by p53 induction.

  18. 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.

  19. The Selective Estrogen Receptor Modulator Raloxifene Inhibits Neutrophil Extracellular Trap Formation

    PubMed Central

    Flores, Roxana; Döhrmann, Simon; Schaal, Christina; Hakkim, Abdul; Nizet, Victor; Corriden, Ross

    2016-01-01

    Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effects on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA), a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs). Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Similar to raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation, but not reactive oxygen species production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production. PMID:28003814

  20. Impaired surface expression of PAF receptors on human neutrophils is dependent upon cell activation.

    PubMed

    Zhou, W; Javors, M A; Olson, M S

    1994-02-01

    The capacity of human neutrophils to bind PAF was rapidly diminished upon cell stimulation with both physiological agonists (N-formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4)) and pharmacologic agonists (phorbol 12-myristate 13-acetate (PMA), A23187). As a consequence, PAF responses in neutrophils were blunted, as monitored by an inhibition of intracellular Ca2+ mobilization. Downregulation of the PAF receptor in neutrophils by diverse agonists was temperature-sensitive and required intact cells. Scatchard analysis of binding data revealed that PAF binding sites were lost without an appreciable change in the affinity of the ligand for the receptor. The binding of the PAF receptor antagonist WEB2086 to neutrophils decreased in parallel with PAF binding. PMA-induced PAF receptor downregulation was staurosporine-sensitive while PAF receptor downregulation by A23187, FMLP, or LTB4 was staurosporine-resistant. Both neutrophil aggregation (a form of intercellular adhesion) and PAF receptor downregulation occurred only at high concentrations of agonists while other signaling processes such as the increase in [Ca2+]i, PKC activation, and PAF synthesis were stimulated at low concentrations of agonists. Furthermore, agonist-induced PAF receptor downregulation was observed only under conditions in which the activated neutrophils were stirred (or shaken) and were allowed to aggregate. Additionally, chelation of extracellular Ca2+ with EGTA minimized cell aggregation and also inhibited PAF receptor downregulation. While the nature of the biochemical signal or the physical changes in the plasma membrane associated with aggregation or that follow aggregation remain to be elucidated it is clear that full expression of cell activation (i.e., neutrophil aggregation) is required for PAF receptor downregulation.

  1. Purification and characterization of an isoform of protein kinase C from bovine neutrophils

    SciTech Connect

    Dianoux, A.C.; Stasia, M.J.; Vignais, P.V. )

    1989-01-24

    Protein kinase C (PKC) from bovine neutrophils was purified 1,420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bovine neutrophil PKC was autophosphorylated in the presence of ({gamma}-{sup 32}P)ATP, provided that the medium was supplemented with Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of M{sub r} 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of M{sub r} 23,000 was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of ({gamma}-{sup 32}P)ATP, Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification. It is suggested that the M{sub r} 23,000 protein might be a natural substrate for bovine neutrophil PKC.

  2. Alarmins Link Neutrophils and Dendritic Cells

    PubMed Central

    Yang, De; de la Rosa, Gonzalo; Tewary, Poonam; Oppenheim, Joost J.

    2009-01-01

    Neutrophils are the first major population of leukocyte to infiltrate infected or injured tissues and are crucial for initiating host innate defense and adaptive immunity. Although the contribution of neutrophils to innate immune defense is mediated predominantly by phagocytosis and killing of microorganisms, neutrophils also participate in the induction of adaptive immune responses. At sites of infection and/or injury, neutrophils release numerous mediators upon degranulation or death, among these are alarmins which have a characteristic dual capacity to mobilize and activate antigen-presenting cells. We describe here how alarmins released by neutrophil degranulation and/or death can link neutrophils to dendritic cells by promoting their recruitment and activation, resulting in the augmentation of innate and adaptive immune responses. PMID:19699678

  3. The Role of Neutrophils in Transplanted Organs.

    PubMed

    Scozzi, D; Ibrahim, M; Menna, C; Krupnick, A S; Kreisel, D; Gelman, A E

    2017-02-01

    Neutrophils are often viewed as nonspecialized effector cells whose presence is a simple indicator of tissue inflammation. There is new evidence that neutrophils exist in subsets and have specialized effector functions that include extracellular trap generation and the stimulation of angiogenesis. The application of intravital imaging to transplanted organs has revealed novel requirements for neutrophil trafficking into graft tissue and has illuminated direct interactions between neutrophils and other leukocytes that promote alloimmunity. Paradoxically, retaining some neutrophilia may be important to induce or maintain tolerance. Neutrophils can stimulate anti-inflammatory signals in other phagocytes and release molecules that inhibit T cell activation. In this article, we will review the available evidence of how neutrophils regulate acute and chronic inflammation in transplanted organs and discuss the possibility of targeting these cells to promote tolerance.

  4. The Role of Neutrophils in Transplanted Organs

    PubMed Central

    Menna, Cecilia; Krupnick, Alexander S.; Kreisel, Daniel; Gelman, Andrew E.

    2016-01-01

    Neutrophils are often viewed as non-specialized effector cells whose presence is a simple indicator of tissue inflammation. There is new evidence that neutrophils exist in subsets and have specialized effector functions that include extracellular trap generation and the stimulation of angiogenesis. The application of intravital imaging to transplanted organs has revealed novel requirements for neutrophil trafficking into graft tissue and illuminated direct interactions between neutrophils and other leukocytes that promote alloimmunity. Paradoxically, retaining some neutrophilia may be important to induce or maintain tolerance. Neutrophils can stimulate anti-inflammatory signals in other phagocytes and release molecules that inhibit T cell activation. Here we will review the available evidence of how neutrophils regulate acute and chronic inflammation in transplanted organs and discuss the possibility of targeting these cells to promote tolerance. PMID:27344051

  5. Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence

    PubMed Central

    Niemiec, Maria J.; Laforce, Brecht; Garrevoet, Jan; Vergucht, Eva; De Rycke, Riet; Cloetens, Peter; Urban, Constantin F.; Vincze, Laszlo

    2016-01-01

    High pressure frozen (HPF), cryo-substituted microtome sections of 2 μm thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1–2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis. PMID:27812122

  6. Two neutrophilic dermatoses captured simultaneously on histology

    PubMed Central

    Wlodek, Christina; Bhatt, Nidhi; Kennedy, Cameron

    2016-01-01

    A number of neutrophilic dermatoses are associated with malignancies and their treatment. These rarely occur together in the same patient. A Caucasian 72-year-old male was treated for acute myeloid leukemia (AML) with chemotherapy including daunorubicin and cytarabine. Within 48 hours of commencing treatment, he developed pyrexia and, two days later, disseminated non-tender pink plaques on the limbs and trunk. A skin biopsy showed a dermal interstitial infiltrate of lymphocytes, histiocytoid cells and predominantly neutrophils. This extended into the subcutis, where a neutrophilic lobular panniculitis was seen. These findings are consistent with Sweet’s syndrome. In addition, a neutrophilic and lymphocytic infiltrate was also present around eccrine coils and lower ducts. The eccrine epithelium showed squamous metaplasia with dyskeratosis and sloughing into the lumen. These latter findings are consistent with neutrophilic eccrine hidradenitis (NEH). These two histologically distinct entities form part of the neutrophilic dermatoses that have been described in oncology patients with reports of concurrent or sequential occurrence of various neutrophilic dermatoses in the same patient. Ours, however, is only the second reported case of simultaneously captured Sweet’s and NEH in the setting of AML. The most likely explanation is that of an epiphenomenon, whereby the neutrophilic infiltrate extended around the sweat glands in the context of the neutrophilic dermatosis. PMID:27648385

  7. Plasticity of neutrophils reveals modulatory capacity

    PubMed Central

    Perobelli, S.M.; Galvani, R.G.; Gonçalves-Silva, T.; Xavier, C.R.; Nóbrega, A.; Bonomo, A.

    2015-01-01

    Neutrophils are widely known as proinflammatory cells associated with tissue damage and for their early arrival at sites of infection, where they exert their phagocytic activity, release their granule contents, and subsequently die. However, this view has been challenged by emerging evidence that neutrophils have other activities and are not so short-lived. Following activation, neutrophil effector functions include production and release of granule contents, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs). Neutrophils have also been shown to produce a wide range of cytokines that have pro- or anti-inflammatory activity, adding a modulatory role for this cell, previously known as a suicide effector. The presence of cytokines almost always implies intercellular modulation, potentially unmasking interactions of neutrophils with other immune cells. In fact, neutrophils have been found to help B cells and to modulate dendritic cell (DC), macrophage, and T-cell activities. In this review, we describe some ways in which neutrophils influence the inflammatory environment in infection, cancer, and autoimmunity, regulating both innate and adaptive immune responses. These cells can switch phenotypes and exert functions beyond cytotoxicity against invading pathogens, extending the view of neutrophils beyond suicide effectors to include functions as regulatory and suppressor cells. PMID:26108096

  8. CFTR targeting during activation of human neutrophils.

    PubMed

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

    2016-12-01

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

  9. 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

  10. 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...

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

    PubMed Central

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

    1986-01-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing. Images PMID:3771800

  12. 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.

  13. Acetate Kinase Isozymes Confer Robustness in Acetate Metabolism

    PubMed Central

    Chan, Siu Hung Joshua; Nørregaard, Lasse; Solem, Christian; Jensen, Peter Ruhdal

    2014-01-01

    Acetate kinase (ACK) (EC no: 2.7.2.1) interconverts acetyl-phosphate and acetate to either catabolize or synthesize acetyl-CoA dependent on the metabolic requirement. Among all ACK entries available in UniProt, we found that around 45% are multiple ACKs in some organisms including more than 300 species but surprisingly, little work has been done to clarify whether this has any significance. In an attempt to gain further insight we have studied the two ACKs (AckA1, AckA2) encoded by two neighboring genes conserved in Lactococcus lactis (L. lactis) by analyzing protein sequences, characterizing transcription structure, determining enzyme characteristics and effect on growth physiology. The results show that the two ACKs are most likely individually transcribed. AckA1 has a much higher turnover number and AckA2 has a much higher affinity for acetate in vitro. Consistently, growth experiments of mutant strains reveal that AckA1 has a higher capacity for acetate production which allows faster growth in an environment with high acetate concentration. Meanwhile, AckA2 is important for fast acetate-dependent growth at low concentration of acetate. The results demonstrate that the two ACKs have complementary physiological roles in L. lactis to maintain a robust acetate metabolism for fast growth at different extracellular acetate concentrations. The existence of ACK isozymes may reflect a common evolutionary strategy in bacteria in an environment with varying concentrations of acetate. PMID:24638105

  14. 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

  15. Neutrophil uptake of vaccinia virus in vitro

    SciTech Connect

    West, B.C.; Eschete, M.L.; Cox, M.E.; King, J.W.

    1987-10-01

    We studied human neutrophils for uptake of vaccinia virus. Uptake was determined radiometrically and by electron microscopy. Vaccinia virus was labeled with /sup 14/C or /sup 3/H, incubated with neutrophils, and quantified in neutrophil pellets in a new radiometric phagocytosis assay. Better results were obtained from assays of (/sup 3/H)thymidine-labeled virus; uptake increased through 1 hr and then plateaued. Phagocytosis of 3H-labeled Staphylococcus aureus was normal. Uptake of virus was serum dependent. Hexose monophosphate shunt activity was measured by two methods. No /sup 14/CO/sub 2/ from (/sup 14/C)1-glucose accompanied uptake of vaccinia virus, in contrast to the respiratory burst accompanying bacterial phagocytosis. Electron microscopy showed intact to slightly digested intraphagolysosomal vaccinia virus. Pock reduction assay showed a decrease in viral content due to neutrophils until 6 hr of incubation, when a modest but significant increase was observed. Thus, neutrophil uptake of vaccinia virus is distinguished from bacterial phagocytosis.

  16. Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    PubMed Central

    Behnen, Martina; Möller, Sonja; Brozek, Antonia; Klinger, Matthias; Laskay, Tamás

    2017-01-01

    The inflammatory microenvironment is commonly characterized by extracellular acidosis (pH < 7.35). Sensitivity to pH, CO2 or bicarbonate concentrations allows neutrophils to react to changes in their environment and to detect inflamed areas in the tissue. One important antimicrobial effector mechanism is the production of neutrophil extracellular traps (NETs), which are released during a programmed reactive oxygen species (ROS)-dependent cell death, the so-called NETosis. Although several functions of neutrophils have been analyzed under acidic conditions, the effect of extracellular acidosis on NETosis remains mainly unexplored and the available experimental results are contradictory. We performed a comprehensive study with the aim to elucidate the effect of extracellular acidosis on ROS-dependent NETosis of primary human neutrophils and to identify the underlying mechanisms. The study was performed in parallel in a CO2–bicabonate-buffered culture medium, which mimics in vivo conditions, and under HEPES-buffered conditions to verify the effect of pH independent of CO2 or bicarbonate. We could clearly show that extracellular acidosis (pH 6.5, 6.0, and 5.5) and intracellular acidification inhibit the release of ROS-dependent NETs upon stimulation of neutrophils with phorbol myristate acetate and immobilized immune complexes. Moreover, our findings suggest that the diminished NET release is a consequence of reduced ROS production and diminished glycolysis of neutrophils under acidic conditions. It was suggested previously that neutrophils can sense the border of inflamed tissue by the pH gradient and that a drop in pH serves as an indicator for the progress of inflammation. Following this hypothesis, our data indicate that an acidic inflammatory environment results in inhibition of extracellular operating effector mechanisms of neutrophils such as release of ROS and NETs. This way the release of toxic components and tissue damage can be avoided. However, we

  17. Human neutrophils in auto-immunity.

    PubMed

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

    2016-04-01

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

  18. Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

    PubMed

    Healy, Laura D; Puy, Cristina; Fernández, José A; Mitrugno, Annachiara; Keshari, Ravi S; Taku, Nyiawung A; Chu, Tiffany T; Xu, Xiao; Gruber, András; Lupu, Florea; Griffin, John H; McCarty, Owen J T

    2017-04-13

    Activated protein C (APC) is a multi-functional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a non-human primate model of E. coli-induced sepsis, pre-treatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

  19. 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.

  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.

  1. 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

  2. Kallolide A acetate pyrazoline.

    PubMed

    Rodríguez-Escudero, Idaliz; Marrero, Jeffrey; Rodríguez, Abimael D

    2012-01-01

    IN THE CRYSTAL STRUCTURE OF KALLOLIDE A ACETATE PYRAZOLINE [SYSTEMATIC NAME: 7-methyl-16-oxo-4,10-bis-(prop-1-en-2-yl)-17,18-dioxa-14,15-diaza-tetra-cyclo-[9.4.2.1(6,9).0(1,12)]octa-deca-6,8,14-trien-5-yl acetate], C(23)H(28)N(2)O(5), there is a 12-member-ed carbon macrocyclic structure. In addition, there is a tris-ubstituted furan ring, an approximately planar γ-lactone ring [maximum deviation of 0.057 (3) Å] and a pyraz-oline ring, the latter in an envelope conformation. The pyrazoline and the γ-lactone rings are fused in a cis configuration. In the crystal, mol-ecules are linked by weak C-H⋯O inter-actions, forming a two-dimensional network parallel to (001). An intra-molecular C-H⋯O hydrogen bond is also present.

  3. Kallolide A acetate pyrazoline

    PubMed Central

    Rodríguez-Escudero, Idaliz; Marrero, Jeffrey; Rodríguez, Abimael D.

    2012-01-01

    In the crystal structure of kallolide A acetate pyrazoline [systematic name: 7-methyl-16-oxo-4,10-bis­(prop-1-en-2-yl)-17,18-dioxa-14,15-diaza­tetra­cyclo­[9.4.2.16,9.01,12]octa­deca-6,8,14-trien-5-yl acetate], C23H28N2O5, there is a 12-member­ed carbon macrocyclic structure. In addition, there is a tris­ubstituted furan ring, an approximately planar γ-lactone ring [maximum deviation of 0.057 (3) Å] and a pyraz­oline ring, the latter in an envelope conformation. The pyrazoline and the γ-lactone rings are fused in a cis configuration. In the crystal, mol­ecules are linked by weak C—H⋯O inter­actions, forming a two-dimensional network parallel to (001). An intra­molecular C—H⋯O hydrogen bond is also present. PMID:22259545

  4. Neutrophil Extracellular Traps and Microcrystals

    PubMed Central

    2017-01-01

    Neutrophil extracellular traps represent a fascinating mechanism by which PMNs entrap extracellular microbes. The primary purpose of this innate immune mechanism is thought to localize the infection at an early stage. Interestingly, the ability of different microcrystals to induce NET formation has been recently described. Microcrystals are insoluble crystals with a size of 1–100 micrometers that have different composition and shape. Microcrystals have it in common that they irritate phagocytes including PMNs and typically trigger an inflammatory response. This review is the first to summarize observations with regard to PMN activation and NET release induced by microcrystals. Gout-causing monosodium urate crystals, pseudogout-causing calcium pyrophosphate dehydrate crystals, cholesterol crystals associated with atherosclerosis, silicosis-causing silica crystals, and adjuvant alum crystals are discussed. PMID:28373994

  5. Emperipolesis of neutrophils by dysmorphic megakaryocytes.

    PubMed

    Parmley, R T; Kim, T H; Austin, R L; Alvarado, C S; Ragab, A H

    1982-12-01

    Neutrophil engulfment by megakaryocytes was observed within 20 to 30% of megakaryocytes from two children: one with metastatic rhabdomyosarcoma, the other with fever of unknown origin. Other cell types and neutrophil precursors were not observed within megakaryocytes. Only late megakaryocytes were involved in the process, and often these cells appeared vacuolated or degenerating at the light and electron microscope level. Ultrastructurally the engulfed neutrophils were intact and were within the open canalicular system of the megakaryocyte cytoplasm. No evidence of neutrophil granule exocytosis could be demonstrated in ultrastructural morphologic and peroxidase preparations; however, many neutrophils appeared to be endocytosing portions of the megakaryocyte cytoplasm. The phenomenon could not be transferred to normal marrow incubated with patient serum or plasma. Thus, our patients differ from previous observations of emperipolesis in: 1) the extreme frequency of the observation; 2) the selective involvement of neutrophils; and 3) the association of the anomaly with dysmorphic and/or disrupted megakaryocytes. These observations are consistent with a neutrophil response to altered and/or injured megakaryocytes.

  6. Characterization of arginase expression by equine neutrophils.

    PubMed

    Lavoie-Lamoureux, Anouk; Martin, James G; Lavoie, Jean-Pierre

    2014-02-15

    Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood neutrophils using quantitative PCR. Arginase expression was also studied by Western blot and enzyme activity assay. The effect of nor-NOHA (1mM), a specific arginase inhibitor, was assessed on arginase activity in vitro and ex vivo on neutrophil's inflammatory gene expression and viability. Results showed that equine neutrophils constitutively express arginase isoform 2, ODC and OAT. Neutrophil ex vivo stimulation did not induce arginase I or influence arginase II mRNA expression. Ex vivo inhibition of arginase activity by nor-NOHA had no effect on neutrophils inflammatory gene expression induced by LPS/fMLP (5h) but significantly reversed the cell loss observed after this stimulation.

  7. Neutrophil Extracellular Traps of Cynoglossus semilaevis: Production Characteristics and Antibacterial Effect

    PubMed Central

    Zhao, Ming-li; Chi, Heng; Sun, Li

    2017-01-01

    Neutrophil extracellular traps (NETs) are structures released by neutrophils as a cellular immune defense against microbial invasion. The process of NETs generation, netosis (NETosis), can take place via either a suicidal mechanism, during which the NETs-releasing cells became dead, or a “live” mechanism, during which the NETs-releasing cells remain vital. NETosis has been studied intensively in mammals in recent years, but very little is known about the NETosis in fish. In this study, we examined NETosis in tongue sole (Cynoglossus semilaevis), a species of teleost with important economic values. We found that following stimulation with phorbol 12-myristate 13-acetate (PMA) and three common fish bacterial pathogens, abundant NETs structures were released by neutrophils that were most likely in a live state. The released NETs captured, but did not kill, the bacterial pathogens; however, the replication of extracellular, but not intracellular, pathogens was inhibited by NETs to significant extents. Reactive oxygen species (ROS), nitric oxide (NO), and myeloperoxidase (MPO) production were observed to be enhanced in NETosing neutrophils, and blocking the production of these factors by inhibitors significantly decreased NETs production induced by PMA and all three bacteria. Taken together, these results indicate for the first time that in teleost there exists a non-cell death pathway of NETosis that produces NETs with antibacterial effects in a ROS-, NO-, and MPO-dependent manner. PMID:28382034

  8. 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.

  9. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid differentially modulate rat neutrophil function in vitro.

    PubMed

    Paschoal, V A; Vinolo, M A R; Crisma, A R; Magdalon, J; Curi, R

    2013-02-01

    Fish oils are used as therapeutic agents in chronic inflammatory diseases. The omega-3 fatty acids (FA) found in these oils are mainly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The anti-inflammatory properties of fish oils are attributed to both omega-3 fatty acids. However, it is unknown whether such effects are due to either EPA or DHA. In this study, the effects of EPA and DHA on rat neutrophil function in vitro were compared. Both EPA and DHA increased the production of H₂O₂ when cells were stimulated or not with lipopolysaccharides (LPS). However, EPA was more potent than DHA in triggering an increase in superoxide release by cells in the basal condition or when stimulated with phorbol myristate acetate (PMA) or zymosan. Only DHA increased the phagocytic capacity and fungicidal activity of neutrophils. Both FA increased the release of tumor necrosis factor-α (TNF-α) in nonstimulated cells, but only EPA increased the production of cytokine-inducing neutrophil chemoattractant-2 (CINC-2) in the absence or presence of LPS, whereas production of interleukin-1 beta (IL-1β) was only increased by DHA in the presence of LPS. In addition, there was no alteration in the production of nitric oxide. In conclusion, we show herein that EPA and DHA can differently modulate aspects of the neutrophil response, which may be relevant for the development of therapies rich in one or other FA depending on the effect required.

  10. The subcellular particulate NADPH-dependent O2.(-)-generating oxidase from human blood monocytes: comparison to the neutrophil system.

    PubMed

    Chaudhry, A N; Santinga, J T; Gabig, T G

    1982-10-01

    Highly purified preparations of normal human monocytes obtained from peripheral blood were shown to contain a subcellular particulate O2.(-)-generating oxidase system. This O2.(-)-generating activity was present in particulate preparations from monocytes that had been previously stimulated with phorbol myristate acetate but was low or absent in control preparations from unstimulated monocytes or stimulated monocytes from a patient with chronic granulomatous disease. In the stimulated preparations from normal monocytes, O2.(-)-generation was linearly proportional to cell protein concentration, insensitive to inhibition by azide, and dependent on NADPH as substrate. These characteristics are similar to the O2.(-)-generating oxidase system from human neutrophils. A significant difference in the apparent Km for NADPH was shown between preparations from stimulated monocytes and neutrophils (monocyte 83 +/- 16 microM, neutrophil 31 +/- 5 microM, mean +/- SE). Additionally, affinity of the stimulated monocyte particulate preparation for NADH was unmeasurably low.

  11. 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

  12. Platelet–neutrophil interactions under thromboinflammatory conditions

    PubMed Central

    Li, Jing; Kim, Kyungho; Barazia, Andrew; Tseng, Alan

    2015-01-01

    Platelets primarily mediate hemostasis and thrombosis, whereas leukocytes are responsible for immune responses. Since platelets interact with leukocytes at the site of vascular injury, thrombosis and vascular inflammation are closely intertwined and occur consecutively. Recent studies using real-time imaging technology demonstrated that platelet–neutrophil interactions on the activated endothelium are an important determinant of microvascular occlusion during thromboinflammatory disease in which inflammation is coupled to thrombosis. Although the major receptors and counter receptors have been identified, it remains poorly understood how heterotypic platelet–neutrophil interactions are regulated under disease conditions. This review discusses our current understanding of the regulatory mechanisms of platelet– neutrophil interactions in thromboinflammatory disease. PMID:25650236

  13. 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.

  14. Neutrophil extracellular traps in cancer progression.

    PubMed

    Cools-Lartigue, Jonathan; Spicer, Jonathan; Najmeh, Sara; Ferri, Lorenzo

    2014-11-01

    Neutrophils are being increasingly recognized as an important element in tumor progression. They have been shown to exert important effects at nearly every stage of tumor progression with a number of studies demonstrating that their presence is critical to tumor development. Novel aspects of neutrophil biology have recently been elucidated and its contribution to tumorigenesis is only beginning to be appreciated. Neutrophil extracellular traps (NETs) are neutrophil-derived structures composed of DNA decorated with antimicrobial peptides. They have been shown to trap and kill microorganisms, playing a critical role in host defense. However, their contribution to tumor development and metastasis has recently been demonstrated in a number of studies highlighting NETs as a potentially important therapeutic target. Here, studies implicating NETs as facilitators of tumor progression and metastasis are reviewed. In addition, potential mechanisms by which NETs may exert these effects are explored. Finally, the ability to target NETs therapeutically in human neoplastic disease is highlighted.

  15. Neutrophil function in pregnancy and rheumatoid arthritis

    PubMed Central

    Crocker, I; Baker, P; Fletcher, J

    2000-01-01

    BACKGROUND—Pregnancy exerts suppressive effects on rheumatoid arthritis (RA). An attenuation in neutrophil function in late pregnancy which may explain this amelioration has previously been reported.
OBJECTIVE—A longitudinal investigation of neutrophil activity in healthy pregnant women (n=9) and pregnant patients with RA (n=9), compared with age matched non-pregnant patients with RA (n=12) and healthy controls (n=22).
METHODS—Neutrophil activation was measured in response to the physiological receptor agonists, n-formyl-methionyl-leucyl-phenylalanine (fMLP) and zymosan activated serum (ZAS). Superoxide anion production (respiratory burst) was determined by lucigenin enhanced chemiluminescence (LUCL); secondary granule lactoferrin release by enzyme linked immunosorbent assay (ELISA); and CD11b, CD18, and CD62L expression by flow cytometric analysis.
RESULTS—Stimulated neutrophil LUCL was significantly reduced in both pregnant women with RA and healthy pregnant women in the second (fMLP 43% and 69%, ZAS 43% and 59%, respectively) and third trimesters (fMLP 24% and 44%, ZAS 32% and 38%, respectively). Responses returned to normal within eight weeks of delivery and unstimulated levels remained unchanged throughout pregnancy. Basal and stimulated CD11b, CD18, and CD62L expression showed no variations throughout gestation for both pregnancy groups. Likewise, stimulated lactoferrin release and plasma lactoferrin remained unchanged. Certain morphological differences in RA neutrophils were highlighted by the flow cytometric analysis. Moreover, resting neutrophils and stimulated cells from patients with RA, including pregnant subjects, showed a marked increase in LUCL, but a reduction in CD11b, CD18, and CD62L. Low dose prednisolone and methylprednisolone had no effect on neutrophil parameters over the period of treatment with non-steroidal anti-inflammatory drugs.
CONCLUSION—The attenuation to neutrophil respiratory burst in both healthy and RA

  16. Photothermal image cytometry of human neutrophils

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitry

    2001-07-01

    Photothermal imaging, when being applied to the study of living cells, provides morpho-functional information about the cell populations. In technical terms, the method is complementary to optical microscopy. The photothermal method was used for cell imaging and quantitative studies. Preliminary results of the studies on living human neutrophils are presented. Differences between normal and pathological neutrophil populations from blood of healthy donors and patients with saracoidosis and pleuritis are demonstrated.

  17. Stability analysis of micropipette aspiration of neutrophils.

    PubMed Central

    Derganc, J; Bozic, B; Svetina, S; Zeks, B

    2000-01-01

    During micropipette aspiration, neutrophil leukocytes exhibit a liquid-drop behavior, i.e., if a neutrophil is aspirated by a pressure larger than a certain threshold pressure, it flows continuously into the pipette. The point of the largest aspiration pressure at which the neutrophil can still be held in a stable equilibrium is called the critical point of aspiration. Here, we present a theoretical analysis of the equilibrium behavior and stability of a neutrophil during micropipette aspiration with the aim to rigorously characterize the critical point. We take the energy minimization approach, in which the critical point is well defined as the point of the stability breakdown. We use the basic liquid-drop model of neutrophil rheology extended by considering also the neutrophil elastic area expansivity. Our analysis predicts that the behavior at large pipette radii or small elastic area expansivity is close to the one predicted by the basic liquid-drop model, where the critical point is attained slightly before the projection length reaches the pipette radius. The effect of elastic area expansivity is qualitatively different at smaller pipette radii, where our analysis predicts that the critical point is attained at the projection lengths that may significantly exceed the pipette radius. PMID:10866944

  18. In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Dobrowolska-Zachwieja, Agnieszka; Grajek, Włodzimierz

    2009-01-01

    Oxidative stress and inflammation are involved in the development of obesity. Beetroot (Beta vulgaris var. rubra) is a food ingredient containing betalain pigments that show antioxidant activity. The in vitro effect of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals has been investigated. Fifteen obese women (aged 45 +/- 9 years, BMI >30 kg/m2) and nine healthy controls (women, aged 29 +/- 11 years, BMI = 22.2 +/- 1.6 kg/m2) were examined. The investigated products were used as concentrates and after transport and digestion in an artificial gastrointestinal tract. Neutrophil oxidant production, in response to phorbol 12-myristate 13-acetate, was characterized by luminol-dependent chemiluminescence and a flow cytometric dichlorofluorescin oxidation assay. Caspase-3 activity, a marker of apoptosis, was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Neutrophils from obese individuals had a significantly higher ROS production compared with the controls (p < 0.05). Beetroot products inhibited neutrophil oxidative metabolism in a concentration-dependent manner. Also observed were the pro-apoptotic effects of beetroot at a concentration range of 0.1-10% in 24 h culture of stimulated neutrophils. These natural products (in both the liquid and solid state) have antioxidant and antiinflammatory capacity, and could be an important adjunct in the treatment of obesity.

  19. Effect of acute and chronic excesses of dietary nitrogen on blood neutrophil functions in cattle.

    PubMed

    Raboisson, D; Caubet, C; Tasca, C; De Marchi, L; Ferraton, J M; Gannac, S; Millet, A; Enjalbert, F; Schelcher, F; Foucras, G

    2014-12-01

    phorbol 12-myristate 13-acetate was not modified, in contrast to OZ stimulation. Decreased ROS production during chronic EDN probably involves the early events leading to ROS production, as OZ acts through membrane receptors and phorbol 12-myristate 13-acetate directly activates protein kinase C. This is the first study to provide evidence that the modifications of neutrophil functions produced by excess nitrogen depend on the intensity and duration of the excess. Further studies, including epidemiological studies during risk periods, are needed to resolve the issues linked to EDN.

  20. 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.

  1. The hederagenin saponin SMG-1 is a natural FMLP receptor inhibitor that suppresses human neutrophil activation.

    PubMed

    Hwang, Tsong-Long; Wang, Chien-Chiao; Kuo, Yao-Haur; Huang, Hui-Chi; Wu, Yang-Chang; Kuo, Liang-Mou; Wu, Yi-Hsiu

    2010-10-15

    The pericarp of Sapindus mukorossi Gaertn is traditionally used as an expectorant in Japan, China, and Taiwan. Activated neutrophils produce high concentrations of the superoxide anion (O(2)(-)) and elastase known to be involved in airway mucus hypersecretion. In the present study, the anti-inflammatory functions of hederagenin 3-O-(3,4-O-di-acetyl-alpha-L-arabinopyranoside)-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-arabinopyranoside (SMG-1), a saponin isolated from S. mukorossi, and its underlying mechanisms were investigated in human neutrophils. SMG-1 potently and concentration-dependently inhibited O(2)(*-) generation and elastase release in N-Formyl-Met-Leu-Phe (FMLP)-activated human neutrophils. Furthermore, SMG-1 reduced membrane-associated p47(phox) expression in FMLP-induced intact neutrophils, but did not alter subcellular NADPH oxidase activity in reconstituted systems. SMG-1 attenuated FMLP-induced increase of cytosolic calcium concentration and phosphorylation of p38 MAPK, ERK, JNK, and AKT. However, SMG-1 displayed no effect on cellular cAMP levels and activity of adenylate cyclase and phosphodiesterase. Significantly, receptor-binding analysis showed that SMG-1 inhibited FMLP binding to its receptor in a concentration-dependent manner. In contrast, neither phorbol myristate acetate-induced O(2)(*-) generation and MAPKs activation nor thapsigargin-caused calcium mobilization was altered by SMG-1. Taken together, our results demonstrate that SMG-1 is a natural inhibitor of the FMLP receptor, which may have the potential to be developed into a useful new therapeutic agent for treating neutrophilic inflammatory diseases.

  2. 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.

  3. Recombinant gamma interferon causes neutrophil migration mediated by the release of a macrophage neutrophil chemotactic factor.

    PubMed Central

    Ribeiro, R. A.; Cunha, F. Q.; Ferreira, S. H.

    1990-01-01

    A dose-dependent neutrophil migration was observed following the injection of purified (Hu IFN-gamma) or recombinant (rIFN-gamma) human gamma interferon into rat peritoneal cavities. This finding contrasts with their inability to cause chemotaxis in vitro in the Boyden chamber. Neutrophil migration into peritoneal cavities and subcutaneous air pouches induced by both preparations of interferon was abolished by pretreatment of the animals with dexamethasone. IFN-gamma-induced neutrophil migration was enhanced when the macrophage population of the peritoneal cavities was increased by previous injection of thioglycollate and reduced by peritoneal lavage. Macrophage monolayers pretreated either with rIFN-gamma or with lipopolysaccharide from E. coli release into the supernatant a factor that stimulates neutrophil recruitment in animals treated with dexamethasone. Dexamethasone blocked this release but did not affect the neutrophil recruitment induced by this factor. These results suggest that IFN-gamma-induced neutrophil migration in vivo may be mediated by the release from resident macrophages of a neutrophil chemotactic factor and that dexamethasone blockade of neutrophil recruitment by IFN-gamma is due to inhibition of the release of this factor. PMID:2119790

  4. Neutrophils in Cancer: Two Sides of the Same Coin.

    PubMed

    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.

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

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

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

  6. 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

  7. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... 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...

  8. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... 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...

  9. 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...

  10. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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 the specifications of the...

  11. Pallidol hexa­acetate ethyl acetate monosolvate

    PubMed Central

    Mao, Qinyong; Taylor, Dennis K.; Ng, Seik Weng; Tiekink, Edward R. T.

    2013-01-01

    The entire mol­ecule of pallidol hexa­acetate {systematic name: (±)-(4bR,5R,9bR,10R)-5,10-bis­[4-(acet­yloxy)phen­yl]-4b,5,9b,10-tetra­hydro­indeno­[2,1-a]indene-1,3,6,8-tetrayl tetra­acetate} is completed by the application of twofold rotational symmetry in the title ethyl acetate solvate, C40H34O12·C4H8O2. The ethyl acetate mol­ecule was highly disordered and was treated with the SQUEEZE routine [Spek (2009 ▶). Acta Cryst. D65, 148–155]; the crystallographic data take into account the presence of the solvent. In pallidol hexa­acetate, the dihedral angle between the fused five-membered rings (r.m.s. deviation = 0.100 Å) is 54.73 (6)°, indicating a significant fold in the mol­ecule. Significant twists between residues are also evident as seen in the dihedral angle of 80.70 (5)° between the five-membered ring and the pendent benzene ring to which it is attached. Similarly, the acetate residues are twisted with respect to the benzene ring to which they are attached [C—O(carb­oxy)—C—C torsion angles = −70.24 (14), −114.43 (10) and −72.54 (13)°]. In the crystal, a three-dimensional architecture is sustained by C—H⋯O inter­actions which encompass channels in which the disordered ethyl acetate mol­ecules reside. PMID:24046702

  12. IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils.

    PubMed Central

    Bober, L A; Waters, T A; Pugliese-Sivo, C C; Sullivan, L M; Narula, S K; Grace, M J

    1995-01-01

    IL-4 is a T-helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL-4 on neutrophilic maturation using the cell line HL-60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL-60 cells with recombinant hu IL-4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL-60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL-4 increased surface expression of the neutrophil-lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL-4 treated HL-60 cells demonstrated enhanced Fc- and complement-mediated phagocytic capacity and increased hexose-monophosphate shunt activity. In addition, IL-4 was capable of sustaining the neutrophil maturation of HL-60 cells that had been pre-treated for 24 h with DMSO. To investigate the effect of IL-4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL-4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti-human IL-4 abolished phagocytic stimulation. Finally, IL-4 treatment also stimulated resting neutrophils to migrate toward zymosan-activated serum (ZAS) and human IL-5. The results demonstrate that IL-4 is a potent maturation factor for myelocytes to become neutrophils and that IL-4 can stimulate resting mature neutrophils. PMID:7529148

  13. The impact of trauma on neutrophil function.

    PubMed

    Hazeldine, Jon; Hampson, Peter; Lord, Janet M

    2014-12-01

    A well described consequence of traumatic injury is immune dysregulation, where an initial increase in immune activity is followed by a period of immune depression, the latter leaving hospitalised trauma patients at an increased risk of nosocomial infections. Here, we discuss the emerging role of the neutrophil, the most abundant leucocyte in human circulation and the first line of defence against microbial challenge, in the initiation and propagation of the inflammatory response to trauma. We review the findings of the most recent studies to have investigated the impact of trauma on neutrophil function and discuss how alterations in neutrophil biology are being investigated as potential biomarkers by which to predict the outcome of hospitalised trauma patients. Furthermore, with trauma-induced changes in neutrophil biology linked to the development of such post-traumatic complications as multiple organ failure and acute respiratory distress syndrome, we highlight an area of research within the field of trauma immunology that is gaining considerable interest: the manipulation of neutrophil function as a means by which to potentially improve patient outcome.

  14. 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.

  15. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence.

    PubMed

    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 ((1)O2(*)) 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.

  16. 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

  17. Changes in neutrophil functions in astronauts.

    PubMed

    Kaur, Indreshpal; Simons, Elizabeth R; Castro, Victoria A; Mark Ott, C; Pierson, Duane L

    2004-09-01

    Exploration class human spaceflight missions will require astronauts with robust immune systems. Innate immunity will be an essential element for the healthcare maintenance of astronauts during these lengthy expeditions. This study investigated neutrophil phagocytosis, oxidative burst, and degranulation of 25 astronauts after four space shuttle missions and in nine healthy control subjects. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch, immediately after landing, and 3 days after landing. The number of neutrophils increased by 85% at landing compared to preflight levels. The mean values for phagocytosis of Escherichia coli and oxidative burst capacity in neutrophils from astronauts on the 5-day mission were not significantly different from those observed in neutrophils from the control subjects. Before and after 9- to 11-day missions, however, phagocytosis and oxidative burst capacities were significantly lower than control mean values. No consistent changes in degranulation or expression of surface markers were observed before or after any of the space missions. This study indicates that neutrophil phagocytic and oxidative functions are affected by factors associated with space flight and this relationship may depend on mission duration.

  18. Effect of Cellulose Acetate Beads on Interleukin-23 Release.

    PubMed

    Nishise, Shoichi; Abe, Yasuhiko; Nomura, Eiki; Sato, Takeshi; Sasaki, Yu; Iwano, Daisuke; Yoshizawa, Kazuya; Yagi, Makoto; Sakuta, Kazuhiro; Ueno, Yoshiyuki

    2016-08-01

    Interleukin (IL)-23, which is released by activated monocytes and neutrophils, promotes production of high levels of IL-17 by T-helper 17 cells. Cellulose acetate (CA) beads are used as carriers for granulocyte and monocyte (GM) adsorptive apheresis using Adacolumn. Contact between blood and CA beads induces cytokine release; however, their inflammatory effects on IL-23 release are unclear. We aimed to clarify the effect of CA beads on IL-23 release in vitro. We incubated peripheral blood with and without CA beads and measured IL-23. Compared to blood samples incubated without CA beads, blood samples incubated with CA beads had significantly decreased amounts of IL-23. In conclusion, CA beads inhibited IL-23 release from adsorbed GMs. The biological effects of this decrease in IL-23 release during GM adsorption to CA beads need further clarification.

  19. Neutrophil maturation rate determines the effects of dipeptidyl peptidase 1 inhibition on neutrophil serine protease activity

    PubMed Central

    Wikell, C; Clifton, S; Shearer, J; Benjamin, A; Peters, S A

    2016-01-01

    Background and Purpose Neutrophil serine proteases (NSPs) are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation. The effects of neutrophil turnover rate on NSP activity following DPP1 inhibition was studied in a rat pharmacokinetic/pharmacodynamic model. Experimental Approach Rats were treated with a DPP1 inhibitor twice daily for up to 14 days; NSP activity was measured in onset or recovery studies, and an indirect response model was fitted to the data to estimate the turnover rate of the response. Key Results Maximum NSP inhibition was achieved after 8 days of treatment and a reduction of around 75% NSP activity was achieved at 75% in vitro DPP1 inhibition. Both the rate of inhibition and recovery of NSP activity were consistent with a neutrophil turnover rate of between 4–6 days. Using human neutrophil turnover rate, it is predicted that maximum NSP inhibition following DPP1 inhibition takes around 20 days in human. Conclusions and Implications Following inhibition of DPP1 in the rat, the NSP activity was determined by the amount of DPP1 inhibition and the turnover of neutrophils and is thus supportive of the role of neutrophil maturation in the activation of NSPs. Clinical trials to monitor the effect of a DPP1 inhibitor on NSPs should take into account the delay in maximal response on the one hand as well as the potential delay in a return to baseline NSP levels following cessation of treatment. PMID:27186823

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

    PubMed Central

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

    2016-01-01

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

  1. Pulmonary lesions induced by Pasteurella haemolytica in neutrophil sufficient and neutrophil deficient calves.

    PubMed Central

    Breider, M A; Walker, R D; Hopkins, F M; Schultz, T W; Bowersock, T L

    1988-01-01

    The role of neutrophils in the development of peracute lung lesions of bovine pneumonic pasteurellosis was investigated. Eight calves were divided into two groups of four calves each. Group I was treated with intravenous phosphate-buffered saline and served as the neutrophil sufficient calves. Group II was treated with intravenous hydroxyurea which produced a state of neutropenia. When peripheral blood neutrophil numbers dropped below 300 cells/microL in group II, all calves were challenged with an intrabronchial bolus of Pasteurella haemolytica in the log phase of growth. An acute inflammatory process occurred in both groups of calves indicated by a rise in body temperature. While pulmonary lesions occurred in both groups by six hours postinoculation, they varied in pathological characteristics. Pulmonary lesions in the neutrophil sufficient calves consisted of fibrinopurulent alveolitis-bronchiolitis with associated alveolar septal necrosis, interlobular edema, and intravascular thrombi. The neutrophil deficient calves had extensive intra-alveolar edema, interlobular edema, intraalveolar hemorrhage, atelectasis, and focal areas of alveolar septal necrosis. These results show that P. haemolytica can induce severe pulmonary tissue damage through both neutrophil dependent and neutrophil independent mechanisms. Images Fig. 1. Fig. 2. PMID:3370555

  2. Purification and characterization of a lipid thiobis ester from human neutrophil cytosol that reversibly deactivates the O2- -generating NADPH oxidase.

    PubMed

    Eklund, E A; Gabig, T G

    1990-05-25

    Intact neutrophils possess a cellular mechanism that efficiently deactivates the microbicidal O2-generating NADPH oxidase during the respiratory burst (Akard, L. P., English, D., and Gabig, T. G. (1988) Blood 72, 322-327). The present studies directed at identifying the molecular mechanism(s) involved in NADPH oxidase deactivation showed that a heat- and trypsin-insensitive species in the cytosolic fraction from normal unstimulated neutrophils was capable of deactivating the membrane-associated NADPH oxidase isolated from opsonized zymosan- or phorbol 12-myristate 13-acetate-stimulated neutrophils. This cytosolic species also deactivated the cell-free-activated oxidase. Deactivation by this cytosolic species occurred in the absence of NADPH-dependent catalytic turnover and was reversible, since NADPH oxidase activity could be subsequently reactivated in the cell-free system. The sedimentable particulate fraction from unstimulated neutrophils did not demonstrate deactivator activity. Deactivator activity was demonstrated in the neutral lipid fraction of neutrophil cytosol extracted with chloroform:methanol. Following complete purification of cytosolic deactivator activity by thin layer chromatography and reversed phase high performance liquid chromatography, the deactivator species was shown to be a lipid thiobis ester compound by mass spectroscopy. Cellular metabolism of this compound in human neutrophils may reveal a unique mechanism for enzymatic control of the NADPH oxidase system and thereby play an important role in regulation of the inflammatory response.

  3. Neutrophil-platelet adhesion: relative roles of platelet P-selectin and neutrophil beta2 (DC18) integrins.

    PubMed

    Brown, K K; Henson, P M; Maclouf, J; Moyle, M; Ely, J A; Worthen, G S

    1998-01-01

    Neutrophils and platelets interact both physically and metabolically during inflammation and thrombosis, but the mechanisms responsible for their adhesion remain incompletely understood. Neutrophil-platelet adhesion was measured after specific stimulation of neutrophils, platelets, or both and quantified by flow cytometry. Specific stimulation of either the neutrophil or the platelet led to a marked increase in the percentage of neutrophils that bound platelets, although platelet stimulation led to a large increase and neutrophil stimulation to only a small increase in the number of platelets per neutrophil. Stimulation of both cells further increased the number of neutrophil-platelet adhesive events and led to large numbers of platelets binding to each neutrophil. Confirming previous observations, blocking antibodies to platelet P-selectin (CD62P) partially inhibited adhesion. However, blockade of the neutrophil beta2 integrin CD11b/CD18 also inhibited the percentage of neutrophils that bound platelets. Combining P-selectin and CD11b/18 blockade further inhibited the stimulated increase in the percentage of neutrophils binding platelets and the increased number of platelets per neutrophil. Both cell adhesion molecules were active even when only a single cell type was primarily activated, supporting physiologically important transcellular activation. These data suggest that: (1) neutrophil-platelet adhesion can be initiated by specific activation of either the neutrophil or the platelet and that specific activation of either cell type leads to distinct patterns of adhesion, and (2) neutrophil-platelet adhesion uses both platelet P-selectin and the neutrophil beta2 integrin CD11b/CD18 when the cells are primarily or secondarily activated.

  4. Neutrophils: important contributors to tumor progression and metastasis.

    PubMed

    Swierczak, Agnieszka; Mouchemore, Kellie A; Hamilton, John A; Anderson, Robin L

    2015-12-01

    The presence of neutrophils in tumors has traditionally been considered to be indicative of a failed immune response against cancers. However, there is now evidence showing that neutrophils can promote tumor growth, and increasingly, the data support an active role for neutrophils in tumor progression to distant metastasis. Neutrophils have been implicated in promoting metastasis in cancer patients, where neutrophil numbers and neutrophil-related factors and functions have been associated with progressive disease. Nevertheless, the role of neutrophils in tumors, both at the primary and secondary sites, remains controversial, with some studies reporting their anti-tumor functions. This review will focus on the data demonstrating a role for neutrophils in both tumor growth and metastasis and will attempt to clarify the discrepancies in the literature.

  5. Effect of magnetic resonance imaging on human respiratory burst of neutrophils.

    PubMed

    Heine, J; Scheinichen, D; Jaeger, K; Herzog, T; Sümpelmann, R; Leuwer, M

    1999-03-05

    It is known that low intensity magnetic fields increase superoxide anion production during the respiratory burst of rat peritoneal neutrophils in vitro. We investigated whether the high intensity magnetic fields (1.5 T) during magnetic resonance imaging can influence the human neutrophil function under in vivo conditions. Blood samples were obtained from 12 patients immediately before and after magnetic resonance imaging (mean time 27.6(+/-11.4 min)). The induced respiratory burst was investigated by the intracellular oxidative transformation of dihydrorhodamine 123 to the fluorescent dye rhodamine 123 via flow cytometry. The respiratory burst was induced either with phorbol 12-myristate 13-acetate, Escherichia coli, N-formyl-methionyl-leucylphenylalanine or priming with tumor necrosis factor followed by FMLP stimulation. There was no significant difference between the respiratory burst before and after magnetic resonance imaging, irrespective of the stimulating agent. Short time exposure to a high intensity magnetic field during magnetic resonance imaging seems not to influence the production of radical species in living neutrophils.

  6. Neutrophil homeostasis and its regulation by danger signaling.

    PubMed

    Wirths, Stefan; Bugl, Stefanie; Kopp, Hans-Georg

    2014-06-05

    Hematopoiesis in general is demand driven and adaptive, but in contrast to erythropoiesis or thrombocytopoiesis, our knowledge on how neutrophil production is adapted to individual needs remains incomplete. Recently, neutrophil homeostasis has been shown to depend on danger receptors, macrophages, and even circadian rhythms. Puzzle pieces for a broader view of neutrophil homeostasis accumulate, and we will herein try to put seemingly contradictory evidence in a perspective of neutrophil homeostasis and emergency granulopoiesis determined by innate immunologic signaling.

  7. Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

    PubMed

    Fine, Noah; Barzilay, Oriyah; Glogauer, Michael

    2017-01-01

    Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through Pattern Recognition Receptor (PPR) interaction with Pathogen-Associated Molecular Patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition, and phagocytosis by neutrophils in whole blood can be greatly enhanced due to the presence of blood serum components and platelets. Powerful and sensitive flow cytometry based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

  8. Antimicrobial Decapeptide KSL-W Enhances Neutrophil Chemotaxis and Function

    DTIC Science & Technology

    2011-12-16

    its antimicrobial activity [25]. Because of the known multifunctional activities associated with many antimicrobial peptides, we became interested in...stated. 2.5. Neutrophil treatment and measuring actin polymerization Purified human neutrophils were treated with HBSS, FMLP (10−7 M and 10−10 M), or...control neutrophils were resuspended in 1 ml of 1× DPBS. 2.7. Actin polymerization F- actin content in unstimulated and FMLP-/KSLW-treated neutrophils

  9. 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

  10. 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

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

    PubMed

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

    1988-07-01

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

  12. The in vitro effects of Newcastle disease virus on the metabolic and antibacterial functions of human neutrophils

    SciTech Connect

    Faden, H.; Humbert, J.; Lee, J.; Sutyla, P.; Ogra, P.L.

    1981-08-01

    Live Newcastle disease virus (NDV) was used to investigate the in vitro effects of a viral infection on phagocytosis, chemiluminescence generation, superoxide production, oxygen consumption, NADPH-oxidase activity, and intracellular killing of bacteria by Ficoll-Hypaque separated human neutrophils. Phagocytosis of oil red O particles by NDV-treated PMN was inhibited by 50%. Chemiluminescence by PMN was inhibited 79% after zymosan stimulation and 86% after tetradeconyl phorbol acetate stimulation. Superoxide generation was inhibited by 68%. Oxygen consumption was inhibited in the presence of NDV by 37% after stimulation with phorbol myristate acetate, while membrane-associated NADPH-enzyme activity was decreased by 19%. The percent of surviving intracellular S. aureus was significantly elevated in NDV-treated PMN after 60 and 120 min of incubation. Purified bacterial neuraminidase markedly suppressed chemiluminescence, while neuraminic acid blocked the effects of the virus. These observations suggest that infections with myxoviruses may suppress a number of vital neutrophil functions. It appears that the effects may be partly mediated by the interaction of viral neuraminidase with the external neutrophil membrane.

  13. Wegener's granulomatosis and autoantibodies to neutrophil antigens

    PubMed Central

    McCluskey, D R; Maxwell, A P; Watt, L

    1988-01-01

    We report five cases of Wegener's granulomatosis all of whom had clinical and histological evidence of disease activity at presentation and in whom autoantibodies to neutrophil antigens were detected. This test may prove useful for the diagnosis of this serious condition and help to monitor disease activity during treatment. PMID:3068870

  14. [Congenital neutrophil defects and periodontal diseases].

    PubMed

    Del Fabbro, M; Francetti, L; Pizzoni, L; Weinstein, R L

    2000-06-01

    An alteration of the immune system function is one of the main factors involved in the development of periodontal disease. Polymorpho-nuclear neutrophil leukocytes (PMN) play a crucial role in the cell-mediated immune response against bacterial challenge. The mechanism of neutralization of pathogen microorganisms by PMNs involves many different steps: adhesion to capillary endothelium in the inflamed region, trans-endothelial migration, chemotaxis, phagocytosis and, ultimately, bacterial killing by oxidative and non-oxidative mechanisms. A defect in one of these steps leads to altered neutrophil function and, consequently, to a higher host susceptibility to periodontal tissue infection. The main intrinsic neutrophil diseases such as neutropenia, leukocyte adhesion deficiency (LAD-1), Chediak-Higashi syndrome, Papillon-Lefèvre syndrome, chronic granulomatous disease (CGD), are often related to severe and early-onset forms of periodontitis, as described by many evidences in the literature. Therefore PMN dysfunctions, both intrinsic and extrinsic, represent an important risk factor for periodontal disease. Studies on the basic molecular mechanisms of such dysfunctions, also in terms of genetic polymorphisms, recently allowed to identify some specific markers related to a higher susceptibility to the development of disease. Many researches have yet to be performed aiming to gain insight on the dynamics of PMN activation and interaction with other cells, in order to improve and modulate neutrophil function and to develop specific approaches for care and prevention of periodontal diseases.

  15. Leukotriene B4 binding to human neutrophils

    SciTech Connect

    Lin, A.H.; Ruppel, P.L.; Gorman, R.R.

    1984-12-01

    (/sup 3/H) Leukotriene B4 (LTB4) binds concentration dependently to intact human polymorphonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4 degrees C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of (/sup 3/H) LTB4 indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 X 10(-9)M and Bmax of 1.96 X 10(4) sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 X 10(-9)M and a Bmax of 45.16 X 10(4) sites per neutrophil. Competitive binding experiments with structural analogues of LTB4 demonstrate that the interaction between LTB4 and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25 degrees C (/sup 3/H) LTB4 is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB4. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific (/sup 3/H) LTB4 binding, suggesting that LTB4 is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB4 in neutrophils are tightly coupled processes.

  16. 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

  17. Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

    PubMed

    Goswami, Debashree; März, Sigrid; Li, Yu-Tung; Artz, Annette; Schäfer, Kerstin; Seelige, Ruth; Pacheco-Blanco, Mariana; Jing, Ding; Bixel, Maria Gabriele; Araki, Masatake; Araki, Kimi; Yamamura, Ken-Ichi; Vestweber, Dietmar

    2017-03-30

    CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

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

    PubMed

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

    2010-09-01

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

  19. Decrease of neutrophils chemiluminescence during exposure to low-power laser infrared radiation

    NASA Astrophysics Data System (ADS)

    Czuba, Zenon P.; Adamek, Mariusz; Krol, Wojciech; Sieron, Aleksander; Cieslar, Grzegorz

    1995-01-01

    The neutrophil is the cell in which phagocyting and transforming of some exogeneous agents results in marked stimulation of nonmitochondrial respiratory chain activity (respiratory burst). In our experiment we focused on determining the level of chemiluminescence (CL) of stimulated neurotrophils during and after irradiation, measuring the photon emission intensity in 6 second's intervals. We used Ga-Al-As pulsed laser (wavelength 904 nm, mean power 8,9 mW, Alpha-Electronics GmbH, Germany) which was placed over the tube containing the suspension of guinea pig peritoneal neurotrophils (2X106 cells/ml). The sensitivity range of used photomultiplier (9514s, THORN EMI, Middlesex, England) was 300-600 nm, which allowed us to measure the CL of neutrophils while being irradiated. The neutrophils were stimulated by phorbol myristate acetate (PMA) and CL intensified by luminol. The decay of luminol-dependent CL of neutrophils may be described by hyperbolic function curve. We switched the laser radiation on for 20 s, 60 s and 300 s and each time we observed the same reaction: the about 20% decrease of intensity of CL immediately after beginning the irradiation. The CL remained on decreased level during the whole period of irradiation reaching immediately the level of CL intensity characteristic for decay curve (20% increase), just after switching off the laser. Only after the longest irradiation time (300 s) we observed CL being higher and inconsistent with decay curve for several minutes. The type of reaction was always the same, regardless to the point of CL decay curve at which laser radiation was applied. The same changes of Cl we obtained irradiating the enzymatic system: horseradish peroxidase (HRP)-luminol - H2O2.

  20. 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

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

    PubMed

    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

    2015-05-27

    Essential oils were obtained by hydrodistillation of the flowers+leaves and stems of Artemisia kotuchovii Kupr. (AKEO(f+l) and AKEO(stm), 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, methyleugenol, 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 AKEO(stm), but not AKEOf+l, inhibited N-formyl-Met-Leu-Phe (fMLF)-stimulated Ca(2+) 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 AKEO(stm) composition, were also tested in human neutrophils and HL-60 cells transfected with N-formyl peptide receptor 1 (FPR1). One component, 6-methyl-3,5-heptadien-2-one (MHDO), inhibited fMLF- and interleukin 8 (IL-8)-stimulated Ca(2+) flux, fMLF-induced chemotaxis, and PMA-induced ROS production in human neutrophils. MHDO also inhibited fMLF-induced Ca(2+) flux in FPR1-HL60 cells. These results suggest that MHDO may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration and ROS production.

  2. DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment.

    PubMed

    Varjú, Imre; Longstaff, Colin; Szabó, László; Farkas, Ádám Zoltán; Varga-Szabó, Veronika Judit; Tanka-Salamon, Anna; Machovich, Raymund; Kolev, Krasimir

    2015-06-01

    In response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10(-9) cm(2). Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

  3. 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.

  4. Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease.

    PubMed

    Silva, Adriana Carolina Rodrigues Almeida; de Almeida, Breno Fernando Martins; Soeiro, Carolina Soares; Ferreira, Wagner Luis; de Lima, Valéria Marçal Félix; Ciarlini, Paulo César

    2013-04-01

    Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production.

  5. Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease

    PubMed Central

    Silva, Adriana Carolina Rodrigues Almeida; de Almeida, Breno Fernando Martins; Soeiro, Carolina Soares; Ferreira, Wagner Luis; de Lima, Valéria Marçal Félix; Ciarlini, Paulo César

    2013-01-01

    Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production. PMID:24082406

  6. Monosodium urate crystals induce extracellular DNA traps in neutrophils, eosinophils, and basophils but not in mononuclear cells.

    PubMed

    Schorn, Christine; Janko, Christina; Latzko, Melanie; Chaurio, Ricardo; Schett, Georg; Herrmann, Martin

    2012-01-01

    Neutrophil extracellular traps (NETs) are fibers of extracellular DNA released from neutrophils due to overwhelming phagocytic stimuli. The function of NETs is to trap and kill microbes to avoid spreading of potential pathogens. NETs are formed after encounter with various gram-positive and -negative bacteria but also in response to mediators causing sterile inflammation like interleukin-8 (IL-8), tumor necrosis factor (TNF), and phorbol myristate acetate (PMA). Here we show the formation of NETs (NETting) in response to monosodium urate (MSU) crystals as further model for sterile inflammation. We identified monocytes, neutrophils, and eosinophils as MSU phagocytosing cells. Basophils did not take up the crystals, instead they upregulated their activation marker CD203c after contact with MSU. Nevertheless, MSU crystals induced extracellular trap formation also in basophils, like in eosinophils and neutrophils, which phagocytose the crystals. In contrast, monocytes do not form NETs despite uptake of the MSU crystals. In contrast to the canonical stimuli like bacteria and PMA, MSU-induced NETosis was not abrogated by plasma. Our data show that MSU crystals induce extracellular DNA trap formation in all three granulocytes lineages (NETs, EETs, and BETs) but not in monocytes, and DNA externalization does not necessitate the uptake of the crystals.

  7. Periodontal Ligament Stem Cells Regulate Apoptosis of Neutrophils

    PubMed Central

    Wang, Qing; Ding, Gang; Xu, Xin

    2017-01-01

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

  8. Isolation and Characterization of Neutrophils with Anti-Tumor Properties.

    PubMed

    Sionov, Ronit Vogt; Assi, Simaan; Gershkovitz, Maya; Sagiv, Jitka Y; Polyansky, Lola; Mishalian, Inbal; Fridlender, Zvi G; Granot, Zvi

    2015-06-19

    Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or naïve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a > 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

  9. Acetate fuels the cancer engine.

    PubMed

    Lyssiotis, Costas A; Cantley, Lewis C

    2014-12-18

    Cancer cells have distinctive nutrient demands to fuel growth and proliferation, including the disproportionate use of glucose, glutamine, and fatty acids. Comerford et al. and Mashimo et al. now demonstrate that several types of cancer are avid consumers of acetate, which facilitates macromolecular biosynthesis and histone modification.

  10. Origins of blood acetate in the rat.

    PubMed Central

    Buckley, B M; Williamson, D H

    1977-01-01

    A novel enzymimc cycling assay for the determination of acetate in biological material is described. Measurements of the acetate concentration in blood and liver samples from rats of various ages and nutritional states with this assay are reported. The contribution of the intestine, the liver and the rest of the body to maintaining the concentration of acetate in the circulation is examined. Evidence is presented that the gut flora constitute the main source of acetate in blood of fed adult rats, though endogenous production of acetate is of significance in other situations. The streptozotocin-diabetic rat has an elevated blood acetate concentration. PMID:597244

  11. 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.

  12. Neutrophil extracellular traps in tissue pathology.

    PubMed

    Nakazawa, Daigo; Kumar, Santosh; Desai, Jyaysi; Anders, Hans-Joachim

    2017-03-01

    Neutrophil extracellular traps (NETs) are innate immune systems against invading pathogens. NETs are characterized as released DNA mixed with cytoplasmic antimicrobial proteins such as myeloperoxidase, proteinase3 and neutrophil elastase. While NETs are thought to have an important role in host defense, recent work has suggested that NETs contribute to tissue injury in non-infectious disease states. Uncontrolled NET formation in autoimmune diseases, metabolic disorders, cancers and thrombotic diseases can exacerbate a disease or even be a major initiator of tissue injury. But spotting NETs in tissues is not easy. Here we review the available histopathological evidence on the presence of NETs in a variety of diseases. We discuss technical difficulties and potential sources of misinterpretation while trying to detect NETs in tissue samples.

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

    PubMed Central

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

    2015-01-01

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

  14. 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.

  15. Autophagy is induced by anti-neutrophil cytoplasmic Abs and promotes neutrophil extracellular traps formation.

    PubMed

    Sha, Li-Li; Wang, Huan; Wang, Chen; Peng, Hong-Ying; Chen, Min; Zhao, Ming-Hui

    2016-11-01

    Dysregulated neutrophil extracellular traps (NETs) formation contributes to the pathogenesis of anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV). Increasing evidence indicates that autophagy is involved in the process of NETs formation. In this study, we aimed to investigate whether ANCA could induce autophagy in the process of NETs formation. Autophagy was detected using live cell imaging, microtubule-associated protein light chain 3B (LC3B) accumulation and Western blotting. The results showed that autophagy vacuolization was detected in neutrophils treated with ANCA-positive IgG by live cell imaging. This effect was enhanced by rapamycin, the autophagy inducer, and weakened by 3-methyladenine (3-MA), the autophagy inhibitor. In line with these results, the autophagy marker, LC3B, showed a punctate distribution pattern in the neutrophils stimulated with ANCA-positive IgG. In the presence of rapamycin, LC3B accumulation was further increased; however, this effect was attenuated by 3-MA. Moreover, incubated with ANCA-positive IgG, the NETosis rate significantly increased compared with the unstimulated group. And, the rate significantly increased or decreased in the neutrophils pretreated with rapamycin or 3-MA, respectively, as compared with the cells incubated with ANCA-positive IgG. Overall, this study demonstrates that autophagy is induced by ANCA and promotes ANCA-induced NETs formation.

  16. Lupus Erythematosus and Neutrophilic Urticarial Dermatosis

    PubMed Central

    Gusdorf, Laurence; Bessis, Didier; Lipsker, Dan

    2014-01-01

    Abstract Neutrophilic urticarial dermatosis (NUD) resembles urticaria clinically but is a neutrophilic dermatosis histopathologically. The majority of patients with NUD have an underlying systemic condition, mainly, autoinflammatory disorders such as cryopyrin-associated periodic syndromes, Schnitzler syndrome, and adult-onset Still disease, but a few also have systemic lupus erythematosus (LE). Here, we confirm these data and we report relevant clinical and histopathological data of 7 patients with LE and NUD. We retrospectively retrieved the medical records of all patients with LE in whom skin biopsy showed NUD in registers of Strasbourg and Montpellier University hospitals since 2000. All were female and aged between 13 and 45 years. Skin lesions were typically rose or red macules or slightly elevated papules occurring in a wide distribution. Individual lesions resolved within 24 hours and were not or only slightly itchy. Every patient had associated signs, most of the time polyarthritis and/or fever. NUD was the presenting mode of LE in 2 patients. NUD was misdiagnosed as a classic lupus flare and led to therapeutic intensification with the introduction of immunosuppressive drugs in 4 patients. Histopathological findings consisted of intense neutrophilic interstitial and perivascular infiltrate with leukocytoclasia and without fibrinoid necrosis of vessel walls. Direct immunofluorescence testing showed a lupus band in 4 patients. Antinuclear antibodies were always positive, anti-dsDNA antibodies were positive in 5 patients, and anti-Ro/SSA antibodies in 6 patients. Immunosuppressive drugs such as prednisone, hydroxychloroquine, mycophenolate mofetil, and methotrexate were never effective to treat NUD. Antihistamines were effective in 1 patient and dapsone or colchicine was effective in 5 patients. NUD is not exceptional in patients with systemic LE and is easily misdiagnosed as an acute LE flare. Furthermore, we show that conventional immunosuppressive LE

  17. Sexy again: the renaissance of neutrophils in psoriasis.

    PubMed

    Schön, Michael P; Broekaert, Sigrid M C; Erpenbeck, Luise

    2017-04-01

    Notwithstanding their prominent presence in psoriatic skin, the functional role of neutrophilic granulocytes still remains somewhat enigmatic. Sparked by exciting scientific discoveries regarding neutrophil functions within the last years, the interest in these short-lived cells of the innate immune system has been boosted recently. While it had been known for some time that neutrophils produce and respond to a number of inflammatory mediators, recent research has linked neutrophils with the pathogenic functions of IL-17, possibly in conjunction with the formation of NETs (neutrophil extracellular traps). Antipsoriatic therapies exert their effects, at least in part, through interference with neutrophils. Neutrophils also appear to connect psoriasis with comorbid diseases. However, directly tampering with neutrophil functions is not trivial as evinced by the failure of therapeutic approaches targeting redundantly regulated cellular communication networks. It has also become apparent that neutrophils link important pathogenic functions of the innate and the adaptive immune system and that they are intricately involved in regulatory networks underlying the pathophysiology of psoriasis. In order to advocate intensified research into the role of this interesting cell population, we here highlight some features of neutrophils and put them into perspective with our current view of the pathophysiology of psoriasis.

  18. Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes.

    PubMed

    Ayres-Sander, Chantal E; Lauridsen, Holly; Maier, Cheryl L; Sava, Parid; Pober, Jordan S; Gonzalez, Anjelica L

    2013-01-01

    During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1β-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.

  19. Targeting neutrophils in ischemic stroke: translational insights from experimental studies

    PubMed Central

    Jickling, Glen C; Liu, DaZhi; Ander, Bradley P; Stamova, Boryana; Zhan, Xinhua; Sharp, Frank R

    2015-01-01

    Neutrophils have key roles in ischemic brain injury, thrombosis, and atherosclerosis. As such, neutrophils are of great interest as targets to treat and prevent ischemic stroke. After stroke, neutrophils respond rapidly promoting blood–brain barrier disruption, cerebral edema, and brain injury. A surge of neutrophil-derived reactive oxygen species, proteases, and cytokines are released as neutrophils interact with cerebral endothelium. Neutrophils also are linked to the major processes that cause ischemic stroke, thrombosis, and atherosclerosis. Thrombosis is promoted through interactions with platelets, clotting factors, and release of prothrombotic molecules. In atherosclerosis, neutrophils promote plaque formation and rupture by generating oxidized-low density lipoprotein, enhancing monocyte infiltration, and degrading the fibrous cap. In experimental studies targeting neutrophils can improve stroke. However, early human studies have been met with challenges, and suggest that selective targeting of neutrophils may be required. Several properties of neutrophil are beneficial and thus may important to preserve in patients with stroke including antimicrobial, antiinflammatory, and neuroprotective functions. PMID:25806703

  20. 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.

  1. Differentiating neutrophils using the optical coulter counter

    NASA Astrophysics Data System (ADS)

    Schonbrun, Ethan; Di Caprio, Giuseppe

    2015-11-01

    We present an optofluidic measurement system that quantifies cell volume, dry mass, and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell's refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

  2. Differentiating neutrophils using the optical coulter counter

    NASA Astrophysics Data System (ADS)

    Schonbrun, E.; Di Caprio, G.

    2015-03-01

    We present an opto-fluidic measurement system that quantifies cell volume, dry mass and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell's refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

  3. Tumor associated macrophages and neutrophils in cancer.

    PubMed

    Galdiero, Maria Rosaria; Bonavita, Eduardo; Barajon, Isabella; Garlanda, Cecilia; Mantovani, Alberto; Jaillon, Sébastien

    2013-11-01

    The tumor microenvironment is a complex framework, in which myeloid cells play important roles in sculpting cancer development from tumor initiation to metastasis. Immune cells are key participants of the tumor microenvironment where they can promote or inhibit cancer formation and development. Plasticity is a widely accepted hallmark of myeloid cells and in particular of the monocyte-macrophage lineage. It includes the ability to display a wide spectrum of activation states in response to distinct signals and classical M1 or alternative M2 macrophages represent a paradigm of this feature. Neutrophils have long been viewed as terminally differentiated effector cells, playing a major role during the acute phase of inflammation and resistance against microbes. Recent evidence questioned this limited point of view, indicating that neutrophils can interact with distinct cell populations and produce a wide number of cytokines and effector molecules. Therefore, macrophages and neutrophils are both integrated in the regulation of the innate and adaptive immune responses in various inflammatory situations, including cancer.

  4. Galectin-1 promotes human neutrophil migration.

    PubMed

    Auvynet, Constance; Moreno, Samadhi; Melchy, Erika; Coronado-Martínez, Iris; Montiel, Jose Luis; Aguilar-Delfin, Irma; Rosenstein, Yvonne

    2013-01-01

    An important step of innate immune response is the recruitment of polymorphonuclear leukocytes (PMN) to injured tissues through chemotactic molecules. Galectins, a family of endogenous lectins, participate in numerous functions such as lymphoid cell migration, homing, cell-cell and cell-matrix interactions. Particularly, galectin-3 (Gal-3) and -9 have been implicated in the modulation of acute and chronic inflammation by inducing the directional migration of monocytes/macrophages and eosinophils, whereas Gal-1 is considered to function as an anti-inflammatory molecule, capable of inhibiting the influx of PMN to the site of injury. In this study, we assessed the effect of Gal-1 on neutrophil recruitment, in the absence of additional inflammatory insults. Contrasting with its capacity to inhibit cell trafficking and modulate the release of mediators described in models of acute inflammation and autoimmunity, we evidenced that Gal-1 has the capacity to induce neutrophil migration both in vitro and in vivo. This effect is not mediated through a G-protein-coupled receptor but potentially through the sialoglycoprotein CD43, via carbohydrate binding and through the p38 mitogen-activated protein kinase pathway. These results suggest a novel biological function for CD43 on neutrophils and highlight that depending on the environment, Gal-1 can act either as chemoattractant or, as a molecule that negatively regulates migration under acute inflammatory conditions, underscoring the potential of Gal-1 as a target for innovative drug development.

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

    PubMed

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

    2006-01-01

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

  6. 'Slings' enable neutrophil rolling at high shear.

    PubMed

    Sundd, Prithu; Gutierrez, Edgar; Koltsova, Ekaterina K; Kuwano, Yoshihiro; Fukuda, Satoru; Pospieszalska, Maria K; Groisman, Alex; Ley, Klaus

    2012-08-16

    Most leukocytes can roll along the walls of venules at low shear stress (1 dyn cm−2), but neutrophils have the ability to roll at tenfold higher shear stress in microvessels in vivo. The mechanisms involved in this shear-resistant rolling are known to involve cell flattening and pulling of long membrane tethers at the rear. Here we show that these long tethers do not retract as postulated, but instead persist and appear as 'slings' at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro, where P-selectin-glycoprotein-ligand-1 (PSGL-1) is found in discrete sticky patches whereas LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The 'step-wise peeling of slings' is distinct from the 'pulling of tethers' reported previously. Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.

  7. 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.

  8. 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.

  9. Carbon-isotopic analysis of dissolved acetate.

    PubMed

    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.

  10. Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

    PubMed Central

    Junger, W G; Hoyt, D B; Davis, R E; Herdon-Remelius, C; Namiki, S; Junger, H; Loomis, W; Altman, A

    1998-01-01

    Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion. PMID:9637711

  11. 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

  12. Ménage-à-Trois: The Ratio of Bicarbonate to CO2 and the pH Regulate the Capacity of Neutrophils to Form NETs

    PubMed Central

    Maueröder, Christian; Mahajan, Aparna; Paulus, Susanne; Gößwein, Stefanie; Hahn, Jonas; Kienhöfer, Deborah; Biermann, Mona H.; Tripal, Philipp; Friedrich, Ralf P.; Munoz, Luis E.; Neurath, Markus F.; Becker, Christoph; Schett, Georg Andreas; Herrmann, Martin; Leppkes, Moritz

    2016-01-01

    In this study, we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET) formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (phorbol-2-myristate-13-acetate), ionomycin, monosodium urate, and LPS. In contrast, an acidic environment impaired NET formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to NET formation. Inflammatory foci may be characterized by an acidic environment. Our data indicate that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover, our findings highlight the necessity for strict pH control during assays of NET formation. PMID:28018350

  13. 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....

  14. Human neutrophil elastase in RSV bronchiolitis.

    PubMed

    Emboriadou, M; Hatzistilianou, Maria; Magnisali, Ch; Sakelaropoulou, A; Exintari, M; Conti, Pio; Aivazis, V

    2007-01-01

    Acute bronchiolitis is the most common lower respiratory tract infection in young children and may be life-threatening in those with underlying cardiac or respiratory conditions. We evaluated the nasal and serum levels of human neutrophil elastase (HNE) in patients with acute respiratory syncytial virus (RSV) bronchiolitis and investigated the correlation of these levels with illness severity. Fifty-one patients (28 boys, 23 girls) with acute bronchiolitis positive for RSV by direct immunoenzyme assay in nasal secretions (Group A) were studied. Thirty healthy children (17 boys, 13 girls) constituted the control group (Group B). Subjects in both groups were matched for age and gender. The ages (mean+/-SE) in Groups A and B were 4.5+/-0.41 and 5.0+/-0.65 mo, respectively. Venous blood and nasal secretions were taken from patients in group A on 1, 5, and 15 days after admission and once from controls (Group B) for determinations of HNE in nasal lavage and serum, as well as white blood counts (WBC). The peripheral blood eosinophil and neutrophil counts were elevated in 22/51 patients (43.1%) and 15/51 patients (29.4%), respectively. In nasal lavage specimens, neutrophils represented>or=75% and eosinophils>2% of all cells in 42/51 (82.0%) patients and 11/51 (21.5%) patients, respectively. There was strong correlation between the level of HNE and the percentage of neutrophils in nasal lavage (r=0.92). The mean nasal HNE concentrations of the patients on 1, 5, and 15 days after admission were higher than those of Group B (p<0.0001, p<0.001, p<0.001, respectively). Mean serum HNE concentrations on 1, 5, and 15 days after admission were higher in Group A than in Group B (p<0.0001, p<0.0001, p<0.0001, respectively). Nasal and serum HNE concentrations showed no correlations with the clinical score of disease severity (r=0.28 and r=0.29, respectively). This study shows that (a) serum and nasal HNE concentrations were significantly higher in RSV bronchiolitis patients than in

  15. Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

    PubMed

    Takeda, Yuji; Fu, Junfen; Suzuki, Kichiya; Sendo, Dai; Nitto, Takeaki; Sendo, Fujiro; Araki, Yoshihiko

    2003-06-10

    GPI-80 is a member of the amidohydrolase family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion. GPI-80 is expressed mainly in human neutrophils. Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. To verify this, we examined whether GPI-80 is expressed on the human promyelocytic leukemia cell line HL-60 following treatment with differentiation inducers. GPI-80 expression was induced in cells treated with dimethyl sulfoxide (DMSO) to stimulate differentiation down the neutrophil pathway. On the other hand, all-trans-retinoic acid (ATRA), another neutrophil-inducing reagent, induced no clear GPI-80 expression. Potent monocyte-inducing reagents such as 1alpha,25-dihydroxyvitamin D(3) or phorbol 12-myristate 13-acetate also had no significant effect on the protein expression. GPI-80-positive cells were found in the well-differentiated CD11b-positive and transferrin-receptor-negative cell population. Granulocyte colony-stimulating factor, which augments neutrophil differentiation of HL-60 cells, up-regulated GPI-80 expression in the presence of DMSO. Granulocyte/macrophage colony-stimulating factor, which is known to suppress the neutrophil maturation of cells, inhibited expression. Adhesion of DMSO-induced cells was regulated by anti-GPI-80 monoclonal antibody, similar to the regulation observed in neutrophils. These results suggest that use of DMSO to induce neutrophil differentiation provides suitable conditions for GPI-80 expression, and that this culture system may be a helpful model for further study of the regulation of GPI-80 expression during myeloid differentiation.

  16. 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...

  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 582.1721 - 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 21 CFR 522.2476 - Trenbolone acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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...

  8. 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...

  9. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives

    PubMed Central

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Macallan, Derek

    2016-01-01

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

  10. Photochemistry of 2-nitrobenzylidene acetals.

    PubMed

    Sebej, Peter; Solomek, Tomás; Hroudná, L'ubica; Brancová, Pavla; Klán, Petr

    2009-11-20

    Photolysis of dihydroxy compounds (diols) protected as 2-nitrobenzylidene acetals (ONBA) and subsequent acid- or base-catalyzed hydrolysis of the 2-nitrosobenzoic acid ester intermediates result in an efficient and high-yielding release of the substrates. We investigated the scope and limitations of ONBA photochemistry and expanded upon earlier described two-step procedures to show that the protected diols of many structural varieties can also be liberated in a one-pot procedure. In view of the fact that the acetals of nonsymmetrically substituted diols are converted into one of the corresponding 2-nitrosobenzoic acid ester isomers with moderate to high regioselectivity, the mechanism of their formation was studied using various experimental techniques. The experimental data were found to be in agreement with DFT-based quantum chemical calculations that showed the preferential cleavage occurs on the acetal C-O bond in the vicinity of more electron-withdrawing (or less electron-donating) groups. The study also revealed considerable complexity in the cleavage mechanism and that the structural variations in the substrate can significantly alter the reaction pathway. This deprotection strategy was found to be also applicable for 2-thioethanol when released from the corresponding monothioacetal in the presence of a reducing agent, such as ascorbic acid.

  11. Neutrophil depletion delays wound repair in aged mice

    PubMed Central

    Nishio, Naomi; Okawa, Yayoi; Sakurai, Hidetoshi

    2008-01-01

    One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice. PMID:19424869

  12. Neutrophils in host defense: new insights from zebrafish

    PubMed Central

    Harvie, Elizabeth A.; Huttenlocher, Anna

    2015-01-01

    Neutrophils are highly motile phagocytic cells that play a critical role in the immune response to infection. Zebrafish (Danio rerio) are increasingly used to study neutrophil function and host-pathogen interactions. The generation of transgenic zebrafish lines with fluorescently labeled leukocytes has made it possible to visualize the neutrophil response to infection in real time by use of optically transparent zebrafish larvae. In addition, the genetic tractability of zebrafish has allowed for the generation of models of inherited neutrophil disorders. In this review, we discuss several zebrafish models of infectious disease, both in the context of immunocompetent, as well as neutrophil-deficient hosts and how these models have shed light on neutrophil behavior during infection. PMID:25717145

  13. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection

    PubMed Central

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J.; Roediger, Ben; Brzoska, Anthony J.; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L.; von Andrian, Ulrich H.; Hickey, Michael J.; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in post-capillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we show that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin lyses perivascular macrophages leading to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin, and indicate that Staphylococcus aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  14. Neutrophils: critical components in experimental animal models of cancer

    PubMed Central

    Hagerling, Catharina; Werb, Zena

    2016-01-01

    Neutrophils have a crucial role in tumor development and metastatic progression. The contribution of neutrophils in tumor development is multifaceted and contradictory. On the one hand, neutrophils prompt tumor inception, promote tumor development by mediating the initial angiogenic switch and facilitate colonization of circulating tumor cells, and on the other hand, have cytotoxic and anti-metastatic capabilities. Our understanding of the role of neutrophils in tumor development has greatly depended on different experimental animal models of cancer. In this review we cover important findings that have been made about neutrophils in experimental animal models of cancer, point to their advantages and limitations, and discuss novel techniques that can be used to expand our knowledge of how neutrophils influence tumor progression. PMID:26976824

  15. Neutrophils and Macrophages: the Main Partners of Phagocyte Cell Systems

    PubMed Central

    Silva, Manuel T.; Correia-Neves, Margarida

    2012-01-01

    Biological cellular systems are groups of cells sharing a set of characteristics, mainly key function and origin. Phagocytes are crucial in the host defense against microbial infection. The previously proposed phagocyte cell systems including the most recent and presently prevailing one, the mononuclear phagocyte system (MPS), grouped mononuclear cells but excluded neutrophils, creating an unacceptable situation. As neutrophils are archetypical phagocytes that must be members of comprehensive phagocyte systems, Silva recently proposed the creation of a myeloid phagocyte system (MYPS) that adds neutrophils to the MPS. The phagocytes grouped in the MYPS include the leukocytes neutrophils, inflammatory monocytes, macrophages, and immature myeloid DCs. Here the justifications behind the inclusion of neutrophils in a phagocyte system is expanded and the MYPS are further characterized as a group of dedicated phagocytic cells that function in an interacting and cooperative way in the host defense against microbial infection. Neutrophils and macrophages are considered the main arms of this system. PMID:22783254

  16. Technical note: proteomic approaches to fundamental questions about neutrophil biology.

    PubMed

    McLeish, Kenneth R; Merchant, Michael L; Klein, Jon B; Ward, Richard A

    2013-10-01

    Proteomics is one of a group of technologies that generates high-throughput, large-scale datasets that can be used to understand cell or organ functions at a systems level. This review will focus on the application of proteomics to the understanding of neutrophil biology. The strengths and weaknesses of common proteomic methods and their application to neutrophils are reviewed, with the goal of evaluating whether the technology is ready to advance our understanding of neutrophil biology.

  17. The Neutrophil Response Induced by an Agonist for Free Fatty Acid Receptor 2 (GPR43) Is Primed by Tumor Necrosis Factor Alpha and by Receptor Uncoupling from the Cytoskeleton but Attenuated by Tissue Recruitment

    PubMed Central

    Björkman, Lena; Mårtensson, Jonas; Winther, Malene; Gabl, Michael; Holdfeldt, André; Uhrbom, Martin; Bylund, Johan; Højgaard Hansen, Anders; Pandey, Sunil K.; Ulven, Trond; Forsman, Huamei

    2016-01-01

    Ligands with improved potency and selectivity for free fatty acid receptor 2 (FFA2R) have become available, and we here characterize the neutrophil responses induced by one such agonist (Cmp1) and one antagonist (CATPB). Cmp1 triggered an increase in the cytosolic concentration of Ca2+, and the neutrophils were then desensitized to Cmp1 and to acetate, a naturally occurring FFA2R agonist. The antagonist CATPB selectively inhibited responses induced by Cmp1 or acetate. The activated FFA2R induced superoxide anion secretion at a low level in naive blood neutrophils. This response was largely increased by tumor necrosis factor alpha (TNF-α) in a process associated with a recruitment of easily mobilizable granules, but neutrophils recruited to an aseptic inflammation in vivo were nonresponding. Superoxide production induced by Cmp1 was increased in latrunculin A-treated neutrophils, but no reactivation of desensitized FFA2R was induced by this drug, suggesting that the cytoskeleton is not directly involved in terminating the response. The functional and regulatory differences between the receptors that recognize short-chain fatty acids and formylated peptides, respectively, imply different roles of these receptors in the orchestration of inflammation and confirm the usefulness of a selective FFA2R agonist and antagonist as tools for the exploration of the precise role of the FFA2R. PMID:27503855

  18. Propagation of thrombosis by neutrophils and extracellular nucleosome networks

    PubMed Central

    Pfeiler, Susanne; Stark, Konstantin; Massberg, Steffen; Engelmann, Bernd

    2017-01-01

    Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis. PMID:27927771

  19. Regulation of the estrous cycle by neutrophils via opioid peptides.

    PubMed

    Sasaki, Soichiro; Tamaki, Yutaka; Nagata, Kisaburo; Kobayashi, Yoshiro

    2011-07-15

    We found previously that neutrophil-depleted mice exhibited significant blockading of both the regular estrous cycle and cyclic changes of steroid hormone levels. In this study, we aimed at elucidation of the underlying mechanism. To examine the possibility that an increase in bacteria in the vaginal vault of neutrophil-depleted mice causes blockading of the estrous cycle, we treated neutrophil-depleted mice with antibiotics but failed to restore the estrous cycle. We then examined another possibility that neutrophils regulate the estrous cycle via opioid peptides, because opioid peptides regulate steroidogenesis in theca and granulosa cells in the ovaries, and because neutrophils contain opioid peptides. In support of this possibility, naloxone, an opioid antagonist, blocked the estrous cycle and a μ opioid receptor agonist restored the estrous cycle in neutrophil-depleted mice. Pro-opiomelanocortin was immunohistochemically detected in peripheral blood neutrophils but not in ones that had infiltrated into the ovaries. i.v. injection of anti-MIP-2 polyclonal Ab caused blockading of the estrous cycle, whereas MIP-2 was detected in the ovaries, suggesting a role of MIP-2 in the regulation of the estrous cycle. Moreover, i.v. injection of MIP-2 decreased the pro-opiomelanocortin signal in peripheral blood neutrophils and caused blockading of the estrous cycle. Together, these results suggest that neutrophils maintain the estrous cycle via opioid peptides.

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

    PubMed Central

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

    2015-01-01

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

  1. Neutrophil Interactions Stimulate Evasive Hyphal Branching by Aspergillus fumigatus

    PubMed Central

    Jorgensen, Julianne; Frydman, Galit H.; Jones, Caroline N.

    2017-01-01

    Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching. PMID:28076396

  2. Exploring inflammatory disease drug effects on neutrophil function.

    PubMed

    Wu, Xiaojie; Kim, Donghyuk; Young, Ashlyn T; Haynes, Christy L

    2014-08-21

    Neutrophils are critical inflammatory cells; thus, it is important to characterize the effects of drugs on neutrophil function in the context of inflammatory diseases. Herein, chemically guided neutrophil migration, known as chemotaxis, is studied in the context of drug treatment at the single cell level using a microfluidic platform, complemented by cell viability assays and calcium imaging. Three representative drugs known to inhibit surface receptor expression, signaling enzyme activity, and the elevation of intracellular Ca(2+) levels, each playing a significant role in neutrophil chemotactic pathways, are used to examine the in vitro drug effects on cellular behaviors. The microfluidic device establishes a stable concentration gradient of chemokines across a cell culture chamber so that neutrophil migration can be monitored under various drug-exposure conditions. Different time- and concentration-dependent regulatory effects were observed by comparing the motility, polarization, and effectiveness of neutrophil chemotaxis in response to the three drugs. Viability assays revealed distinct drug capabilities in reducing neutrophil viability while calcium imaging clarified the role of Ca(2+) in the neutrophil chemotaxis. This study provides mechanistic insight into the drug effects on neutrophil function, facilitating comparison of current and potential pharmaceutical approaches.

  3. Neutrophils and Immunity: From Bactericidal Action to Being Conquered

    PubMed Central

    Teng, Tie-Shan

    2017-01-01

    The neutrophil is the major phagocyte and the final effector cell of the innate immunity, with a primary role in the clearance of extracellular pathogens. Using the broad array of cytokines, extracellular traps, and effector molecules as the humoral arm, neutrophils play a crucial role in the host defense against pathogen infections. On the other hand, the pathogen has the capacity to overcome neutrophil-mediated host defense to establish infection causing human disease. Pathogens, such as S. aureus, have the potential to thwart neutrophil chemotaxis and phagocytosis and thereby succeed in evading killing by neutrophils. Furthermore, S. aureus surviving within neutrophils promotes neutrophil cytolysis, resulting in the release of host-derived molecules that promote local inflammation. Here, we provide a detailed overview of the mechanisms by which neutrophils kill the extracellular pathogens and how pathogens evade neutrophils degradation. This review will provide insights that might be useful for the development of novel therapies against infections caused by antibiotic resistant pathogens. PMID:28299345

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

    PubMed

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

    2014-10-01

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

  5. Neutrophilic Skin Lesions in Autoimmune Connective Tissue Diseases

    PubMed Central

    Hau, Estelle; Vignon Pennamen, Marie-Dominique; Battistella, Maxime; Saussine, Anne; Bergis, Maud; Cavelier-Balloy, Benedicte; Janier, Michel; Cordoliani, Florence; Bagot, Martine; Rybojad, Michel; Bouaziz, Jean-David

    2014-01-01

    Abstract The pathophysiology of neutrophilic dermatoses (NDs) and autoimmune connective tissue diseases (AICTDs) is incompletely understood. The association between NDs and AICTDs is rare; recently, however, a distinctive subset of cutaneous lupus erythematosus (LE, the prototypical AICTD) with neutrophilic histological features has been proposed to be included in the spectrum of lupus. The aim of our study was to test the validity of such a classification. We conducted a monocentric retrospective study of 7028 AICTDs patients. Among these 7028 patients, a skin biopsy was performed in 932 cases with mainly neutrophilic infiltrate on histology in 9 cases. Combining our 9 cases and an exhaustive literature review, pyoderma gangrenosum, Sweet syndrome (n = 49), Sweet-like ND (n = 13), neutrophilic urticarial dermatosis (n = 6), palisaded neutrophilic granulomatous dermatitis (n = 12), and histiocytoid neutrophilic dermatitis (n = 2) were likely to occur both in AICTDs and autoinflammatory diseases. Other NDs were specifically encountered in AICTDs: bullous LE (n = 71), amicrobial pustulosis of the folds (n = 28), autoimmunity-related ND (n = 24), ND resembling erythema gyratum repens (n = 1), and neutrophilic annular erythema (n = 1). The improvement of AICTDS neutrophilic lesions under neutrophil targeting therapy suggests possible common physiopathological pathways between NDs and AICTDs. PMID:25546688

  6. Energy Metabolism of Human Neutrophils during Phagocytosis

    PubMed Central

    Borregaard, Niels; Herlin, Troels

    1982-01-01

    Detailed quantitative studies were performed on the generation and utilization of energy by resting and phagocytosing human neutrophils. The ATP content was 1.9 fmol/cell, was constant during rest, and was not influenced by the presence or absence of glucose in the medium. The intracellular content of phosphocreatine was less than 0.2 fmol/cell. In the presence of glucose, ATP was generated almost exclusively from lactate produced from glucose taken up from the surrounding medium. The amount of lactate produced could account for 85% of the glucose taken up by the cells, and the intracellular glycosyl store, glycogen, was not drawn upon. The rate of ATP generation as calculated from the rate of lactate production was 1.3 fmol/cell/min. During phagocytosis, there was no measurable increase in glucose consumption or lactate production, and the ATP content fell rapidly to 0.8 fmol/cell. This disappearance of ATP was apparently irreversible since no corresponding increase in ADP or AMP was observed. It therefore appears that this phagocytosis-induced fall in ATP concentration represents all the extra energy utilized in human neutrophils in the presence of glucose. In the absence of glucose, the rate of ATP generation in the resting cell was considerably smaller, 0.75 fmol/cell per min, as calculated from the rate of glycolysis, which is sustained exclusively by glycogenolysis. Under this condition, however, phagocytosis induces significant enhancement of glycogenolysis and the rate of lactate production is increased by 60%, raising the rate of ATP generation to 1.2 fmol/cell per min. Nonetheless, the ATP content drops significantly from 1.9 to 1.0 fmol/cell. Neutrophils from patients with chronic granulomatous disease have the same rate of glycolysis and the same ATP content as normal cells, thus confirming that the defective respiration of these cells does not affect their energy metabolism. PMID:7107894

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

    PubMed

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

    2015-04-01

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

  8. Neutrophil extracellular traps in neuropathy with anti-neutrophil cytoplasmic autoantibody-associated microscopic polyangiitis.

    PubMed

    Takeuchi, Hiroki; Kawasaki, Teruaki; Shigematsu, Kazuo; Kawamura, Kazuyuki; Oka, Nobuyuki

    2017-04-01

    To clarify the roles of neutrophils in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitic neuropathy, we studied neutrophil extracellular traps (NETs) in peripheral nerve vasculitis. Stored nerve samples from 17 patients with microscopic polyangiitis (MPA) were immunohistochemically analyzed using antibodies for citrullinated histone H3 (citH3) and various neutrophil enzymes. We defined merged citH3 and extracellularly released myeloperoxidase (MPO) as NET formation. We also compared NET formation between MPO-ANCA-positive/negative MPA and rheumatoid arthritis (RA)-associated vasculitic neuropathy. NETs were identified mostly in vasculitic small arterioles of 6 of 12 MPO-ANCA-positive MPA patients, and their frequency was higher (p < 0.05) than in ANCA-negative patients. NETs were not found in vasculitic neuropathy with RA or patients with chronic inflammatory demyelinating polyradiculoneuropathy. NETs were also observed in the peripheral nervous system of MPA patients as well as in the lung and kidney. These results suggest that NETs may be involved in the pathogenesis of MPA neuropathy.

  9. Monocyte and neutrophil isolation and migration assays.

    PubMed

    Yona, Simon; Hayhoe, Richard; Avraham-Davidi, Inbal

    2010-02-01

    This unit describes methods for isolating mouse monocytes and neutrophils, as well as in vitro protocols for measuring cell migration and polarization. The method employed here for the isolation of naïve phagocytes overcomes many of the difficulties previously encountered concerning phagocyte activation. Three in vitro protocols are provided for the analysis of cell migration, one requiring no specialized equipment, one requiring the modified Boyden chamber, and the other employing a flow chamber, which measures cell adhesion, rolling, and migration. Finally, a method is provided for imaging polarized cells by confocal microscopy.

  10. Neutrophils Discriminate between Lipopolysaccharides of Different Bacterial Sources and Selectively Release Neutrophil Extracellular Traps

    PubMed Central

    Pieterse, Elmar; Rother, Nils; Yanginlar, Cansu; Hilbrands, Luuk B.; van der Vlag, Johan

    2016-01-01

    The release of neutrophil extracellular traps (NETs), either during “suicidal” or “vital” NETosis, represents an important strategy of neutrophils to combat Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, is a reported stimulus for NET formation. Although it is widely acknowledged that the structural diversity in LPS structures can elicit heterogeneous immune responses, species- and serotype-specific differences in the capacity of LPS to trigger NET formation have not yet been investigated. In the present study, we compared the NET-inducing potential of LPS derived from Escherichia coli (serotypes O55:B5, O127:B8, O128:B12, O111:B4, and O26:B6), Salmonella enterica (serotype enteritidis), and Pseudomonas aeruginosa (serotype 10), under platelet-free and platelet-rich conditions in vitro, and in whole blood ex vivo. Here, we demonstrate that under serum- and platelet-free conditions, mimicking tissue circumstances, neutrophils discriminate between LPS of different bacterial sources and selectively release NETs only in response to LPS derived from E. coli O128:B12 and P. aeruginosa 10, which both induced “suicidal” NETosis in an autophagy- and reactive oxygen species (ROS)-dependent, but TLR4-independent manner. Intriguingly, in whole blood cultures ex vivo, or in vitro in the presence of platelets, all LPS serotypes induced “vital” NET formation. This platelet-dependent release of NETs occurred rapidly without neutrophil cell death and was independent from ROS formation and autophagy but required platelet TLR4 and CD62P-dependent platelet–neutrophil interactions. Taken together, our data reveal a complex interplay between neutrophils and LPS, which can induce both “suicidal” and “vital” NETosis, depending on the bacterial origin of LPS and the presence or absence of platelets. Our findings suggest that LPS sensing by neutrophils may be a critical determinant for

  11. Differential stimulation of luminol-enhanced chemiluminescence (CL) and arachidonic acid metabolism in rat peritoneal neutrophils

    SciTech Connect

    Sturm, R.J.; Adams, L.M.; Cullinan, C.A.; Berkenkopf, J.W.; Weichman, B.M.

    1986-03-05

    Phorbol 12-myristate, 13-acetate (PMA) induced the production of radical oxygen species (ROS) from rat peritoneal neutrophils as assessed by CL. ROS generation occurred in a time- (maximum at 13.5 min) and dose- (concentration range of 1.7-498 nM) related fashion. However, 166 nM PMA did not induce either cyclooxygenase (CO) or lipoxygenase (LPO) product formation by 20 min post-stimulation. Conversely, A23187, at concentrations between 0.1 and 10 ..mu..M, stimulated both pathways of arachidonic acid metabolism, but had little or no effect upon ROS production. When suboptimal concentrations of PMA (5.5 nM) and A23187 (0.1-1 ..mu..M) were coincubated with the neutrophils, a synergistic ROS response was elicited. However, arachidonic acid metabolism in the presence of PMA was unchanged relative to A12187 alone. Nordihydroguaiaretic acid (NDGA) inhibited both PMA-induced CL (IC/sub 50/ = 0.9 ..mu..M) and A23187-induced arachidonic acid metabolism (IC/sub 50/ = 1.7 ..mu..M and 6.0 ..mu..M for LPO and CO, respectively). The mixed LPO-CO inhibitor, BW755C, behaved in a qualitatively similar manner to NDGA, whereas the CO inhibitors, indomethacin, piroxicam and naproxen had no inhibitory effect on ROS generation at concentrations as high as 100 ..mu..M. These results suggest that NDGA and BW755C may inhibit CL and arachidonic acid metabolism by distinct mechanisms in rat neutrophils.

  12. Characterization of C1 inhibitor binding to neutrophils.

    PubMed Central

    Chang, N S; Boackle, R J; Leu, R W

    1991-01-01

    In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH. PMID:2045131

  13. Nucleosomes and neutrophil activation in sickle cell disease painful crisis.

    PubMed

    Schimmel, Marein; Nur, Erfan; Biemond, Bart J; van Mierlo, Gerard J; Solati, Shabnam; Brandjes, Dees P; Otten, Hans-Martin; Schnog, John-John; Zeerleder, Sacha

    2013-11-01

    Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ(0)-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ(+-)thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome.

  14. Effect of clozapine on neutrophil kinetics in rabbits.

    PubMed

    Iverson, Suzanne; Kautiainen, Antti; Ip, Julia; Uetrecht, Jack P

    2010-07-19

    Clozapine is an atypical antipsychotic drug effective in the treatment of refractory schizophrenia; however, its use is limited due to its propensity to cause agranulocytosis in some patients. Little is known about the mechanism of idiosyncratic drug-induced agranulocytosis, in part because of the lack of a valid animal model. Clozapine is oxidized by activated human neutrophils and bone marrow cells to a reactive nitrenium ion by the myeloperoxidase-hydrogen peroxide system of neutrophils. This reactive metabolite has been shown in vitro to induce the apoptosis of neutrophils and bone marrow cells. While in vitro studies demonstrated the toxic potential of clozapine upon oxidation, it is not clear if similar conditions occur in vivo. In response to the difficulties encountered with detecting apoptotic neutrophils in vivo, we conducted a series of studies in rabbits using two fluorescent cell-labeling techniques to study the effect of clozapine treatment on neutrophil kinetics, that is, their rates of production and removal from circulation. The fluorescein dye, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), was used as a general cell label to measure the half-life of neutrophils in blood. In addition, the thymidine analogue, 5-bromo-2-deoxyuridine (BrdU), was used to label dividing cells, thus enabling the measurement of the efflux of neutrophils from the bone marrow. Clozapine, indeed, increased the rate of both the release of neutrophils from the bone marrow and their subsequent disappearance from circulation. Failure of the bone marrow to compensate for a shorter neutrophil half-life could lead to agranulocytosis. Alternatively, the damage to neutrophils caused by clozapine could, in some patients, lead to an immune-mediated response against neutrophils resulting in agranulocytosis.

  15. Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism.

    PubMed Central

    Entman, M L; Youker, K; Shappell, S B; Siegel, C; Rothlein, R; Dreyer, W J; Schmalstieg, F C; Smith, C W

    1990-01-01

    Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils. Images PMID:1970581

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

    PubMed

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

    2016-09-15

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

  17. Alteration of Neutrophil Reactive Oxygen Species Production by Extracts of Devil's Claw (Harpagophytum)

    PubMed Central

    Muzila, Mbaki; Wright, Helen; Roberts, Helen; Grant, Melissa; Nybom, Hilde; Sehic, Jasna; Ekholm, Anders

    2016-01-01

    Harpagophytum, Devil's Claw, is a genus of tuberiferous xerophytic plants native to southern Africa. Some of the taxa are appreciated for their medicinal effects and have been traditionally used to relieve symptoms of inflammation. The objectives of this pilot study were to investigate the antioxidant capacity and the content of total phenols, verbascoside, isoverbascoside, and selected iridoids, as well as to investigate the capacity of various Harpagophytum taxa in suppressing respiratory burst in terms of reactive oxygen species produced by human neutrophils challenged with phorbol myristate acetate (PMA), opsonised Staphylococcus aureus, and Fusobacterium nucleatum. Harpagophytum plants were classified into different taxa according to morphology, and DNA analysis was used to confirm the classification. A putative new variety of H. procumbens showed the highest degree of antioxidative capacity. Using PMA, three Harpagophytum taxa showed anti-inflammatory effects with regard to the PBS control. A putative hybrid between H. procumbens and H. zeyheri in contrast showed proinflammatory effect on the response of neutrophils to F. nucleatum in comparison with treatment with vehicle control. Harpagophytum taxa were biochemically very variable and the response in suppressing respiratory burst differed. Further studies with larger number of subjects are needed to corroborate anti-inflammatory effects of different taxa of Harpagophytum. PMID:27429708

  18. Utilization of acetate by Beggiatoa.

    PubMed

    Burton, S D; Morita, R Y; Miller, W

    1966-03-01

    Burton, Sheril D. (Institute of Marine Science, University of Alaska, College), Richard Y. Morita, and Wayne Miller. Utilization of acetate by Beggiatoa. J. Bacteriol. 91:1192-1200. 1966.-A proposed system which would permit acetate incorporation into four-carbon compounds without the presence of key enzymes of the citric acid cycle or glyoxylate cycle is described. In this system, acetyl-coenzyme A (CoA) is condensed with glyoxylate to form malate, which, in turn, is converted to oxaloacetate. Oxaloacetate then reacts with glutamate to produce alpha-ketoglutarate, which is subsequently converted to isocitrate. Cleavage of isocitrate produces glyoxylate and succinate. Thus, the proposed system is similar to the glyoxylate bypass in that malate is produced from glyoxylate and acetyl-CoA, but differs from both the citric acid cycle and the glyoxylate bypass, since citrate and fumarate are not involved. Fumarase, aconitase, catalase, citritase, pyruvate kinase, enolase, phosphoenolpyruvate carboxylase, lactic dehydrogenase, alpha-ketoglutarate dehydrogenase, and condensing enzyme were not detectable in crude extracts of Beggiatoa. Succinate was oxidized by a soluble enzyme not associated with an electron-transport particle. Isocitrate was identified as the sole compound labeled when C(14)O(2) was added to a reduced nicotinamide adenine dinucleotide, CO(2) generating system (crystalline glucose-6-phosphate dehydrogenase and glucose-6-phosphate) in the presence of alpha-ketoglutarate.

  19. Simulation model for flow of neutrophils in pulmonary capillary network.

    PubMed

    Shirai, Atsushi; Fujita, Ryo; Hayase, Toshiyuki

    2005-01-01

    The concentration of neutrophils in the pulmonary microvasculature is higher than in systemic large vessels. It is thought that the high concentration of neutrophils facilitates their effective recruitment to sites of inflammation. Thus, in order to understand the role of neutrophils in the immune system, it is important to clarify their flow characteristics in the pulmonary microvasculature. In previous studies, we numerically investigated the motion of a neutrophil through a single capillary segment modeled by a moderate axisymmetric constriction in a straight pipe, developing a mathematical model for the prediction of the transit time of the cell through the segment. In the present study, this model was extended for application to network simulation of the motion of neutrophils. First, we numerically investigated shape recovery of a neutrophil after expulsion from a narrow capillary segment. This process was modeled in two different phases: elastic recovery and viscous recovery. The resulting model was combined with the previously developed models to simulate motion of the cells and plasma flow in a capillary network. A numerical simulation of the motion of neutrophils and plasma flow in a simple lattice capillary network showed that neutrophils were widely dispersed in the network with an increased concentration.

  20. Intergrin-dependent neutrophil migration in the injured mouse cornea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an early responder to an inflammatory stimulus, neutrophils must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, neutrophils recruited from the peripher...

  1. Promoting metastasis: neutrophils and T cells join forces.

    PubMed

    Fridlender, Zvi G; Albelda, Steven M; Granot, Zvi

    2015-07-01

    The role neutrophils play in cancer is a matter of debate as both pro- and anti-tumor functions have been documented. In a recent publication in Nature, Coffelt et al. identify a new mechanism where neutrophils and T cells cooperate to generate metastasis-supporting immune suppression.

  2. Transepithelial migration of neutrophils into the lung requires TREM-1

    PubMed Central

    Klesney-Tait, Julia; Keck, Kathy; Li, Xiaopeng; Gilfillan, Susan; Otero, Karel; Baruah, Sankar; Meyerholz, David K.; Varga, Steven M.; Knudson, Cory J.; Moninger, Thomas O.; Moreland, Jessica; Zabner, Joseph; Colonna, Marco

    2012-01-01

    Acute respiratory infections are responsible for more than 4 million deaths each year. Neutrophils play an essential role in the innate immune response to lung infection. These cells have an armamentarium of pattern recognition molecules and antimicrobial agents that identify and eliminate pathogens. In the setting of infection, neutrophil triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory signaling. Here we demonstrate for the first time that TREM-1 also plays an important role in transepithelial migration of neutrophils into the airspace. We developed a TREM-1/3–deficient mouse model of pneumonia and found that absence of TREM-1/3 markedly increased mortality following Pseudomonas aeruginosa challenge. Unexpectedly, TREM-1/3 deficiency resulted in increased local and systemic cytokine production. TREM-1/3–deficient neutrophils demonstrated intact bacterial killing, phagocytosis, and chemotaxis; however, histologic examination of TREM-1/3–deficient lungs revealed decreased neutrophil infiltration of the airways. TREM-1/3–deficient neutrophils effectively migrated across primary endothelial cell monolayers but failed to migrate across primary airway epithelia grown at the air-liquid interface. These data define a new function for TREM-1 in neutrophil migration across airway epithelial cells and suggest that it amplifies inflammation through targeted neutrophil migration into the lung. PMID:23241959

  3. Impaired neutrophil directional chemotactic accuracy in chronic periodontitis patients

    PubMed Central

    Roberts, Helen M; Ling, Martin R; Insall, Robert; Kalna, Gabriela; Spengler, Julia; Grant, Melissa M; Chapple, Iain LC

    2015-01-01

    Aim To investigate the chemotactic accuracy of peripheral blood neutrophils from patients with chronic periodontitis compared with matched healthy controls, before and after non-surgical periodontal therapy. Material & Methods Neutrophils were isolated from patients and controls (n = 18) by density centrifugation. Using the Insall chamber and video microscopy, neutrophils were analysed for directional chemotaxis towards N-formyl-methionyl-leucyl-phenylalanine [fMLP (10 nM), or CXCL8 (200 ng/ml)]. Circular statistics were utilized for the analysis of cell movement. Results Prior to treatment, neutrophils from patients with chronic periodontitis had significantly reduced speed, velocity and chemotactic accuracy compared to healthy controls for both chemoattractants. Following periodontal treatment, patient neutrophils continued to display reduced speed in response to both chemoattractants. However, velocity and accuracy were normalized for the weak chemoattractant CXCL8 while they remained significantly reduced for fMLP. Conclusions Chronic periodontitis is associated with reduced neutrophil chemotaxis, and this is only partially restored by successful treatment. Dysfunctional neutrophil chemotaxis may predispose patients with periodontitis to their disease by increasing tissue transit times, thus exacerbating neutrophil-mediated collateral host tissue damage. PMID:25360483

  4. Transepithelial migration of neutrophils into the lung requires TREM-1.

    PubMed

    Klesney-Tait, Julia; Keck, Kathy; Li, Xiaopeng; Gilfillan, Susan; Otero, Karel; Baruah, Sankar; Meyerholz, David K; Varga, Steven M; Knudson, Cory J; Moninger, Thomas O; Moreland, Jessica; Zabner, Joseph; Colonna, Marco

    2013-01-01

    Acute respiratory infections are responsible for more than 4 million deaths each year. Neutrophils play an essential role in the innate immune response to lung infection. These cells have an armamentarium of pattern recognition molecules and antimicrobial agents that identify and eliminate pathogens. In the setting of infection, neutrophil triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory signaling. Here we demonstrate for the first time that TREM-1 also plays an important role in transepithelial migration of neutrophils into the airspace. We developed a TREM-1/3-deficient mouse model of pneumonia and found that absence of TREM-1/3 markedly increased mortality following Pseudomonas aeruginosa challenge. Unexpectedly, TREM-1/3 deficiency resulted in increased local and systemic cytokine production. TREM-1/3-deficient neutrophils demonstrated intact bacterial killing, phagocytosis, and chemotaxis; however, histologic examination of TREM-1/3-deficient lungs revealed decreased neutrophil infiltration of the airways. TREM-1/3-deficient neutrophils effectively migrated across primary endothelial cell monolayers but failed to migrate across primary airway epithelia grown at the air-liquid interface. These data define a new function for TREM-1 in neutrophil migration across airway epithelial cells and suggest that it amplifies inflammation through targeted neutrophil migration into the lung.

  5. Human neutrophil leukocyte elastase activity is inhibited by Phenol Red

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neutrophil elastase (NE) activity in urine, sputum and nasal mucous is used as an indicator of inflammation due to viral or bacterial infection. However, bovine nasal mucous neutrophils collected, lysed and stored in Dulbecco's minimal medium containing Phenol Red, showed no NE activity with methox...

  6. How Neutrophil Extracellular Traps Become Visible

    PubMed Central

    2016-01-01

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

  7. Dexamethasone Inhibits S. aureus-Induced Neutrophil Extracellular Pathogen-Killing Mechanism, Possibly through Toll-Like Receptor Regulation

    PubMed Central

    Wan, Ting; Zhao, Yingying; Fan, Fangli; Hu, Renjian; Jin, Xiuming

    2017-01-01

    Neutrophils release neutrophil extracellular traps (NETs) in a pathogen-killing process called NETosis. Excessive NETs formation, however, is implicated in disease pathogenesis. Therefore, to understand how NETosis is regulated, we examined the effect of dexamethasone (DXM), an anti-inflammatory drug, on this process and the role of toll-like receptors (TLRs). We stimulated human neutrophils with phorbol 12-myristate 13-acetate (PMA) or Staphylococcus aureus (S. aureus) and quantified NETs formation. We also examined the effect of DXM on the bactericidal effect of NETs and the role of reactive oxygen species (ROS) and nuclear factor (NF)-κB in DXM-regulated NETosis. DXM significantly inhibited S. aureus-induced NETosis and extracellular bacterial killing. ROS production and NF-κB activation were not involved in DXM-regulated NETosis. TLR2 and TLR4, but not TLR5 or TLR6, modified S. aureus-induced NETs formation. Neither DXM nor TLRs were involved in PMA-induced NETosis. Furthermore, TLR2 and TLR4 agonists rescued DXM-inhibited NETosis, and neither TLR2 nor TLR4 antagonists could further inhibit NETosis reduction induced by DXM, indicating that DXM may inhibit NETosis by regulating TLR2 and TLR4. In conclusion, the mechanisms of S. aureus- and PMA-induced NETosis are different. DXM decreases NETs formation independently of oxidant production and NF-κB phosphorylation and possibly via a TLR-dependent mechanism. PMID:28232829

  8. Dexamethasone Inhibits S. aureus-Induced Neutrophil Extracellular Pathogen-Killing Mechanism, Possibly through Toll-Like Receptor Regulation.

    PubMed

    Wan, Ting; Zhao, Yingying; Fan, Fangli; Hu, Renjian; Jin, Xiuming

    2017-01-01

    Neutrophils release neutrophil extracellular traps (NETs) in a pathogen-killing process called NETosis. Excessive NETs formation, however, is implicated in disease pathogenesis. Therefore, to understand how NETosis is regulated, we examined the effect of dexamethasone (DXM), an anti-inflammatory drug, on this process and the role of toll-like receptors (TLRs). We stimulated human neutrophils with phorbol 12-myristate 13-acetate (PMA) or Staphylococcus aureus (S. aureus) and quantified NETs formation. We also examined the effect of DXM on the bactericidal effect of NETs and the role of reactive oxygen species (ROS) and nuclear factor (NF)-κB in DXM-regulated NETosis. DXM significantly inhibited S. aureus-induced NETosis and extracellular bacterial killing. ROS production and NF-κB activation were not involved in DXM-regulated NETosis. TLR2 and TLR4, but not TLR5 or TLR6, modified S. aureus-induced NETs formation. Neither DXM nor TLRs were involved in PMA-induced NETosis. Furthermore, TLR2 and TLR4 agonists rescued DXM-inhibited NETosis, and neither TLR2 nor TLR4 antagonists could further inhibit NETosis reduction induced by DXM, indicating that DXM may inhibit NETosis by regulating TLR2 and TLR4. In conclusion, the mechanisms of S. aureus- and PMA-induced NETosis are different. DXM decreases NETs formation independently of oxidant production and NF-κB phosphorylation and possibly via a TLR-dependent mechanism.

  9. Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis

    PubMed Central

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

    2016-01-01

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

  10. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

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

  11. Coexistence of chronic neutrophilic leukemia with multiple myeloma.

    PubMed

    Dinçol, Günçağ; Nalçaci, Meliha; Doğan, Oner; Aktan, Melih; Küçükkaya, Reyhan; Ağan, Mehmet; Dinçol, Koray

    2002-03-01

    A case report of simultaneous presentation of chronic neutrophilic leukemia and multiple myeloma (IgG kappa) in a 71-year-old male is described. The patient showed mature neutrophilic leukocytosis, hepatosplenomegaly, high neutrophil alkaline phosphatase score, hyperuricemia, neutrophils with toxic granulation and Döhle bodies, absence of Philadelphia chromosome and of the bcr-abl fusion gene. Moreover, a monoclonal IgG kappa paraproteinemia (36.93 g l(-1)) was detected. Bence-Jones proteinuria was 3.84 g l(-1). The bone marrow was grossly hypercellular with marked myeloid hyperplasia and aggregates of plasma cells. The patient died of severe bronchopneumonia after the transformation of chronic neutrophilic leukemia to acute myelomonocytic leukemia, 1.5 years following diagnosis.

  12. Enzymatic production of glycerol acetate from glycerol.

    PubMed

    Oh, Seokhyeon; Park, Chulhwan

    2015-02-01

    In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.

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

    PubMed

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

    2007-07-27

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

  14. Role of the endothelial surface layer in neutrophil recruitment.

    PubMed

    Marki, Alex; Esko, Jeffrey D; Pries, Axel R; Ley, Klaus

    2015-10-01

    Neutrophil recruitment in most tissues is limited to postcapillary venules, where E- and P-selectins are inducibly expressed by venular endothelial cells. These molecules support neutrophil rolling via binding of PSGL-1 and other ligands on neutrophils. Selectins extend ≤ 38 nm above the endothelial plasma membrane, and PSGL-1 extends to 50 nm above the neutrophil plasma membrane. However, endothelial cells are covered with an ESL composed of glycosaminoglycans that is ≥ 500 nm thick and has measurable resistance against compression. The neutrophil surface is also covered with a surface layer. These surface layers would be expected to completely shield adhesion molecules; thus, neutrophils should not be able to roll and adhere. However, in the cremaster muscle and in many other models investigated using intravital microscopy, neutrophils clearly roll, and their rolling is easily and quickly induced. This conundrum was thought to be resolved by the observation that the induction of selectins is accompanied by ESL shedding; however, ESL shedding only partially reduces the ESL thickness (to 200 nm) and thus is insufficient to expose adhesion molecules. In addition to its antiadhesive functions, the ESL also presents neutrophil arrest-inducing chemokines. ESL heparan sulfate can also bind L-selectin expressed by the neutrophils, which contributes to rolling and arrest. We conclude that ESL has both proadhesive and antiadhesive functions. However, most previous studies considered either only the proadhesive or only the antiadhesive effects of the ESL. An integrated model for the role of the ESL in neutrophil rolling, arrest, and transmigration is needed.

  15. 21 CFR 184.1005 - Acetic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Acetic acid. 184.1005 Section 184.1005 Food and... Substances Affirmed as GRAS § 184.1005 Acetic acid. (a) Acetic acid (C2H4O2, CAS Reg. No. 64-19-7) is known as ethanoic acid. It occurs naturally in plant and animal tissues. It is produced by fermentation...

  16. 21 CFR 184.1005 - Acetic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Acetic acid. 184.1005 Section 184.1005 Food and... Substances Affirmed as GRAS § 184.1005 Acetic acid. (a) Acetic acid (C2H4O2, CAS Reg. No. 64-19-7) is known as ethanoic acid. It occurs naturally in plant and animal tissues. It is produced by fermentation...

  17. 21 CFR 184.1005 - Acetic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Acetic acid. 184.1005 Section 184.1005 Food and... Substances Affirmed as GRAS § 184.1005 Acetic acid. (a) Acetic acid (C2H4O2, CAS Reg. No. 64-19-7) is known as ethanoic acid. It occurs naturally in plant and animal tissues. It is produced by fermentation...

  18. 21 CFR 184.1005 - Acetic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Acetic acid. 184.1005 Section 184.1005 Food and... Substances Affirmed as GRAS § 184.1005 Acetic acid. (a) Acetic acid (C2H4O2, CAS Reg. No. 64-19-7) is known as ethanoic acid. It occurs naturally in plant and animal tissues. It is produced by fermentation...

  19. Visceral Leishmaniasis Patients Display Altered Composition and Maturity of Neutrophils as well as Impaired Neutrophil Effector Functions

    PubMed Central

    Yizengaw, Endalew; Getahun, Mulusew; Tajebe, Fitsumbrhan; Cruz Cervera, Edward; Adem, Emebet; Mesfin, Getnet; Hailu, Asrat; Van der Auwera, Gert; Yardley, Vanessa; Lemma, Mulualem; Skhedy, Ziv; Diro, Ermias; Yeshanew, Arega; Melkamu, Roma; Mengesha, Bewketu; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Takele, Yegnasew; Kropf, Pascale

    2016-01-01

    Immunologically, active visceral leishmaniasis (VL) is characterized by profound immunosuppression, severe systemic inflammatory responses, and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication, and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis; however, their role in human VL is poorly understood. In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase, and elastase, all contained in neutrophils’ granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analyzed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species, and phagocytose bacterial particles, but not Leishmania parasites. Our results suggest that impaired effector functions, increased activation, and immaturity of neutrophils play a key role in the pathogenesis of VL. PMID:27965662

  20. 21 CFR 184.1721 - Sodium acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 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...

  1. 21 CFR 184.1721 - Sodium acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 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...

  2. 21 CFR 184.1721 - Sodium acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 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...

  3. 21 CFR 184.1721 - Sodium acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 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...

  4. Immune modulation in the guinea pig using cortisone acetate.

    PubMed

    Scipioni, R L; Baggs, R B; Kraus, A L

    1991-01-01

    Cortisone acetate was administered to a group of guinea pigs (Cavia porcellus) at 0 (control), 20 (low) or 200 (high) mg/kg. Steroid was given daily for two individual 7 day periods, separated by 7 days of no treatment. The effects of this steroid on body weight gain, thymic weight, total and differential leukocyte counts, serum antibody titer against a bacterin, dermal hypersensitivity response to a sensitizing agent and histological evaluation of lymphoid and other tissues were evaluated. Significant differences in body weight gain (p less than 0.05) and thymic weight (p less than .01) were noted. For total leukocyte count, no significant difference among treatment groups at individual time points was noted (p greater than .10), while significant differences were seen in lymphocyte and neutrophil counts (p less than .01). A significant difference in antibody titer among the treatment groups was observed (p less than .01). For the dermal hypersensitivity response, there was no consistent pattern among the treatment groups in gross (macroscopic) skin reactions. Microscopically, differences were seen in the inflammatory response among the treatment groups. Histologically, steroid related changes were seen in thymus, spleen, lymph node and liver. At necropsy, 24 of 40 animals had lesions of focal necrotizing hepatitis. Three affected animals died and remaining animals showed no clinical illness. The cause of the necrotizing hepatitis could not be determined by culture, special stains, electron microscopy, serology or by attempts at transmission with affected liver samples.

  5. Science review: Cell membrane expression (connectivity) regulates neutrophil delivery, function and clearance

    PubMed Central

    Seely, Andrew JE; Pascual, José L; Christou, Nicolas V

    2003-01-01

    As the principal cellular component of the inflammatory host defense and contributor to host injury after severe physiologic insult, the neutrophil is inherently coupled to patient outcome in both health and disease. Extensive research has focused on the mechanisms that regulate neutrophil delivery, function, and clearance from the inflammatory microenvironment. The neutrophil cell membrane mediates the interaction of the neutrophil with the extracellular environment; it expresses a complex array of adhesion molecules and receptors for various ligands, including mediators, cytokines, immunoglobulins, and membrane molecules on other cells. This article presents a review and analysis of the evidence that the neutrophil membrane plays a central role in regulating neutrophil delivery (production, rolling, adhesion, diapedesis, and chemotaxis), function (priming and activation, microbicidal activity, and neutrophil-mediated host injury), and clearance (apoptosis and necrosis). In addition, we review how change in neutrophil membrane expression is synonymous with change in neutrophil function in vivo. Employing a complementary analysis of the neutrophil as a complex system, neutrophil membrane expression may be regarded as a measure of neutrophil connectivity, with altered patterns of connectivity representing functionally distinct neutrophil states. Thus, not only does the neutrophil membrane mediate the processes that characterize the neutrophil lifecycle, but characterization of neutrophil membrane expression represents a technology with which to evaluate neutrophil function. PMID:12930553

  6. Extractive fermentation of acetic acid

    SciTech Connect

    Busche, R.M.

    1991-12-31

    In this technoeconomic evaluation of the manufacture of acetic acid by fermentation, the use of the bacterium: Acetobacter suboxydans from the old vinegar process was compared with expected performance of the newer Clostridium thermoaceticum bacterium. Both systems were projected to operate as immobilized cells in a continuous, fluidized bed bioreactor, using solvent extraction to recover the product. Acetobacter metabolizes ethanol aerobically to produce acid at 100 g/L in a low pH medium. This ensures that the product is in the form of a concentrated extractable free acid, rather than as an unextractable salt. Unfortunately, yields from glucose by way of the ethanol fermentation are poor, but near the biological limits of the organisms involved. Conversely, C. thermoaceticum is a thermophilic anaerobe that operates at high fermentation rates on glucose at neutral pH to produce acetate salts directly in substantially quantitative yields. However, it is severely inhibited by product, which restricts concentration to a dilute 20 g/L. An improved Acetobacter system operating with recycled cells at 50 g/L appears capable of producing acid at $0.38/lb, as compared with a $0.29/lb price for synthetic acid. However, this system has only a limited margin for process improvement. The present Clostridium system cannot compete, since the required selling price would be $0.42/lb. However, if the organism could be adapted to tolerate higher product concentrations at acid pH, selling price could be reduced to $0.22/lb, or about 80% of the price of synthetic acid.

  7. Elevated fecal calprotectin levels during necrotizing enterocolitis are associated with activated neutrophils extruding neutrophil extracellular traps

    PubMed Central

    MacQueen, BC; Christensen, RD; Yost, CC; Lambert, DK; Baer, VL; Sheffield, MJ; Gordon, PV; Cody, MJ; Gerday, E; Schlaberg, R; Lowe, J; Shepherd, JG

    2016-01-01

    BACKGROUND Neonates with necrotizing enterocolitis (NEC) have higher calprotectin levels in stool than do healthy neonates. However, it is not known whether high stool calprotectin at the onset of bowel symptoms identifies neonates who truly have NEC vs. other bowel disorders. STUDY DESIGN Neonates were eligible for this study when an x-ray was ordered to “rule-out NEC”. Stool calprotectin was quantified at that time and in a follow-up stool. Each episode was later categorized as NEC or not NEC. The location of calprotectin in the bowel was determined by immunohistochemistry. RESULTS Neonates with NEC had higher initial and follow-up stool calprotectin levels than did neonates without NEC. Calprotectin in bowel from neonates with NEC was within neutrophil extracellular traps (NETs). CONCLUSION At the onset of signs concerning for NEC, fecal calprotectin is likely to be higher in neonates with NEC. Calprotectin in their stools is exported from neutrophils via NETs. PMID:27388941

  8. A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization

    SciTech Connect

    Stasia, M.J.; Dianoux, A.C.; Vignais, P.V. )

    1989-12-12

    In {sup 32}P{sub i}-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. The apparent molecular mass of the purified protein range between 20 and 23 kDa. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of our discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by ({gamma}-{sup 32}P)ATP in the presence of bovine neutrophil PKC supplemented with Ca{sup 2+}, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. IEF of the {sup 32}P-labeled 23-kDa protein followed by autoradiography revealed for discrete bands with distinct isoelectric points similar to those of the bands stained by Coomassie blue after IEF on nonlabeled 23-kDa protein. The bands of the 23-kDa protein resolved by IEF and transfered to nitrocellulose showed ability to bind ({sup 35}S)GTP-{gamma}-S. The immunoreactivity of antibodies raised in rabbits against the bovine neutrophil 23-kDa protein was demonstrated on immunoblots after SDS-PAGE. The 23-kDa protein differed also from several other proteins of similar molecular mass that have been identified in neutrophils, namely, calmodulin, the small subunit of the low-potential cytochrome b, and a low molecular weight protein which is ADP-ribosylated by the botulinum toxin.

  9. [Neutrophils and monocytes in gingival epithelium

    PubMed

    Meng, H X; Zheng, L P

    1994-06-01

    Neutrophils and monocytes of gingival epithellium in health gingiva(H),marginal gingivitis(MG),juvenile periodontitis(JP),adult periodontitis(AP) and subgingival bacteria were quantitated and analyzed,The results showed that the numbers of PMN within either pocket epithelium or oral gingival epithelium in JP were significantly lower than in AP and G.The amounts of PMN in AP were much larger than other three groups.Positive correlation between the number of PMN in sulcular pocket epitelium and the motile bacteri of subgingival plaque was demonstrated by correlation analysis.Monocytes mainly presented in deep pocket and junctional epithelum which were stained by NAE method,however very few Langhans cells were seen in these areas.

  10. On the maturation rate of the neutrophil.

    PubMed

    Zajicek, G; Shohat, M; Polliack, A

    1984-05-01

    Fifty-three maturing bone marrow cells of the granulocyte cell series stained with Giemsa stain and magnified 1,000 times were scanned by a "computerized microscope" consisting of a LSI-11/23 microprocessor and a black-and-white video camera attached to a "frame grabber ." Each sampled cell was digitized into 70 X 70 pixels, each pixel representing 0.04 micron of the real image. The pixel gray values ranged between 0 and 255. Zero stood for white, 255 represented black, while the numbers in between stood for the various shades of gray. The cells represented six different stages of granulocytic maturation: myeloblast, promyelocyte, myelocyte, metamyelocyte , band form, and polymorphonuclear granulocyte. A discriminant analysis program selected 19 features best distinguishing between the six different cell types and computed five canonical discriminant functions defining a Space in which maturation was studied. In the Space, distance between two cells serves as a measure of similarity. The closer two cells are, the more similar they are and vice versa. This measure was applied here to express the degree of similarity between the neutrophil maturation classes, and since they represent states in the neutrophil life history, it is applicable also as a yardstick for the quantitation of differentiation. In the Space, the life history of a cell is represented by a trajectory originating in the myeloblast and terminating in the granulocyte state. Displacement along the trajectory represents cell maturation that is expressed relatively to the least differentiated state of the myeloblast. The further a cell from this state the more mature it is. The same yardstick also serves for differentiation rate estimates represented in the Space by displacement velocities that are derived from the known "transit times" of a cell in each state. The methodology is also applied for cell production estimates. Unlike other "computerized microscopes" serving for cell classification, the

  11. Chemotactic and Phagocytic Activity of Blood Neutrophils in Allergic Asthma.

    PubMed

    Mosca, Tainá; Menezes, Maria C S; Silva, Ademir Veras; Stirbulov, Roberto; Forte, Wilma C N

    2015-01-01

    Allergic asthma is a chronic inflammatory airway disease, and has been considered a T helper-2-biased response. Studies suggest that neutrophils may be associated with exacerbation and asthma severity. We sought to evaluate the chemotactic activity and phagocytic capacity by peripheral blood neutrophils from individuals with controlled and uncontrolled allergic asthma, and compare the results with non-asthmatic controls groups. Blood neutrophils were isolated from 95 patients: 24 with controlled asthma, 24 uncontrolled asthma, 24 healthy subjects and 23 patients with IgE-mediated allergies other than asthma. The neutrophil chemotaxis, stimulated with LPS, autologous serum or homologous serum, was determined using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by NBT test. The phagocytic digestion phase and chemotaxis by neutrophils from asthmatic patients was higher than in non-asthmatic controls (p  < 0.05). Autologous serum-induced neutrophil chemotaxis in patients with uncontrolled asthma was greater (p  < 0.05) than in other study groups. The ingestion phase of phagocytosis showed similar values in asthmatics and non-asthmatics. We conclude that the blood neutrophil from controlled and uncontrolled asthmatic patients exhibit activation markers, particularly phagocytic digestion and chemotactic activities.

  12. Potentiation and inhibition of migration of human neutrophils by auranofin.

    PubMed Central

    Elferink, J G; de Koster, B M

    1993-01-01

    OBJECTIVES--As auranofin resembles some neutrophil activating sulphur containing compounds, it was decided to investigate whether it had activating effects on neutrophil migration in addition to the published inhibitory effects. METHODS--The Boyden chamber assay was used to determine the migration velocity of human neutrophils. The difference between chemotaxis and chemokinesis was established with a chequerboard assay. RESULTS--Low concentrations of auranofin stimulated human neutrophil migration; concentrations of auranofin higher than 1 mumol/l were inhibitory. Inhibitors of leukotriene formation, or of protein kinase C, had the same effect on auranofin induced potentiation of migration as on fMLP activated migration. Auranofin, at a concentration of 100 nmol/l, caused a transient increase in the cGMP level of neutrophils. The auranofin induced increase in migration was strongly inhibited by methylene blue and by LY83583, two inhibitors of cGMP accumulation. CONCLUSIONS--The auranofin induced enhancement of migration is partly due to a chemokinetic effect, but mainly due to a chemotactic effect. The potentiating effect of auranofin on migration is not specifically due to the ability of the drug to inhibit protein kinase C activity or to generate leukotrienes. These results suggest that the enhancement of neutrophil migration by low levels of auranofin is related to the enhancement of cGMP levels in neutrophils. PMID:8215623

  13. Flow cytometric study of in vitro neutrophil activation by biomaterials.

    PubMed

    Gorbet, M B; Yeo, E L; Sefton, M V

    1999-03-05

    Neutrophil activation for adherent and nonadherent cells, as measured by flow cytometry, was not strongly dependent on material surface chemistry. We had hypothesized that material-induced neutrophil activation was an important parameter associated with material failure. All materials tested [cellophane, an acrylonitrile copolymer (AN69), Pellethane, nylon, polyethylene terephthalate, low density polyethylene, and polydimethylsiloxane] activated isolated human neutrophils, which were resuspended in plasma or serum, to similar extents based on L-selectin shedding, CD11b upregulation, and stimulation of the oxidative burst after 30-min exposure. Inhibition of complement activation by sCR1 unexpectedly had little effect if any on nonadherent neutrophils. However, neutrophil adhesion, but not the level of activation of the adherent cells, was strongly dependent on complement activation. Pretreatment with albumin did not inhibit adhesion or reduce neutrophil activation, but plasma pretreatment resulted in increased activation for nonadherent and adherent cells. More adhesion and a higher level of activation of adherent cells was observed following pretreatment with fibrinogen, a ligand of CD11b. Taken together these results suggest that upon contact with a material, neutrophil activation may occur though mechanisms that are not mediated by complement. For example, the presence of plasma proteins such as fibrinogen at the interface may trigger activation and the release of other activating agents. Although the material differences are small, the extent of activation may be significant and warrant further study of the mechanism and consequences of that activation.

  14. Review of the neutrophil response to Bordetella pertussis infection.

    PubMed

    Eby, Joshua C; Hoffman, Casandra L; Gonyar, Laura A; Hewlett, Erik L

    2015-12-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10-14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28-35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors.

  15. Review of the neutrophil response to Bordetella pertussis infection

    PubMed Central

    Eby, Joshua C.; Hoffman, Casandra L.; Gonyar, Laura A.; Hewlett, Erik L.

    2015-01-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10–14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28–35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors. PMID:26432818

  16. Age-Appropriate Functions and Dysfunctions of the Neonatal Neutrophil

    PubMed Central

    Lawrence, Shelley Melissa; Corriden, Ross; Nizet, Victor

    2017-01-01

    Neonatal and adult neutrophils are distinctly different from one another due to well-defined and documented deficiencies in neonatal cells, including impaired functions, reduced concentrations of microbicidal proteins and enzymes necessary for pathogen destruction, and variances in cell surface receptors. Neutrophil maturation is clearly demonstrated throughout pregnancy from the earliest hematopoietic precursors in the yolk sac to the well-developed myeloid progenitor cells in the bone marrow around the seventh month of gestation. Notable deficiencies of neonatal neutrophils are generally correlated with gestational age and clinical condition, so that the least functional neutrophils are found in the youngest, sickest neonates. Interruption of normal gestation secondary to preterm birth exposes these shortcomings and places the neonate at an exceptionally high rate of infection and sepsis-related mortality. Because the fetus develops in a sterile environment, neonatal adaptive immune responses are deficient from lack of antigen exposure in utero. Newborns must therefore rely on innate immunity to protect against early infection. Neutrophils are a vital component of innate immunity since they are the first cells to respond to and defend against bacterial, viral, and fungal infections. However, notable phenotypic and functional disparities exist between neonatal and adult cells. Below is review of neutrophil ontogeny, as well as a discussion regarding known differences between preterm and term neonatal and adult neutrophils with respect to cell membrane receptors and functions. Our analysis will also explain how these variations decrease with postnatal age. PMID:28293548

  17. Phenol-Soluble Modulin α Peptide Toxins from Aggressive Staphylococcus aureus Induce Rapid Formation of Neutrophil Extracellular Traps through a Reactive Oxygen Species-Independent Pathway

    PubMed Central

    Björnsdottir, Halla; Dahlstrand Rudin, Agnes; Klose, Felix P.; Elmwall, Jonas; Welin, Amanda; Stylianou, Marios; Christenson, Karin; Urban, Constantin F.; Forsman, Huamei; Dahlgren, Claes; Karlsson, Anna; Bylund, Johan

    2017-01-01

    Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence. PMID:28337204

  18. Phenol-Soluble Modulin α Peptide Toxins from Aggressive Staphylococcus aureus Induce Rapid Formation of Neutrophil Extracellular Traps through a Reactive Oxygen Species-Independent Pathway.

    PubMed

    Björnsdottir, Halla; Dahlstrand Rudin, Agnes; Klose, Felix P; Elmwall, Jonas; Welin, Amanda; Stylianou, Marios; Christenson, Karin; Urban, Constantin F; Forsman, Huamei; Dahlgren, Claes; Karlsson, Anna; Bylund, Johan

    2017-01-01

    Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.

  19. Chorionic plate vessels as an origin of amniotic fluid neutrophils.

    PubMed

    Lee, Soong Deok; Kim, Mi Ran; Hwang, Pil Gyu; Shim, Soon-Sup; Yoon, Bo Hyun; Kim, Chong Jai

    2004-07-01

    The present study was conducted to investigate the potential anatomical source of amniotic fluid neutrophils. Microdissection of neutrophils from the chorioamnion of the fetal membranes and the amnion of the chorionic plates of 10 preterm placentas with acute chorioamnionitis was performed and the genotypes of the neutrophils were compared with those of the mother and fetus using polymerase chain reaction of nine autosomal STR loci. In separate analyses, we reviewed eight cases of fetal autopsies with increased amniotic fluid neutrophils for the presence of neutrophils in the alveoli, and also analyzed the relationship between the amniotic fluid white blood cell (WBC) count and the histological pattern of placental inflammation. The genotypes of all of the neutrophils found in the chorioamnion of the fetal membrane matched those of the mother (n = 10). The genotypes of neutrophils found in the chorionic plate were of mixed maternal and fetal origin (n = 4). In the autopsy series of the fetuses with amniotic fluid WBC (n = 8), only five cases showed neutrophils in the alveolar space, while all the placentas had chorioamnionitis. There was no significant difference in amniotic fluid WBC count between the cases with or without acute membranitis, while among the cases with placental inflammation, those with inflammation of the chorionic plate had a significantly higher amniotic fluid WBC count than both the membranitis-only cases (P < 0.001) and the membranitis and funisitis cases (P < 0.05). These results imply that fetal vasculature at the chorionic plate is the main source of amniotic fluid neutrophils, especially in the cases without funisitis.

  20. Evidence for chemokine synergy during neutrophil migration in ARDS

    PubMed Central

    Williams, Andrew E; José, Ricardo J; Mercer, Paul F; Brealey, David; Parekh, Dhruv; Thickett, David R; O'Kane, Cecelia; McAuley, Danny F; Chambers, Rachel C

    2017-01-01

    Background Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterised by pulmonary oedema, respiratory failure and severe inflammation. ARDS is further characterised by the recruitment of neutrophils into the lung interstitium and alveolar space. Objectives The factors that regulate neutrophil infiltration into the inflamed lung and our understanding of the pathomechanisms in ARDS remain incomplete. This study aimed at determining the role of the chemokine (C-C motif) ligand (CCL)2 and CCL7 in ARDS. Methods CCL2 and CCL7 protein levels were measured in bronchoalveolar lavage (BAL) fluid obtained from lipopolysaccharide(LPS)-challenged human volunteers and two separate cohorts of patients with ARDS. Neutrophil chemotaxis to ARDS BAL fluid was evaluated and the contribution of each was assessed and compared with chemokine (C-X-C motif) ligand 8 (CXCL8). Chemokine receptor expression on neutrophils from blood or BAL fluid of patients with ARDS was analysed by flow cytometry. Results CCL2 and CCL7 were significantly elevated in BAL fluid recovered from LPS-challenged volunteers and patients with ARDS. BAL fluid from patients with ARDS was highly chemotactic for human neutrophils and neutralising either CCL2 or CCL7 attenuated the neutrophil chemotactic response. Moreover, CCL2 and CCL7 synergised with CXCL8 to promote neutrophil migration. Furthermore, neutrophils isolated from the blood or BAL fluid differentially regulated the cell surface expression of chemokine (C-X-C motif) receptor 1 and C-C chemokine receptor type 2 during ARDS. Conclusion This study highlights important inflammatory chemokines involved in regulating neutrophil migration, which may have potential value as therapeutic targets for the treatment of ARDS. PMID:27496101

  1. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    NASA Astrophysics Data System (ADS)

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

    2010-05-01

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the KD of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β2-integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

  2. Tetramethylpyrazine inhibits neutrophil activation following permanent cerebral ischemia in rats.

    PubMed

    Chang, Cheng-Yi; Kao, Tsung-Kuei; Chen, Wen-Ying; Ou, Yen-Chuan; Li, Jian-Ri; Liao, Su-Lan; Raung, Shue-Ling; Chen, Chun-Jung

    2015-07-31

    Experimental studies have demonstrated the beneficial effects of tetramethylpyrazine (TMP) against ischemic stroke and highlighted its crucial role in anti-inflammatory activity. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its anti-inflammatory action against ischemic brain injury. We report a global inhibitory effect of TMP on inflammatory cell intracerebral activation and infiltration in a rat model of permanent cerebral ischemia. The results of immunohistochemistry, enzymatic assay, flow cytometric analysis, and cytological analysis revealed that intraperitoneal TMP administration reduced neuronal loss, macrophage/microglia activation, brain parenchyma infiltrative neutrophils, and circulating neutrophils after cerebral ischemia. Biochemical studies of cultured neutrophils further demonstrated that TMP attenuated neutrophil migration, endothelium adhesion, spontaneous nitric oxide (NO) production, and stimuli-activated NO production after cerebral ischemia. In parallel with these anti-neutrophil phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules, including plasma high-mobility group box-1 protein (HMGB1) and neutrophil toll-like receptor-4 (TLR4), Akt, extracellular signal-regulated kinase (ERK), and inducible nitric oxide synthase. Another finding in this study was that the anti-neutrophil effect of TMP was accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in neutrophils after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous anti-inflammatory defense capacity and the attenuation of pro-inflammatory responses via targeting of circulating neutrophils by elevating Nrf2/HO-1 expression and inhibiting HMGB1/TLR4, Akt, and ERK signaling might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

  3. Manufacturing Ethyl Acetate From Fermentation Ethanol

    NASA Technical Reports Server (NTRS)

    Rohatgi, Naresh K.; Ingham, John D.

    1991-01-01

    Conceptual process uses dilute product of fermentation instead of concentrated ethanol. Low-concentration ethanol, extracted by vacuum from fermentation tank, and acetic acid constitutes feedstock for catalytic reaction. Product of reaction goes through steps that increases ethyl acetate content to 93 percent by weight. To conserve energy, heat exchangers recycle waste heat to preheat process streams at various points.

  4. Electron transfer induced fragmentation of acetic acid

    NASA Astrophysics Data System (ADS)

    Ferreira da Silva, F.; Meneses, G.; Almeida, D.; Limão-Vieira, P.

    2014-04-01

    We present negative ion formation driven by electron transfer in atom (K) molecule (acetic acid) collisions. Acetic acid has been found in the interstellar medium, is also considered a biological related compound and as such studying low energy electron interactions will bring new insights as far as induced chemistry is concerned.

  5. CELLULOSE NITRATE-ACETATE MIXED ESTERS

    DTIC Science & Technology

    cellulose acetate . The degree of polymerization of the products, as estimated from viscosity data, shows the occurrence of chain degradation for both...mixed esters showed tensile strength at least comparable to that of films of cellulose nitrate or cellulose acetate . The impact sensitivity of the

  6. Quantifying and localizing actin-free barbed ends in neutrophils.

    PubMed

    Glogauer, Michael

    2007-01-01

    We describe here a permeablization method that retains coupling between N-formylmethionyl-leucyl-phenylalanine (fMLP) receptor stimulation and barbed-end actin nucleation in neutrophils. Using fluorescently-tagged actin monomers, we are able to quantify and localize actin-free barbed ends generated downstream of chemoattractant receptors. Partial permeabilization of the neutrophils with the mild detergent n-octyl-beta-glucopyranoside maintains signaling from membrane receptor to the actin cytoskeleton while allowing for the introduction of inhibitors and activators of signal transduction pathways implicated in regulating actin cytoskeleton dynamics. This is a useful assay for studying signal transduction to the actin cytoskeleton in neutrophils.

  7. Cytokine-induced neutrophil-derived interleukin-8.

    PubMed Central

    Strieter, R. M.; Kasahara, K.; Allen, R. M.; Standiford, T. J.; Rolfe, M. W.; Becker, F. S.; Chensue, S. W.; Kunkel, S. L.

    1992-01-01

    During acute inflammation, the first line of cellular response for host defense is the neutrophil. In addition to the historic role of the neutrophil as a phagocyte, recent studies have identified this cell as an important source of a number of cytokines. In this study, we provide evidence that the neutrophil is a significant source of interleukin-8 (IL-8). Neutrophils freshly isolated from whole blood were not found to constitutively express IL-8 mRNA. In contrast, when these leukocytes were cultured on plastic they were activated, leading to the significant expression of de novo steady-state levels of IL-8 mRNA. In addition, when neutrophils were treated with cycloheximide, there was evidence for "superinduction" of steady-state levels of IL-8 mRNA and inhibition of antigenic IL-8 production. Neutrophils were subsequently stimulated with lipopolysaccharide (LPS), tumor necrosis factor-alpha, or interleukin-1-beta and were found to express IL-8 mRNA and antigen in both a time- and dose-dependent manner. Furthermore, neutrophils stimulated with traditional chemotactic/activating factors, such as the split product of the fifth component of complement (C5a), formylmethionyleucylphenylalanine (fMLP), and leukotriene B4 (LTB4) in a dose-dependent manner did not produce significant antigenic IL-8, as compared with unstimulated controls. In contrast, when neutrophils were exposed to either of these neutrophil agonists in the presence of LPS, the production of antigenic IL-8 was significantly elevated, as compared with either of the stimuli alone, suggesting a synergistic response. These data would suggest that the neutrophil can no longer be viewed as only a phagocyte or warehouse for proteolytic enzymes, but is a pivotal effector cell that is able to respond to mediators in its environment and generate cytokines. This latter neutrophil response may be important for either the elicitation of additional neutrophils or to orchestrate the conventional immune response at

  8. Low molecular weight heparins prevent the induction of autophagy of activated neutrophils and the formation of neutrophil extracellular traps.

    PubMed

    Manfredi, Angelo A; Rovere-Querini, Patrizia; D'Angelo, Armando; Maugeri, Norma

    2017-02-01

    The protection exerted by neutrophils against invading microbes is partially mediated via the generation of neutrophil extracellular traps (NETs). In sterile conditions NETs are damaging species, enriched in autoantigens and endowed with the ability to damage the vessel wall and bystander tissues, to promote thrombogenesis, and to impair wound healing. To identify and reposition agents that can be used to modulate the formation of NETs is a priority in the research agenda. Low molecular weight heparins (LMWH) are currently used, mostly on an empirical basis, in conditions in which NETs play a critical role, such as pregnancy complications associated to autoimmune disease. Here we report that LMWHs induce a profound change in the ability of human neutrophils to generate NETs and to mobilize the content of the primary granules in response to unrelated inflammatory stimuli, such as IL-8, PMA and HMGB1. Autophagy consistently accompanies NET generation in our system and autophagy inhibitors, 3-MA and wortmannin, prevent NET generation. Pretreatment with LMWH in vitro critically jeopardizes neutrophil ability to activate autophagy, a mechanism that might contribute to neutrophil unresponsiveness. Finally, we verified that treatment of healthy volunteers with a single prophylactic dose of parnaparin abrogated the ability of neutrophils to activate autophagy and to generate NETs. Together, these results support the contention that neutrophils, and NET generation in particular, might represent a preferential target of the anti-inflammatory action of LMWH.

  9. Neutrophil surface presentation of the anti-neutrophil cytoplasmic antibody-antigen proteinase 3 depends on N-terminal processing

    PubMed Central

    von Vietinghoff, S; Eulenberg, C; Wellner, M; Luft, F C; Kettritz, R

    2008-01-01

    The neutrophil serine protease proteinase 3 (PR3) is a main autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis. PR3 surface presentation on neutrophilic granulocytes, the main effector cells, is pathogenically important. PR3 is presented by the NB1 (CD177) glycoprotein, but how the presentation develops during neutrophil differentiation is not known. An N-terminally unprocessed PR3 (proPR3) is produced early during neutrophil development and promotes myeloid cell differentiation. We therefore investigated if PR3 presentation depended on NB1 during neutrophil differentiation and if PR3 and proPR3 could both be presented by NB1. In contrast to mature neutrophils, differentiating neutrophils showed an early NB1-independent PR3 surface display that was recognized by only two of four monoclonal anti-PR3 antibodies and occurred in parallel with proPR3, but not PR3 secretion, suggesting that the NB1-independent surface PR3 was proPR3. PR3 gene expression preceeded NB1. When the NB1 receptor was detected on the surface, a mode of PR3 surface display similar to mature neutrophils developed together with the degranulation system. Ectopic expression studies showed that NB1 was a sufficient receptor for PR3 but not proPR3. ProPR3 display on the plasma membrane may influence the bone marrow microenvironment. NB1-mediated PR3 presentation depended on PR3 N-terminal processing implicating the PR3–N-terminus as NB1-binding site. PMID:18462208

  10. Depomedroxyprogesterone acetate for hot flashes.

    PubMed

    Barton, Debra; Loprinzi, Charles; Quella, Susan; Sloan, Jeff; Pruthi, Sandya; Novotny, Paul

    2002-12-01

    To evaluate the efficacy of a long-acting preparation of medroxyprogesterone acetate for hot flash management, 3 men receiving androgen ablation therapy for prostate cancer and 15 women with a history of breast cancer were treated as part of clinical practice with three biweekly intramuscular injections of 500 mg depomedroxyprogesterone. A review of hot flash diaries and patient charts were completed to evaluate the effectiveness and tolerability of these injections for managing hot flashes. Treatment was associated with an approximate 90% decrease in hot flashes (95% CI 82-97%). Daily hot flash frequency decreased from a mean of 10.9 on the first day of treatment (95% CI 8.0-13.8 hot flashes per day) to a mean of 1.1 hot flashes 6 weeks later (95% CI 0.5-1.8 hot flashes) and to a mean of 0.7 hot flashes 12 weeks following therapy initiation (95% CI 0.1-1.2). Improvement in the hot flashes remained for months after discontinuing the injections in many patients. Reported side effects were minimal. This experience suggests that treatment with depomedroxyprogesterone may be an effective and well-tolerated option for the treatment of hot flashes.

  11. Vesicles protect activated acetic acid.

    PubMed

    Todd, Zoe R; House, Christopher H

    2014-10-01

    Abstract Methyl thioacetate, or activated acetic acid, has been proposed to be central to the origin of life and an important energy currency molecule in early cellular evolution. We have investigated the hydrolysis of methyl thioacetate under various conditions. Its uncatalyzed rate of hydrolysis is about 3 orders of magnitude faster (K=0.00663 s(-1); 100°C, pH 7.5, concentration=0.33 mM) than published rates for its catalyzed production, making it unlikely to accumulate under prebiotic conditions. However, our experiments showed that methyl thioacetate was protected from hydrolysis when inside its own hydrophobic droplets. Further, we found that methyl thioacetate protection from hydrolysis was also possible in droplets of hexane and in the membranes of nonanoic acid vesicles. Thus, the hydrophobic regions of prebiotic vesicles and early cell membranes could have offered a refuge for this energetic molecule, increasing its lifetime in close proximity to the reactions for which it would be needed. This model of early energy storage evokes an additional critical function for the earliest cell membranes.

  12. Predominant contribution of syntrophic acetate oxidation to thermophilic methane formation at high acetate concentrations.

    PubMed

    Hao, Li-Ping; Lü, Fan; He, Pin-Jing; Li, Lei; Shao, Li-Ming

    2011-01-15

    To quantify the contribution of syntrophic acetate oxidation to thermophilic anaerobic methanogenesis under the stressed condition induced by acidification, the methanogenic conversion process of 100 mmol/L acetate was monitored simultaneously by using isotopic tracing and selective inhibition techniques, supplemented with the analysis of unculturable microorganisms. Both quantitative methods demonstrated that, in the presence of aceticlastic and hydrogenotrophic methanogens, a large percentage of methane (up to 89%) was initially derived from CO(2) reduction, indicating the predominant contribution of the syntrophic acetate oxidation pathway to acetate degradation at high acid concentrations. A temporal decrease of the fraction of hydrogenotrophic methanogenesis from more than 60% to less than 40% reflected the gradual prevalence of the aceticlastic methanogenesis pathway along with the reduction of acetate. This apparent discrimination of acetate methanization pathways highlighted the importance of the syntrophic acetate-oxidizing bacteria to initialize methanogenesis from high organic loadings.

  13. Neutrophils scan for activated platelets to initiate inflammation

    PubMed Central

    Sreeramkumar, Vinatha; Adrover, José M.; Ballesteros, Ivan; Cuartero, Maria Isabel; Rossaint, Jan; Bilbao, Izaskun; Nácher, Maria; Pitaval, Christophe; Radovanovic, Irena; Fukui, Yoshinori; McEver, Rodger P.; Filippi, Marie-Dominique; Lizasoain, Ignacio; Ruiz-Cabello, Jesús; Zarbock, Alexander; Moro, María A.; Hidalgo, Andrés

    2014-01-01

    Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thrombo-inflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and suggest that their bipolarity allows integration of signals present at both the endothelium and the circulation before inflammation proceeds. PMID:25477463

  14. NETopathies? Unraveling the Dark Side of Old Diseases through Neutrophils.

    PubMed

    Mitsios, Alexandros; Arampatzioglou, Athanasios; Arelaki, Stella; Mitroulis, Ioannis; Ritis, Konstantinos

    2016-01-01

    Neutrophil extracellular traps (NETs) were initially described as an antimicrobial mechanism of neutrophils. Over the last decade, several lines of evidence support the involvement of NETs in a plethora of pathological conditions. Clinical and experimental data indicate that NET release constitutes a shared mechanism, which is involved in a different degree in various manifestations of non-infectious diseases. Even though the backbone of NETs is similar, there are differences in their protein load in different diseases, which represent alterations in neutrophil protein expression in distinct disorder-specific microenvironments. The characterization of NET protein load in different NET-driven disorders could be of significant diagnostic and/or therapeutic value. Additionally, it will provide further evidence for the role of NETs in disease pathogenesis, and it will enable the characterization of disorders in which neutrophils and NET-dependent inflammation are of critical importance.

  15. Neutrophil extracellular traps in dermatology: Caught in the NET.

    PubMed

    Hoffmann, Jochen H O; Enk, Alexander H

    2016-10-01

    Neutrophil, or polymorphonuclear granulocytes (PMN) constitute the most abundant type of leucocytes in peripheral human blood. One of the major advances in the last decade was the discovery of neutrophil extracellular trap (NET) formation: a process by which neutrophils externalize web-like chromatin strands decorated with antimicrobial peptides. These structures were soon implicated in immune defense and auto-immunity alike and now link neutrophils to the pathogenesis of a variety of diseases of dermatological relevance. Currently, NET formation is mainly subdivided into suicidal and vital NETosis. Controversy exists regarding the capacity of NETs to kill pathogens, and little is known about the way NETs are formed in vivo. Here, we discuss the current terminology, methods for NET quantification, pathways leading to NET formation, and the role of NETs in systemic and cutaneous immune defense and auto-immunity, with a focus on psoriasis and systemic lupus erythematosus.

  16. NETopathies? Unraveling the Dark Side of Old Diseases through Neutrophils

    PubMed Central

    Mitsios, Alexandros; Arampatzioglou, Athanasios; Arelaki, Stella; Mitroulis, Ioannis; Ritis, Konstantinos

    2017-01-01

    Neutrophil extracellular traps (NETs) were initially described as an antimicrobial mechanism of neutrophils. Over the last decade, several lines of evidence support the involvement of NETs in a plethora of pathological conditions. Clinical and experimental data indicate that NET release constitutes a shared mechanism, which is involved in a different degree in various manifestations of non-infectious diseases. Even though the backbone of NETs is similar, there are differences in their protein load in different diseases, which represent alterations in neutrophil protein expression in distinct disorder-specific microenvironments. The characterization of NET protein load in different NET-driven disorders could be of significant diagnostic and/or therapeutic value. Additionally, it will provide further evidence for the role of NETs in disease pathogenesis, and it will enable the characterization of disorders in which neutrophils and NET-dependent inflammation are of critical importance. PMID:28123386

  17. Fatty acids as modulators of neutrophil recruitment, function and survival.

    PubMed

    Rodrigues, Hosana G; Takeo Sato, Fabio; Curi, Rui; Vinolo, Marco A R

    2016-08-15

    Neutrophils are well-known to act in the destruction of invading microorganisms. They have also been implicated in the activation of other immune cells including B- and T-lymphocytes and in the resolution of inflammation and tissue regeneration. Neutrophils are produced in the bone marrow and released into the circulation from where they migrate to tissues to perform their effector functions. Neutrophils are in constant contact with fatty acids that can modulate their function, activation and fate (survival or cell death) through different mechanisms. In this review, the effects of fatty acids pertaining to five classes, namely, long-chain saturated fatty acids (LCSFAs), short-chain fatty acids (SCFAs), and omega-3 (n-3), omega-6 (n-6) and omega-9 (n-9) unsaturated fatty acids, on neutrophils and the relevance of these effects for disease development are discussed.

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

    PubMed

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

    2001-08-01

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

  19. Free p-Cresol Alters Neutrophil Function in Dogs.

    PubMed

    Bosco, Anelise Maria; Pereira, Priscila Preve; Almeida, Breno Fernando Martins; Narciso, Luis Gustavo; Dos Santos, Diego Borba; Santos-Neto, Álvaro José Dos; Ferreira, Wagner Luis; Ciarlini, Paulo César

    2016-05-01

    To achieve a clearer understanding of the mechanisms responsible for neutrophil dysfunction recently described in dogs with chronic renal failure (CRF), the plasma concentrations of free p-cresol in healthy dogs (n = 20) and those with CRF (n = 20) were compared. The degree of correlation was determined between plasma levels of p-cresol and markers of oxidative stress and function of neutrophils in these dogs. The effect of this compound on oxidative metabolism and apoptosis was assessed in neutrophils isolated from 16 healthy dogs incubated in RPMI 1640 supplemented with p-cresol (0.405 mg/L) and compared with medium supplemented with uremic plasma (50%). To achieve this, the plasma concentration of p-cresol was quantified by liquid phase high-performance liquid chromatography. The neutrophil oxidative metabolism was determined using the probes hydroethidine and 2',7'-dichlorofluorescein diacetate and apoptosis was measured using Annexin V-PE by capillary flow cytometry. Compared with the healthy dogs, uremic dogs presented higher concentrations of free p-cresol, greater oxidative stress, and neutrophils primed for accelerated apoptosis. The free p-cresol induced in neutrophils from healthy dogs increased apoptosis and decreased reactive oxygen species production. We conclude that the health status presented during uremia concomitant with the increase in plasma free p-cresol can contribute to the presence of immunosuppression in dogs with CRF.

  20. Wolbachia endosymbionts induce neutrophil extracellular trap formation in human onchocerciasis

    PubMed Central

    Tamarozzi, Francesca; Turner, Joseph D.; Pionnier, Nicolas; Midgley, Angela; Guimaraes, Ana F.; Johnston, Kelly L.; Edwards, Steven W.; Taylor, Mark J.

    2016-01-01

    The endosymbiotic bacteria, Wolbachia, induce neutrophilic responses to the human helminth pathogen Onchocerca volvulus. The formation of Neutrophil Extracellular Traps (NETs), has been implicated in anti-microbial defence, but has not been identified in human helminth infection. Here, we demonstrate NETs formation in human onchocerciasis. Extracellular NETs and neutrophils were visualised around O. volvulus in nodules excised from untreated patients but not in nodules from patients treated with the anti-Wolbachia drug, doxycycline. Whole Wolbachia or microspheres coated with a synthetic Wolbachia lipopeptide (WoLP) of the major nematode Wolbachia TLR2/6 ligand, peptidoglycan associated lipoprotein, induced NETosis in human neutrophils in vitro. TLR6 dependency of Wolbachia and WoLP NETosis was demonstrated using purified neutrophils from TLR6 deficient mice. Thus, we demonstrate for the first time that NETosis occurs during natural human helminth infection and demonstrate a mechanism of NETosis induction via Wolbachia endobacteria and direct ligation of Wolbachia lipoprotein by neutrophil TLR2/6. PMID:27752109

  1. Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment

    PubMed Central

    Kim, Jaehong; Bae, Jong-Sup

    2016-01-01

    Distinct tumor microenvironment forms in each progression step of cancer and has diverse capacities to induce both adverse and beneficial consequences for tumorigenesis. It is now known that immune cells can be activated to favor tumor growth and progression, most probably influenced by the tumor microenvironment. Tumor-associated macrophages and tumor-associated neutrophils can exert protumoral functions, enhancing tumor cell invasion and metastasis, angiogenesis, and extracellular matrix remodeling, while inhibiting the antitumoral immune surveillance. Considering that neutrophils in inflammatory environments recruit macrophages and that recruited macrophages affect neutrophil functions, there may be various degrees of interaction between tumor-associated macrophages and tumor-associated neutrophils. Platelets also play an important role in the recruitment and regulation of monocytic and granulocytic cells in the tumor tissues, suggesting that platelet function may be essential for generation of tumor-associated macrophages and tumor-associated neutrophils. In this review, we will explore the biology of tumor-associated macrophages and tumor-associated neutrophils and their possible interactions in the tumor microenvironment. Special attention will be given to the recruitment and activation of these tumor-associated cells and to the roles they play in maintenance of the tumor microenvironment and progression of tumors. PMID:26966341

  2. Nitric oxide regulates neutrophil migration through microparticle formation.

    PubMed

    Nolan, Sarah; Dixon, Rachel; Norman, Keith; Hellewell, Paul; Ridger, Victoria

    2008-01-01

    The role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation.

  3. Blood baseline neutrophil count predicts bevacizumab efficacy in glioblastoma

    PubMed Central

    Bertaut, Aurélie; Truntzer, Caroline; Madkouri, Rachid; Kaderbhai, Coureche Guillaume; Derangère, Valentin; Vincent, Julie; Chauffert, Bruno; Aubriot-Lorton, Marie Hélene; Farah, Wahlid; Mourier, Klaus Luc; Boidot, Romain; Ghiringhelli, Francois

    2016-01-01

    Bevacizumab is used to treat glioblastoma; however, no current biomarker predicts its efficacy. We used an exploratory cohort of patients treated with the radiochemotherapy then bevacizumab or chemotherapy at recurrence (N = 265). Bevacizumab use increased median overall survival (OS) 18.7 vs 11.3 months, p = 0.0014). In multivariate analysis, age, initial surgery, neutrophil count, Karnofsky status >70% and bevacizumab administration were independent prognostic factors of survival. We found an interaction between bevacizumab use and baseline neutrophil count. The cut-off value for the neutrophil count was set at 6000/mm3. Only patients with a high neutrophil count benefited from the bevacizumab treatment (17.3 vs 8.8 months p < 0.0001). We validated this result using data from the TEMAVIR trial, which tested the efficacy of neoadjuvant bevacizumab plus irinotecan versus radiochemotherapy in the first-line treatment of glioblastoma. Transcriptomic data from TCGA underlined that CSF3 expression, the gene encoding G-CSF, the growth factor for neutrophils, correlated with VEGF-A-dependent angiogenesis. In another independent cohort (BELOB trial), which compared lomustine versus lomustine plus bevacizumab at recurrence, bevacizumab only benefited patients with high CSF3 expression in the tumor. These data suggest that only patients with a high peripheral neutrophil count before bevacizumab treatment benefited from this therapy. PMID:27487142

  4. Influence of suspension on the oxidative burst by rat neutrophils

    NASA Technical Reports Server (NTRS)

    Miller, E. S.; Koebel, D. A.; Davis, S. A.; Klein, J. B.; McLeish, K. R.; Goldwater, D.; Sonnenfeld, G.

    1994-01-01

    The influence of spaceflight on the oxidative burst of neutrophils is not known. The present study was designed to evaluate the influence of antiorthostatic suspension, a ground-based modeling system designed to simulate certain aspects of weightlessness that occur after spaceflight, on the capacity of rat neutrophils to express the oxidative burst, an important host defense mechanism against microbial pathogens. Rats were suspended in whole body harnesses in the antiorthostatic orientation for a 3- or 7-day period. Control rats were suspended orthostatically or allowed to remain in vivarium cages without the attachment of any suspension materials. After suspension, peripheral blood was harvested and neutrophils were isolated by density gradient centrifugation. The enriched neutrophil preparations were stimulated with N-formyl-methionyl-leucine-phenylalanine and phorbol myristic acid to induce the oxidative burst. It was found that neutrophils isolated from suspended animals released the same levels of superoxide anion as did vivarium control animals that were not suspended, indicating that whole body suspension did not alter this aspect of rat neutrophil function.

  5. Granule Protein Processing and Regulated Secretion in Neutrophils

    PubMed Central

    Sheshachalam, Avinash; Srivastava, Nutan; Mitchell, Troy; Lacy, Paige; Eitzen, Gary

    2014-01-01

    Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines, and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First, we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking, and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection. PMID:25285096

  6. Genomic modulators of gene expression in human neutrophils

    PubMed Central

    Naranbhai, Vivek; Fairfax, Benjamin P.; Makino, Seiko; Humburg, Peter; Wong, Daniel; Ng, Esther; Hill, Adrian V. S.; Knight, Julian C.

    2015-01-01

    Neutrophils form the most abundant leukocyte subset and are central to many disease processes. Technical challenges in transcriptomic profiling have prohibited genomic approaches to date. Here we map expression quantitative trait loci (eQTL) in peripheral blood CD16+ neutrophils from 101 healthy European adults. We identify cis-eQTL for 3281 neutrophil-expressed genes including many implicated in neutrophil function, with 450 of these not previously observed in myeloid or lymphoid cells. Paired comparison with monocyte eQTL demonstrates nuanced conditioning of genetic regulation of gene expression by cellular context, which relates to cell-type-specific DNA methylation and histone modifications. Neutrophil eQTL are markedly enriched for trait-associated variants particularly autoimmune, allergy and infectious disease. We further demonstrate how eQTL in PADI4 and NOD2 delineate risk variant function in rheumatoid arthritis, leprosy and Crohn's disease. Taken together, these data help advance understanding of the genetics of gene expression, neutrophil biology and immune-related diseases. PMID:26151758

  7. Effect of Prototheca zopfii on neutrophil function from bovine milk.

    PubMed

    Cunha, Luciane T; Pugine, Silvana P; Valle, Claudia R; Ribeiro, Andrea R; Costa, Ernane J X; De Melo, Mariza P

    2006-12-01

    This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H(2)O(2)) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative "California Mastitis Test" (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H(2)O(2) production was measured using the phenol red method. Catalase activity was measured following H(2)O(2) reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey's test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H(2)O(2) production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.

  8. Roles of lung epithelium in neutrophil recruitment during pneumococcal pneumonia.

    PubMed

    Yamamoto, Kazuko; Ahyi, Ayele-Nati N; Pepper-Cunningham, Zachary A; Ferrari, Joseph D; Wilson, Andrew A; Jones, Matthew R; Quinton, Lee J; Mizgerd, Joseph P

    2014-02-01

    Epithelial cells line the respiratory tract and interface with the external world. Epithelial cells contribute to pulmonary inflammation, but specific epithelial roles have proven difficult to define. To discover unique epithelial activities that influence immunity during infection, we generated mice with nuclear factor-κB RelA mutated throughout all epithelial cells of the lung and coupled this approach with epithelial cell isolation from infected and uninfected lungs for cell-specific analyses of gene induction. The RelA mutant mice appeared normal basally, but in response to pneumococcus in the lungs they were unable to rapidly recruit neutrophils to the air spaces. Epithelial cells expressed multiple neutrophil-stimulating cytokines during pneumonia, all of which depended on RelA. Cytokine expression by nonepithelial cells was unaltered by the epithelial mutation of RelA. Epithelial cells were the predominant sources of CXCL5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas nonepithelial cells were major sources for other neutrophil-activating cytokines. Epithelial RelA mutation decreased whole lung levels of CXCL5 and GM-CSF during pneumococcal pneumonia, whereas lung levels of other neutrophil-recruiting factors were unaffected. Defective neutrophil recruitment in epithelial mutant mice could be rescued by administration of CXCL5 or GM-CSF. These results reveal a specialized immune function for the pulmonary epithelium, the induction of CXCL5 and GM-CSF, to accelerate neutrophil recruitment in the infected lung.

  9. The Dual Role of Neutrophils in Inflammatory Bowel Diseases

    PubMed Central

    Wéra, Odile; Lancellotti, Patrizio; Oury, Cécile

    2016-01-01

    Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, are characterised by aberrant immunological responses leading to chronic inflammation without tissue regeneration. These two diseases are considered distinct entities, and there is some evidence that neutrophil behaviour, above all other aspects of immunity, clearly separate them. Neutrophils are the first immune cells recruited to the site of inflammation, and their action is crucial to limit invasion by microorganisms. Furthermore, they play an essential role in proper resolution of inflammation. When these processes are not tightly regulated, they can trigger positive feedback amplification loops that promote neutrophil activation, leading to significant tissue damage and evolution toward chronic disease. Defective chemotaxis, as observed in Crohn’s disease, can also contribute to the disease through impaired microbe elimination. In addition, through NET production, neutrophils may be involved in thrombo-embolic events frequently observed in IBD patients. While the role of neutrophils has been studied in different animal models of IBD for many years, their contribution to the pathogenesis of IBD remains poorly understood, and no molecules targeting neutrophils are used and validated for the treatment of these pathologies. Therefore, it is crucial to improve our understanding of their mode of action in these particular conditions in order to provide new therapeutic avenues for IBD. PMID:27999328

  10. Marathon Race Affects Neutrophil Surface Molecules: Role of Inflammatory Mediators.

    PubMed

    Santos, Vinicius Coneglian; Sierra, Ana Paula Renno; Oliveira, Rodrigo; Caçula, Kim Guimarães; Momesso, César Miguel; Sato, Fabio Takeo; Silva, Maysa Braga Barros; Oliveira, Heloisa Helena; Passos, Maria Elizabeth Pereira; de Souza, Diego Ribeiro; Gondim, Olivia Santos; Benetti, Marino; Levada-Pires, Adriana Cristina; Ghorayeb, Nabil; Kiss, Maria Augusta Peduti Dal Molin; Gorjão, Renata; Pithon-Curi, Tânia Cristina; Cury-Boaventura, Maria Fernanda

    2016-01-01

    The fatigue induced by marathon races was observed in terms of inflammatory and immunological outcomes. Neutrophil survival and activation are essential for inflammation resolution and contributes directly to the pathogenesis of many infectious and inflammatory conditions. The aim of this study was to investigate the effect of marathon races on surface molecules related to neutrophil adhesion and extrinsic apoptosis pathway and its association with inflammatory markers. We evaluated 23 trained male runners at the São Paulo International Marathon 2013. The following components were measured: hematological and inflammatory mediators, muscle damage markers, and neutrophil function. The marathon race induced an increased leukocyte and neutrophil counts; creatine kinase (CK), lactate dehydrogenase (LDH), CK-MB, interleukin (IL)-6, IL-10, and IL-8 levels. C-reactive protein (CRP), IL-12, and tumor necrosis factor (TNF)-α plasma concentrations were significantly higher 24 h and 72 h after the marathon race. Hemoglobin and hematocrit levels decreased 72 h after the marathon race. We also observed an increased intercellular adhesion molecule-1 (ICAM-1) expression and decreasedTNF receptor-1 (TNFR1) expression immediately after and 24 h after the marathon race. We observed an increased DNA fragmentation and L-selectin and Fas receptor expressions in the recovery period, indicating a possible slow rolling phase and delayed neutrophil activation and apoptosis. Marathon racing affects neutrophils adhesion and survival in the course of inflammation, supporting the "open-window" post-exercise hypothesis.

  11. Modeling the Mechanosensitivity of Neutrophils Passing through a Narrow Channel.

    PubMed

    Wu, Tenghu; Feng, James J

    2015-12-01

    Recent experiments have found that neutrophils may be activated after passing through microfluidic channels and filters. Mechanical deformation causes disassembly of the cytoskeleton and a sudden drop of the elastic modulus of the neutrophil. This fluidization is followed by either activation of the neutrophil with protrusion of pseudopods or a uniform recovery of the cytoskeleton network with no pseudopod. The former occurs if the neutrophil traverses the narrow channel at a slower rate. We propose a chemo-mechanical model for the fluidization and activation processes. Fluidization is treated as mechanical destruction of the cytoskeleton by sufficiently rapid bending. Loss of the cytoskeleton removes a pathway by which cortical tension inhibits the Rac protein. As a result, Rac rises and polarizes through a wave-pinning mechanism if the chemical reaction rate is fast enough. This leads to recovery and reinforcement of the cytoskeleton at the front of the neutrophil, and hence protrusion and activation. Otherwise the Rac signal returns to a uniform pre-deformation state and no activation occurs. Thus, mechanically induced neutrophil activation is understood as the competition between two timescales: that of chemical reaction and that of mechanical deformation. The model captures the main features of the experimental observation.

  12. Marathon Race Affects Neutrophil Surface Molecules: Role of Inflammatory Mediators

    PubMed Central

    2016-01-01

    The fatigue induced by marathon races was observed in terms of inflammatory and immunological outcomes. Neutrophil survival and activation are essential for inflammation resolution and contributes directly to the pathogenesis of many infectious and inflammatory conditions. The aim of this study was to investigate the effect of marathon races on surface molecules related to neutrophil adhesion and extrinsic apoptosis pathway and its association with inflammatory markers. We evaluated 23 trained male runners at the São Paulo International Marathon 2013. The following components were measured: hematological and inflammatory mediators, muscle damage markers, and neutrophil function. The marathon race induced an increased leukocyte and neutrophil counts; creatine kinase (CK), lactate dehydrogenase (LDH), CK-MB, interleukin (IL)-6, IL-10, and IL-8 levels. C-reactive protein (CRP), IL-12, and tumor necrosis factor (TNF)-α plasma concentrations were significantly higher 24 h and 72 h after the marathon race. Hemoglobin and hematocrit levels decreased 72 h after the marathon race. We also observed an increased intercellular adhesion molecule-1 (ICAM-1) expression and decreasedTNF receptor-1 (TNFR1) expression immediately after and 24 h after the marathon race. We observed an increased DNA fragmentation and L-selectin and Fas receptor expressions in the recovery period, indicating a possible slow rolling phase and delayed neutrophil activation and apoptosis. Marathon racing affects neutrophils adhesion and survival in the course of inflammation, supporting the “open-window” post-exercise hypothesis. PMID:27911915

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

    PubMed Central

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

    2016-01-01

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

  14. The interaction of Acanthamoeba castellanii cysts with macrophages and neutrophils.

    PubMed

    Hurt, Michael; Proy, Vincent; Niederkorn, Jerry Y; Alizadeh, Hassan

    2003-06-01

    Acanthamoeba castellanii, a free-living amoeba, causes a sight-threatening form of keratitis. Even after extensive therapies, corneal damage can be severe, often requiring corneal transplantation to restore vision. However, A. castellanii cysts are not eliminated from the conjunctiva and stroma of humans and can excyst, resulting in infection of the corneal transplant. The aim of this study was to determine whether elements of the innate immune apparatus, neutrophils and macrophages, were capable of detecting and eliminating A. castellanii cysts and to examine the mechanism by which they kill the cysts. Results show that neither innate immune cell is attracted chemotactically to intact cysts, yet both were attracted to lysed cysts. Both macrophages and neutrophils were capable of killing significant numbers of cysts, yet neutrophils were 3-fold more efficient than macrophages. Activation of macrophages with lipopolysaccharide and interferon-gamma did not increase their cytolytic ability. Conditioned medium isolated from macrophages did not lyse the cysts; however, prevention of phagocytosis by cytochalasin D inhibited 100% of macrophage-mediated killing of the cysts. Conditioned medium from neutrophils did kill significant numbers of the cysts, and this killing was blocked by quercetin, a potent inhibitor of myeloperoxidase (MPO). These results indicate that neither macrophages nor neutrophils are chemoattracted to intact cysts, yet both are capable of killing the cysts. Macrophages killed the cysts by phagocytosis, whereas neutrophils killed cysts through the secretion of MPO.

  15. Conversion to eslicarbazepine acetate monotherapy

    PubMed Central

    French, Jacqueline; Jacobson, Mercedes P.; Pazdera, Ladislav; Gough, Mallory; Cheng, Hailong; Grinnell, Todd; Blum, David

    2016-01-01

    Objective: To assess the efficacy and safety of eslicarbazepine acetate (ESL) monotherapy. Methods: This post hoc pooled analysis of 2 randomized double-blind studies (093-045 and -046) included adults with partial-onset seizures medically uncontrolled by 1 or 2 antiepileptic drugs (AEDs). Following the baseline period (8 weeks), eligible patients were randomized 2:1 to receive ESL 1,600 mg or 1,200 mg once daily for 18 weeks; the primary endpoint was study exit by meeting predefined exit criteria (signifying worsening seizure control). In each study, treatment was considered effective if the upper 95% confidence limit for exit rate was lower than the historical control threshold (65.3%). Results: Pooled exit rates were as follows: ESL 1,600 mg = 20.6% (95% confidence interval: 15.6%–26.8%); ESL 1,200 mg = 30.8% (23.0%–40.5%). Use of 2 baseline AEDs or rescue medication, US location, epilepsy duration ≥20 years, and higher maximum baseline seizure frequency were associated with higher exit risks. Median percent reductions in standardized seizure frequency between baseline and the 18-week double-blind period were as follows: ESL 1,600 mg = 43.2%; ESL 1,200 mg = 35.7%; baseline carbamazepine use was associated with smaller reductions. Safety profiles were similar between ESL doses. Conclusions: Exit rates for ESL monotherapy (1,600 mg and 1,200 mg once daily) were lower than the historical control threshold, irrespective of baseline AED use and region, with no additional safety concerns identified. Clinical factors and location clearly influence treatment responses in conversion-to-monotherapy trials. Classification of evidence: This pooled analysis provides Class IV evidence that for adults with medically uncontrolled partial-onset seizures, ESL monotherapy is well tolerated and effective. PMID:26911639

  16. Dictyostelium amoebae and neutrophils can swim.

    PubMed

    Barry, Nicholas P; Bretscher, Mark S

    2010-06-22

    Animal cells migrating over a substratum crawl in amoeboid fashion; how the force against the substratum is achieved remains uncertain. We find that amoebae and neutrophils, cells traditionally used to study cell migration on a solid surface, move toward a chemotactic source while suspended in solution. They can swim and do so with speeds similar to those on a solid substrate. Based on the surprisingly rapidly changing shape of amoebae as they swim and earlier theoretical schemes for how suspended microorganisms can migrate (Purcell EM (1977) Life at low Reynolds number. Am J Phys 45:3-11), we suggest the general features these cells use to gain traction with the medium. This motion requires either the movement of the cell's surface from the cell's front toward its rear or protrusions that move down the length of the elongated cell. Our results indicate that a solid substratum is not a prerequisite for these cells to produce a forward thrust during movement and suggest that crawling and swimming are similar processes, a comparison we think is helpful in understanding how cells migrate.

  17. Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils

    PubMed Central

    Fridlender, Zvi G.; Sun, Jing; Mishalian, Inbal; Singhal, Sunil; Cheng, Guanjun; Kapoor, Veena; Horng, Wenhwai; Fridlender, Gil; Bayuh, Rachel; Worthen, G. Scott; Albelda, Steven M.

    2012-01-01

    The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations. PMID:22348096

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

    PubMed

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

    2016-04-15

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

  19. Passive mechanical behavior of human neutrophils: power-law fluid.

    PubMed Central

    Tsai, M A; Frank, R S; Waugh, R E

    1993-01-01

    The mechanical behavior of the neutrophil plays an important role in both the microcirculation and the immune system. Several laboratories in the past have developed mechanical models to describe different aspects of neutrophil deformability. In this study, the passive mechanical properties of normal human neutrophils have been further characterized. The cellular mechanical properties were assessed by single cell micropipette aspiration at fixed aspiration pressures. A numerical simulation was developed to interpret the experiments in terms of cell mechanical properties based on the Newtonian liquid drop model (Yeung and Evans, Biophys. J., 56: 139-149, 1989). The cytoplasmic viscosity was determined as a function of the ratio of the initial cell size to the pipette radius, the cortical tension, aspiration pressure, and the whole cell aspiration time. The cortical tension of passive neutrophils was measured to be about 2.7 x 10(-5) N/m. The apparent viscosity of neutrophil cytoplasm was found to depend on aspiration pressure, and ranged from approximately 500 Pa.s at an aspiration pressure of 98 Pa (1.0 cm H2O) to approximately 50 Pa.s at 882 Pa (9.0 cm H2O) when tested with a 4.0-micron pipette. These data provide the first documentation that the neutrophil cytoplasm exhibits non-Newtonian behavior. To further characterize the non-Newtonian behavior of human neutrophils, a mean shear rate gamma m was estimated based on the numerical simulation. The apparent cytoplasmic viscosity appears to decrease as the mean shear rate increases. The dependence of cytoplasmic viscosity on the mean shear rate can be approximated as a power-law relationship described by mu = mu c(gamma m/gamma c)-b, where mu is the cytoplasmic viscosity, gamma m is the mean shear rate, mu c is the characteristic viscosity at characteristic shear rate gamma c, and b is a material coefficient. When gamma c was set to 1 s-1, the material coefficients for passive neutrophils were determined to be mu c

  20. Fragrance material review on 4-methylbenzyl acetate.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2012-09-01

    A toxicologic and dermatologic review of 4-methylbenzyl acetate when used as a fragrance ingredient is presented. 4-Methylbenzyl acetate is a member of the fragrance structural group Aryl Alkyl Alcohol Simple Acid Esters (AAASAE). The AAASAE fragrance ingredients are prepared by reacting an aryl alkyl alcohol with a simple carboxylic acid (a chain of 1-4 carbons) to generate formate, acetate, propionate, butyrate, isobutyrate and carbonate esters. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for 4-methylbenzyl acetate were evaluated, then summarized, and includes: physical properties, skin irritation, skin sensitization, and elicitation data. A safety assessment of the entire AAASAE will be published simultaneously with this document. Please refer to Belsito et al. (2012) for an overall assessment of the safe use of this material and all AAASAE in fragrances.

  1. [Experimental study of proflavine acetate phototransformation processes].

    PubMed

    Zholdakova, Z I; Sinitsyna, O O; Lebedev, A T; Kharchevnikova, N V

    2009-01-01

    Changes in proflavine acetate phototransformation processes upon exposure to visible-range irradiation were studied by high performance liquid chromatography. Proflavine acetate was offered as a photosensitizer during photodynamic water disinfection. Dye transformation products upon time-varying exposure to irradiation were identified. By using structure-activity relationships and information from toxicity databases, the authors evaluated the hazard of the identified products and identified the most hazardous ones.

  2. Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin

    NASA Technical Reports Server (NTRS)

    Konstantopoulos, K.; Neelamegham, S.; Burns, A. R.; Hentzen, E.; Kansas, G. S.; Snapp, K. R.; Berg, E. L.; Hellums, J. D.; Smith, C. W.; McIntire, L. V.; Simon, S. I.

    1998-01-01

    BACKGROUND: After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS: Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS: The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

  3. A novel immunomodulatory function of neutrophils on rhinovirus-activated monocytes in vitro

    PubMed Central

    Tang, Francesca S M; Hansbro, Philip M; Burgess, Janette K; Ammit, Alaina J; Baines, Katherine J; Oliver, Brian G

    2016-01-01

    Background Rhinovirus (RV) infections are the major precipitant of asthma exacerbations. While neutrophilic lung inflammation occurs during such infections, its role remains unclear. Neutrophilic inflammation is associated with increased asthma severity and steroid refractory disease. Neutrophils are vital for controlling infections but also have immunomodulatory functions. Previously, we found that neutrophils respond to viral mimetics but not replication competent RV. We aimed to investigate if neutrophils are activated and/or modulate immune responses of monocytes during RV16 infection. Methods Primary human monocytes and autologous neutrophils were cocultured with or without RV16, in direct contact or separated by transwells. RV16-stimulated monocytes were also exposed to lysed neutrophils, neutrophil membrane components or soluble neutrophil intracellular components. Interleukin 6 (IL-6) and C-X-C motif (CXC)L8 mRNA and proteins were measured by quantitative PCR and ELISA at 24 hours. Results RV16 induced IL-6 and CXCL8 in monocytes, but not neutrophils. RV16-induced IL-6 and CXCL8 from monocytes was reduced in the presence of live neutrophils. Transwell separation abolished the inhibitory effects. Lysed neutrophils inhibited RV16-induced IL-6 and CXCL8 from monocytes. Neutrophil intracellular components alone effectively inhibited RV16-induced monocyte-derived IL-6 and CXCL8. Neutrophil intracellular components reduced RV16-induced IL-6 and CXCL8 mRNA in monocytes. Conclusions Cell contact between monocytes and neutrophils is required, and preformed neutrophil mediator(s) are likely to be involved in the suppression of cytokine mRNA and protein production. This study demonstrates a novel regulatory function of neutrophils on RV-activated monocytes in vitro, challenging the paradigm that neutrophils are predominantly proinflammatory. PMID:27287090

  4. Motility and Adhesiveness in Human Neutrophils

    PubMed Central

    Smith, C. Wayne; Hollers, James C.; Patrick, Richard A.; Hassett, Clare

    1979-01-01

    Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness. Images PMID:372238

  5. Attachment and ingestion of gonococci human neutrophils.

    PubMed

    Dilworth, J A; Hendley, J O; Mandell, G L

    1975-03-01

    Previous studies have indirectly shown that type 1 gonococci are more resistant to phagocytosis by human neutrophils (PMN) than type 3 gonococci. Using phase contrast, fluorescent, and light microscopy, we directly quantitated PMN-gonococcal interaction, with emphasis on separating ingestion from attachment. PMN monolayers were incubated on slides with type 1 or type 3 gonococcal fluorescent antibody (FA). After methanol fixation, the FA-stained gonococci associated with PMN were cointed. Since the live PMN excludes FA, the FA-stained gonococci represent only extracellular gonococci. Methylene blue was then added to the smae slide to stain both ingested and surface attached gonococci. Using these methods, intracellular and extracellular cell-associated gonococci were quantitated under varying conditions. The numbers of methylene blue-stained cell-associated gonococci that were ingested were: with normal serum, 3.7 plus or minus 4.1 per cent for type 1 and 56.2 plus or minus 3.7 percent for type 3 (P smaller than 0.001); with heat-inactivated serum, 1.0 plus or minus 3.0 per cent for type 1 and 52.6 plus or minus 3.7 per cent for type 3 (P smaller than 0.001); with higher-titer anti-gonococcal antibody serum, 4.8 plus or minus 4.3 percent for type 1 and 64.0 plus or minus 1.6 per cent for type 3 (P smaller than 0.001). Thus, most type 3 organisms were ingested, but most type 1 gonococci were bound on the PMN surface.

  6. Attachment and ingestion of gonococci human neutrophils.

    PubMed Central

    Dilworth, J A; Hendley, J O; Mandell, G L

    1975-01-01

    Previous studies have indirectly shown that type 1 gonococci are more resistant to phagocytosis by human neutrophils (PMN) than type 3 gonococci. Using phase contrast, fluorescent, and light microscopy, we directly quantitated PMN-gonococcal interaction, with emphasis on separating ingestion from attachment. PMN monolayers were incubated on slides with type 1 or type 3 gonococcal fluorescent antibody (FA). After methanol fixation, the FA-stained gonococci associated with PMN were cointed. Since the live PMN excludes FA, the FA-stained gonococci represent only extracellular gonococci. Methylene blue was then added to the smae slide to stain both ingested and surface attached gonococci. Using these methods, intracellular and extracellular cell-associated gonococci were quantitated under varying conditions. The numbers of methylene blue-stained cell-associated gonococci that were ingested were: with normal serum, 3.7 plus or minus 4.1 per cent for type 1 and 56.2 plus or minus 3.7 percent for type 3 (P smaller than 0.001); with heat-inactivated serum, 1.0 plus or minus 3.0 per cent for type 1 and 52.6 plus or minus 3.7 per cent for type 3 (P smaller than 0.001); with higher-titer anti-gonococcal antibody serum, 4.8 plus or minus 4.3 percent for type 1 and 64.0 plus or minus 1.6 per cent for type 3 (P smaller than 0.001). Thus, most type 3 organisms were ingested, but most type 1 gonococci were bound on the PMN surface. Images PMID:46842

  7. Standardization of blood smears prepared in transparent acetate: an alternative method for the microscopic diagnosis of malaria

    PubMed Central

    2014-01-01

    Background Due to students’ initial inexperience, slides are frequently broken and blood smears are damaged in microscopy training, leading to the need for their constant replacement. To minimize this problem a method of preparing blood smears on transparent acetate sheets was developed with the goal of implementing appropriate and more readily available teaching resources for the microscopic diagnosis of malaria. Methods Acetate sheets derived from polyester were used to standardize the preparation and staining of thin and thick blood smears on transparent acetate sheets. Thick and thin blood smears were also prepared using the conventional method on glass slides. The staining was conducted using Giemsa staining for the thick and thin smears. Results Microscopic examination (1,000x) of the thin and thick blood smears prepared on transparent acetate produced high-quality images for both the parasites and the blood cells. The smears showed up on a clear background and with minimal dye precipitation. It was possible to clearly identify the main morphological characteristics of Plasmodium, neutrophils and platelets. After 12 months of storage, there was no change in image quality or evidence of fungal colonization. Conclusion Preparation of thin and thick blood smears in transparent acetate for the microscopic diagnosis of malaria does not compromise the morphological and staining characteristics of the parasites or blood cells. It is reasonable to predict the applicability of transparent acetate in relevant situations such as the training of qualified professionals for the microscopic diagnosis of malaria and the preparation of positive specimens for competency assessment (quality control) of professionals and services involved in the diagnosis of malaria. PMID:24938886

  8. The Mechanics of Neutrophils: Synthetic Modeling of Three Experiments

    PubMed Central

    Herant, Marc; Marganski, William A.; Dembo, Micah

    2003-01-01

    Much experimental data exist on the mechanical properties of neutrophils, but so far, they have mostly been approached within the framework of liquid droplet models. This has two main drawbacks: 1), It treats the cytoplasm as a single phase when in reality, it is a composite of cytosol and cytoskeleton; and 2), It does not address the problem of active neutrophil deformation and force generation. To fill these lacunae, we develop here a comprehensive continuum-mechanical paradigm of the neutrophil that includes proper treatment of the membrane, cytosol, and cytoskeleton components. We further introduce two models of active force production: a cytoskeletal swelling force and a polymerization force. Armed with these tools, we present computer simulations of three classic experiments: the passive aspiration of a neutrophil into a micropipette, the active extension of a pseudopod by a neutrophil exposed to a local stimulus, and the crawling of a neutrophil inside a micropipette toward a chemoattractant against a varying counterpressure. Principal results include: 1), Membrane cortical tension is a global property of the neutrophil that is affected by local area-increasing shape changes. We argue that there exists an area dilation viscosity caused by the work of unfurling membrane-storing wrinkles and that this viscosity is responsible for much of the regulation of neutrophil deformation. 2), If there is no swelling force of the cytoskeleton, then it must be endowed with a strong cohesive elasticity to prevent phase separation from the cytosol during vigorous suction into a capillary tube. 3), We find that both swelling and polymerization force models are able to provide a unifying fit to the experimental data for the three experiments. However, force production required in the polymerization model is beyond what is expected from a simple short-range Brownian ratchet model. 4), It appears that, in the crawling of neutrophils or other amoeboid cells inside a micropipette

  9. The mechanics of neutrophils: synthetic modeling of three experiments.

    PubMed

    Herant, Marc; Marganski, William A; Dembo, Micah

    2003-05-01

    Much experimental data exist on the mechanical properties of neutrophils, but so far, they have mostly been approached within the framework of liquid droplet models. This has two main drawbacks: 1), It treats the cytoplasm as a single phase when in reality, it is a composite of cytosol and cytoskeleton; and 2), It does not address the problem of active neutrophil deformation and force generation. To fill these lacunae, we develop here a comprehensive continuum-mechanical paradigm of the neutrophil that includes proper treatment of the membrane, cytosol, and cytoskeleton components. We further introduce two models of active force production: a cytoskeletal swelling force and a polymerization force. Armed with these tools, we present computer simulations of three classic experiments: the passive aspiration of a neutrophil into a micropipette, the active extension of a pseudopod by a neutrophil exposed to a local stimulus, and the crawling of a neutrophil inside a micropipette toward a chemoattractant against a varying counterpressure. Principal results include: 1), Membrane cortical tension is a global property of the neutrophil that is affected by local area-increasing shape changes. We argue that there exists an area dilation viscosity caused by the work of unfurling membrane-storing wrinkles and that this viscosity is responsible for much of the regulation of neutrophil deformation. 2), If there is no swelling force of the cytoskeleton, then it must be endowed with a strong cohesive elasticity to prevent phase separation from the cytosol during vigorous suction into a capillary tube. 3), We find that both swelling and polymerization force models are able to provide a unifying fit to the experimental data for the three experiments. However, force production required in the polymerization model is beyond what is expected from a simple short-range Brownian ratchet model. 4), It appears that, in the crawling of neutrophils or other amoeboid cells inside a micropipette

  10. Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis.

    PubMed

    Mejía, Susana P; Cano, Luz E; López, Juan A; Hernandez, Orville; González, Ángel

    2015-05-01

    Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

  11. Neutrophil-related factors as biomarkers in EAE and MS

    PubMed Central

    Rumble, Julie M.; Huber, Amanda K.; Krishnamoorthy, Gurumoorthy; Srinivasan, Ashok; Giles, David A.; Zhang, Xu; Wang, Lu

    2015-01-01

    A major function of T helper (Th) 17 cells is to induce the production of factors that activate and mobilize neutrophils. Although Th17 cells have been implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE), little attention has been focused on the role of granulocytes in those disorders. We show that neutrophils, as well as monocytes, expand in the bone marrow and accumulate in the circulation before the clinical onset of EAE, in response to systemic up-regulation of granulocyte colony-stimulating factor (G-CSF) and the ELR+ CXC chemokine CXCL1. Neutrophils comprised a relatively high percentage of leukocytes infiltrating the central nervous system (CNS) early in disease development. G-CSF receptor deficiency and CXCL1 blockade suppressed myeloid cell accumulation in the blood and ameliorated the clinical course of mice that were injected with myelin-reactive Th17 cells. In relapsing MS patients, plasma levels of CXCL5, another ELR+ CXC chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS. PMID:25559893

  12. Asymmetric Localization of Calpain 2 during Neutrophil Chemotaxis

    PubMed Central

    Nuzzi, Paul A.; Senetar, Melissa A.

    2007-01-01

    Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis. PMID:17192410

  13. Studying Neutrophil Migration In Vivo Using Adoptive Cell Transfer.

    PubMed

    Miyabe, Yoshishige; Kim, Nancy D; Miyabe, Chie; Luster, Andrew D

    2016-01-01

    Adoptive cell transfer experiments can be used to study the roles of cell trafficking molecules on the migratory behavior of specific immune cell populations in vivo. Chemoattractants and their G protein-coupled seven-transmembrane-spanning receptors regulate migration of cells in vivo, and dysregulated expression of chemoattractants and their receptors is implicated in autoimmune and inflammatory diseases. Inflammatory arthritides, such as rheumatoid arthritis (RA), are characterized by the recruitment of inflammatory cells into joints. The K/BxN serum transfer mouse model of inflammatory arthritis shares many similar features with RA. In this autoantibody-induced model of arthritis, neutrophils are the critical immune cells necessary for the development of joint inflammation and damage. We have used adoptive neutrophil transfer to define the contributions of chemoattractant receptors, cytokines, and activation receptors expressed on neutrophils that critically regulate their entry into the inflamed joint. In this review, we describe the procedure of neutrophil adoptive transfer to study the influence of neutrophil-specific receptors or mediators upon the their recruitment into the joint using the K/BxN model of inflammatory arthritis as a model of how adoptive cell transfer studies can be used to study immune cell migration in vivo.

  14. Neutrophil biology and the next generation of myeloid growth factors.

    PubMed

    Dale, David C

    2009-01-01

    Neutrophils are the body's critical phagocytic cells for defense against bacterial and fungal infections; bone marrow must produce approximately 10 x 10(9) neutrophils/kg/d to maintain normal blood neutrophil counts. Production of neutrophils depends on myeloid growth factors, particularly granulocyte colony-stimulating factor (G-CSF). After the original phase of development, researchers modified these growth factors to increase their size and delay renal clearance, increase their biologic potency, and create unique molecules for business purposes. Pegylated G-CSF is a successful product of these efforts. Researchers have also tried to identify small molecules to serve as oral agents that mimic the parent molecules, but these programs have been less successful. In 2006, the European Medicines Agency established guidelines for the introduction of new biologic medicinal products claimed to be similar to reference products that had previously been granted marketing authorization in the European community, called bio-similars. Globally, new and copied versions of G-CSF and other myeloid growth factors are now appearing. Some properties of the myeloid growth factors are similar to other agents, offering opportunities for the development of alternative drugs and treatments. For example, recent research shows that hematopoietic progenitor cells can be mobilized with a chemokine receptor antagonist, chemotherapy, G-CSF, and granulocyte macrophage colony-stimulating factor. Advances in neutrophil biology coupled with better understanding and development of myeloid growth factors offer great promise for improving the care of patients with cancer and many other disorders.

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

    SciTech Connect

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

    1987-11-15

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

  16. Marine Natural Product Inhibitors of Neutrophil-Associated Inflammation

    PubMed Central

    Chen, Chun-Yu; Tsai, Yung-Fong; Chang, Wen-Yi; Yang, Shun-Chin; Hwang, Tsong-Long

    2016-01-01

    Neutrophils are widely recognized to play an important role in acute inflammatory responses, and recent evidence has expanded their role to modulating chronic inflammatory and autoimmune diseases. Reactive oxygen species (ROS) and microbicidal compounds released from neutrophils that are recruited to the site of inflammation contribute to the pathogenesis of multiple inflammation-associated diseases such as chronic obstructive pulmonary disease, atherosclerosis, and hepatitis. Marine organisms are a valuable source of bioactive compounds with potential for industrial and pharmaceutical application. Marine natural products that inhibit neutrophil activation could be used as drugs for the treatment of inflammatory diseases. Numerous studies investigating marine natural products have reported novel anti-inflammatory agents. Nevertheless, the detailed mechanisms underlying their actions, which could facilitate our understanding of the molecular events occurring in neutrophils, have not been reported in most of the associated research studies. Therefore, in this review, we will present marine products that inhibit neutrophil-associated inflammation. Furthermore, we will be limiting the detailed discussion to agents with well-investigated molecular targets. PMID:27472345

  17. CXCL5 Drives Neutrophil Recruitment in TH17-Mediated GN

    PubMed Central

    Disteldorf, Erik M.; Krebs, Christian F.; Paust, Hans-Joachim; Turner, Jan-Eric; Nouailles, Geraldine; Tittel, André; Meyer-Schwesinger, Catherine; Stege, Gesa; Brix, Silke; Velden, Joachim; Wiech, Thorsten; Helmchen, Udo; Steinmetz, Oliver M.; Peters, Anett; Bennstein, Sabrina B.; Kaffke, Anna; Llanto, Chrystel; Lira, Sergio A.; Mittrücker, Hans-Willi; Stahl, Rolf A.K.; Kurts, Christian; Kaufmann, Stefan H.E.

    2015-01-01

    Neutrophil trafficking to sites of inflammation is essential for the defense against bacterial and fungal infections, but also contributes to tissue damage in TH17-mediated autoimmunity. This process is regulated by chemokines, which often show an overlapping expression pattern and function in pathogen- and autoimmune-induced inflammatory reactions. Using a murine model of crescentic GN, we show that the pathogenic TH17/IL-17 immune response induces chemokine (C-X-C motif) ligand 5 (CXCL5) expression in kidney tubular cells, which recruits destructive neutrophils that contribute to renal tissue injury. By contrast, CXCL5 was dispensable for neutrophil recruitment and effective bacterial clearance in a murine model of acute bacterial pyelonephritis. In line with these findings, CXCL5 expression was highly upregulated in the kidneys of patients with ANCA-associated crescentic GN as opposed to patients with acute bacterial pyelonephritis. Our data therefore identify CXCL5 as a potential therapeutic target for the restriction of pathogenic neutrophil infiltration in TH17-mediated autoimmune diseases while leaving intact the neutrophil function in protective immunity against invading pathogens. PMID:24904089

  18. Leukocyte subsets and neutrophil function after short-term spaceflight

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Sams, C. F.; Mehta, S. K.; Kaur, I.; Jones, M. L.; Feeback, D. L.; Pierson, D. L.

    1999-01-01

    Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

  19. Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

    PubMed Central

    Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

    2013-01-01

    Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

  20. The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation

    PubMed Central

    Volmering, Stephanie; Block, Helena; Boras, Mark; Lowell, Clifford A.; Zarbock, Alexander

    2016-01-01

    SUMMARY Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton’s tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation. PMID:26777396

  1. Doped with Sodium Acetate and Metallic Sodium

    NASA Astrophysics Data System (ADS)

    Tada, Satoki; Isoda, Yukihiro; Udono, Haruhiko; Fujiu, Hirofumi; Kumagai, Shunji; Shinohara, Yoshikazu

    2014-06-01

    We have investigated the thermoelectric properties of p-type Na-doped Mg2 Si0.25Sn0.75 solid solutions prepared by liquid-solid reaction and hot-pressing methods. Na was introduced into Mg2Si0.25Sn0.75 by using either sodium acetate (CH3COONa) or metallic sodium (2 N). The samples doped with sodium acetate consisted of phases with antifluorite structure and a small amount of MgO as revealed by x-ray diffraction, whereas the sample doped with metallic sodium contained the Sn, MgO, and Mg2SiSn phases. The hole concentrations of Mg1.975Na0.025Si0.25Sn0.75 doped by sodium acetate and metallic sodium were 1.84 × 1025 m-3 and 1.22 × 1025 m-3, respectively, resulting in resistivities of 4.96 × 10-5 Ω m (sodium acetate) and 1.09 × 10-5 Ω m (metallic sodium). The Seebeck coefficients were 198 μV K-1 (sodium acetate) and 241 μV K-1 (metallic sodium). The figures of merit for Mg1.975Na0.025Si0.25Sn0.75 were 0.40 × 10-3 K-1 (sodium acetate) and 0.25 × 10-3 K-1 (metallic sodium) at 400 K. Thus, sodium acetate is a suitable Na dopant for Mg2Si1- x Sn x .

  2. 21 CFR 177.1350 - Ethylene-vinyl acetate copolymers.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ethylene-vinyl acetate copolymers. 177.1350... Use Food Contact Surfaces § 177.1350 Ethylene-vinyl acetate copolymers. Ethylene-vinyl acetate... with the following prescribed conditions: (a)(1) Ethylene-vinyl acetate copolymers consist of...

  3. In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes.

    PubMed

    Santos, T C; Marques, A P; Silva, S S; Oliveira, J M; Mano, J F; Castro, A G; Reis, R L

    2007-10-31

    Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.

  4. Enhanced survival of Leishmania major in neutrophil granulocytes in the presence of apoptotic cells

    PubMed Central

    Hellberg, Lars; Köhl, Jörg; Laskay, Tamás

    2017-01-01

    Neutrophil granulocytes are the first leukocytes that encounter and phagocytose Leishmania major (L. major) parasites in the infected skin. The parasites can nonetheless survive within neutrophils. However, the mechanisms enabling the survival of Leishmania within neutrophils are still elusive. Previous findings indicated that human neutrophils can engulf apoptotic cells. Since apoptotic neutrophils are abundant in infected tissues, we hypothesized that the uptake of apoptotic cells results in diminished anti-leishmanial activity and, consequently, contributes to enhanced survival of the parasites at the site of infection. In the present study, we demonstrated that L. major-infected primary human neutrophils acquire enhanced capacity to engulf apoptotic cells. This was associated with increased expression of the complement receptors 1 and 3 involved in phagocytosis of apoptotic cells. Next, we showed that ingestion of apoptotic cells affects neutrophil antimicrobial functions. We observed that phagocytosis of apoptotic cells by neutrophils downregulates the phosphorylation of p38 MAPK and PKCδ, the kinases involved in activation of NADPH oxidase and hence reactive oxygen species (ROS) production. In line, uptake of apoptotic cells inhibits TNF- and L. major-induced ROS production by neutrophils. Importantly, we found that the survival of Leishmania in neutrophils is strongly enhanced in neutrophils exposed to apoptotic cells. Together, our findings reveal that apoptotic cells promote L. major survival within neutrophils by downregulating critical antimicrobial functions. This suggests that the induction of enhanced uptake of apoptotic cells represents a novel evasion mechanism of the parasites that facilitates their survival in neutrophil granulocytes. PMID:28187163

  5. Recapitulation of in vivo-like neutrophil transendothelial migration using a microfluidic platform.

    PubMed

    Wu, Xiaojie; Newbold, Molly A; Haynes, Christy L

    2015-08-07

    Neutrophil transendothelial migration (TEM) is an essential physiological process that regulates the recruitment of neutrophils in response to inflammatory signals. Herein, a versatile hydrogel scaffold is embedded in a microfluidic platform that supports an endothelial cell layer cultured in the vertical direction and highly stable chemical gradients; this construct is employed to mimic the in vivo neutrophil TEM process. We found that the number of neutrophils migrating across the endothelial cell layer is dependent on the presented chemoattractant concentration and the spatial profile of the chemical gradient. Endothelial cells play a critical role in neutrophil TEM by promoting neutrophil morphological changes as well as expressing surface receptor molecules that are indispensable for inducing neutrophil attachment and migration. Furthermore, the microfluidic device also supports competing chemoattractant gradients to facilitate neutrophil TEM studies in complex microenvironments that more accurately model the in vivo system than simplified microenvironments without the complexity of chemical gradients. This work demonstrates that combinations of any two different chemoattractants induce more significant neutrophil migration than a single chemoattractant in the same total amount, indicating synergistic effects between distinct chemoattractants. The in vitro reconstitution of neutrophil TEM successfully translates planar neutrophil movement into in vivo-like neutrophil recruitment and accelerates understanding of cellular interactions between neutrophils and endothelial cells within the complicated physiological milieu.

  6. Assessment of Antioxidant Activity of Spray Dried Extracts of Psidium guajava Leaves by DPPH and Chemiluminescence Inhibition in Human Neutrophils

    PubMed Central

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

    2014-01-01

    This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β-cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH• method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells. PMID:24822200

  7. Tested Demonstrations: Buffer Capacity of Various Acetic Acid-Sodium Acetate Systems: A Lecture Experiment.

    ERIC Educational Resources Information Center

    Donahue, Craig J.; Panek, Mary G.

    1985-01-01

    Background information and procedures are provided for a lecture experiment which uses indicators to illustrate the concept of differing buffer capacities by titrating acetic acid/sodium acetate buffers with 1.0 molar hydrochloric acid and 1.0 molar sodium hydroxide. A table with data used to plot the titration curve is included. (JN)

  8. Acetylation of Starch with Vinyl Acetate in Imidazolium Ionic Liquids and Characterization of Acetate Distribution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch was acetylated with vinyl acetate in different 1-butyl-3-methylimidazolium (BMIM) salts as solvent in effort to produce starches with different acetylation patterns. Overall degree of substitution was much higher for basic anions such as acetate and dicyanimide (dca) than for neutral anions ...

  9. Acetic acid production from food wastes using yeast and acetic acid bacteria micro-aerobic fermentation.

    PubMed

    Li, Yang; He, Dongwei; Niu, Dongjie; Zhao, Youcai

    2015-05-01

    In this study, yeast and acetic acid bacteria strains were adopted to enhance the ethanol-type fermentation resulting to a volatile fatty acids yield of 30.22 g/L, and improve acetic acid production to 25.88 g/L, with food wastes as substrate. In contrast, only 12.81 g/L acetic acid can be obtained in the absence of strains. The parameters such as pH, oxidation reduction potential and volatile fatty acids were tested and the microbial diversity of different strains and activity of hydrolytic ferment were investigated to reveal the mechanism. The optimum pH and oxidation reduction potential for the acetic acid production were determined to be at 3.0-3.5 and -500 mV, respectively. Yeast can convert organic matters into ethanol, which is used by acetic acid bacteria to convert the organic wastes into acetic acid. The acetic acid thus obtained from food wastes micro-aerobic fermentation liquid could be extracted by distillation to get high-pure acetic acid.

  10. Ice-structuring mechanism for zirconium acetate.

    PubMed

    Deville, Sylvain; Viazzi, Céline; Guizard, Christian

    2012-10-23

    The control of ice nucleation and growth is critical in many natural and engineering situations. However, very few compounds are able to interact directly with the surface of ice crystals. Ice-structuring proteins, found in certain fish, plants, and insects, bind to the surface of ice, thereby controlling their growth. We recently revealed the ice-structuring properties of zirconium acetate, which are similar to those of ice-structuring proteins. Because zirconium acetate is a salt and therefore different from proteins having ice-structuring properties, its ice-structuring mechanism remains unelucidated. Here we investigate this ice-structuring mechanism through the role of the concentration of zirconium acetate and the ice crystal growth velocity. We then explore other compounds presenting similar functional groups (acetate, hydroxyl, or carboxylic groups). On the basis of these results, we propose that zirconium acetate adopts a hydroxy-bridged polymer structure that can bind to the surface of the ice crystals through hydrogen bonding, thereby slowing down the ice crystal growth.

  11. A mammalian acetate switch regulates stress erythropoiesis

    PubMed Central

    Xu, Min; Nagati, Jason S.; Xie, Jian; Li, Jiwen; Walters, Holly; Moon, Young-Ah; Gerard, Robert D.; Huang, Chou-Long; Comerford, Sarah A.; Hammer, Robert E.; Horton, Jay D.; Chen, Rui; Garcia, Joseph A.

    2014-01-01

    Endocrine erythropoietin (Epo), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor Hypoxia Inducible Factor 2 (HIF-2). We previously reported that the lysine acetyltransferase Cbp is required for HIF-2α acetylation and efficient HIF-2 dependent Epo induction during hypoxia. We now show these processes require acetate-dependent acetyl CoA synthetase 2 (Acss2). In Hep3B hepatoma cells and in Epo-generating organs of hypoxic or acutely anemic mice, acetate levels increase and Acss2 is required for HIF-2α acetylation, Cbp/HIF-2α complex formation and recruitment to the Epo enhancer, and efficient Epo induction. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an Acss2-dependent manner. In acquired and genetic chronic anemia mouse models, acetate supplementation also increases Epo expression and resting hematocrits. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and Epo induction during pathophysiological states marked by tissue hypoxia. PMID:25108527

  12. Neutrophil activation in ivermectin-treated onchocerciasis patients.

    PubMed Central

    Njoo, F L; Hack, C E; Oosting, J; Stilma, J S; Kijlstra, A

    1993-01-01

    Ivermectin is a safe and effective drug for onchocerciasis treatment. In certain individuals, however, therapy is accompanied by adverse reactions. The mechanisms underlying these reactions are not yet known. The aim of the present study was to investigate whether neutrophils are involved in the development of these adverse reactions. Elastase and lactoferrin, two markers for the release of neutrophil azurophilic and specific granule contents respectively, were measured by radioimmunoassays in plasma of onchocerciasis patients with varying degrees of side effects, as well as in control subjects before and 1 and 2 days after ivermectin treatment. A considerable increase of elastase levels after treatment was observed, whereas lactoferrin levels did not change. The percentage of patients with elevated elastase levels was significantly correlated with the degree of side effects. These findings suggest that neutrophil activation may be involved in the development of adverse reactions in these patients. PMID:8222324

  13. Neutrophil Fates in Bronchiectasis and Alpha-1 Antitrypsin Deficiency.

    PubMed

    Russell, Derek W; Gaggar, Amit; Solomon, George M

    2016-04-01

    The neutrophil is a powerful cellular defender of the vulnerable interface between the environment and pulmonary tissues. This cell's potent weapons are carefully calibrated in the healthy state to maximize effectiveness in fighting pathogens while minimizing tissue damage and allowing for repair of what damage does occur. The three related chronic airway disorders of cystic fibrosis, non-cystic fibrosis bronchiectasis, and alpha-1 antitrypsin deficiency all demonstrate significant derangements of this homeostatic system that result in their respective pathologies. An important shared feature among them is the inefficient resolution of chronic inflammation that serves as a central means for neutrophil-driven lung damage resulting in disease progression. Examining the commonalities and divergences between these diseases in the light of their immunopathology is informative and may help guide us toward future therapeutics designed to modulate the neutrophil's interplay with the pulmonary environment.

  14. Flexibility of single microvilli on live neutrophils and lymphocytes

    NASA Astrophysics Data System (ADS)

    Yao, Da-Kang; Shao, Jin-Yu

    2007-08-01

    We measured the flexural stiffness of single microvilli on live human neutrophils and lymphocytes using 40-nm fluorescent beads. The beads were bound to the tips of the microvilli by anti- L -selectin antibodies. Digital bead images were acquired with an exposure time of 3s at high magnification. Using a Gaussian point spread function, we obtained an analytical expression that relates the image profile to the flexural stiffness. We found that the flexural stiffnesses were 7 and 4pN/μm for single microvilli on human neutrophils and lymphocytes, respectively. We also verified with live cells that 75% of neutrophil L -selectin and 72% of lymphocyte L -selectin were on the microvillus tips. Our results indicate that the leukocyte microvilli in contact with the endothelium or other surfaces will bend easily under physiological shear stresses.

  15. Effect of laser irradiation on neutrophils metabolism in stress

    NASA Astrophysics Data System (ADS)

    Brill, Gregory E.; Grigoriev, Sergei N.; Romanova, Tatyana P.; Petrisheva, Svetlana G.

    1994-02-01

    In experiments on male mice of CBA line the alteration of neutrophils cytochemical profile in peripheral blood He-Ne laser irradiation in vitro (4 mW/cm2, 15 min) and modification of metabolic disturbances in polymorphonuclear leucocytes in stress by laser radiation were studied. It was found that direct laser irradiation of blood results in the decrease of glycogen and lipids content, the increase of ATP-ase, succinate dehydrogenase and myeloperoxidase activity, rise of lysosomal cationic proteins level, and membrane oxidase systems of neutrophils stimulation. In short-term immobilization stress conditions transcutaneous laser irradiation in vivo (19 mW/cm2, 15 min) prevents the development of stress induced changes of metabolism and function of neutrophils.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Mukaida, N

    2000-12-01

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

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

    PubMed

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

    2016-03-11

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

  19. An elucidation of neutrophil functions against Mycobacterium tuberculosis infection.

    PubMed

    Morris, Devin; Nguyen, Thien; Kim, John; Kassissa, Christine; Khurasany, Melissa; Luong, Jennifer; Kasko, Sarah; Pandya, Shalin; Chu, Michael; Chi, Po-Ting; Ly, Judy; Lagman, Minette; Venketaraman, Vishwanath

    2013-01-01

    We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb) infection, with particular reference to glutathione (GSH). We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH) resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection.

  20. An Elucidation of Neutrophil Functions against Mycobacterium tuberculosis Infection

    PubMed Central

    Morris, Devin; Nguyen, Thien; Kim, John; Kassissa, Christine; Khurasany, Melissa; Luong, Jennifer; Kasko, Sarah; Pandya, Shalin; Chu, Michael; Chi, Po-Ting; Lagman, Minette; Venketaraman, Vishwanath

    2013-01-01

    We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb) infection, with particular reference to glutathione (GSH). We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH) resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection. PMID:24312131

  1. Fer kinase limits neutrophil chemotaxis toward end target chemoattractants.

    PubMed

    Khajah, Maitham; Andonegui, Graciela; Chan, Ronald; Craig, Andrew W; Greer, Peter A; McCafferty, Donna-Marie

    2013-03-01

    Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer(DR/DR)) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer(DR/DR) mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer(DR/DR) mice (p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer(DR/DR) recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Purified Fer(DR/DR) neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WT using an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB(4)). Increased WKYMVm-induced chemotaxis of Fer(DR/DR) neutrophils correlated with sustained PI3K activity and reduced reliance on the p38 MAPK pathway compared with WT neutrophils. Together, these data identify Fer as a novel inhibitory kinase for neutrophil chemotaxis toward end target chemoattractants through modulation of PI3K activity.

  2. Neutrophil Extracellular Traps and Its Implications in Inflammation: An Overview

    PubMed Central

    Delgado-Rizo, Vidal; Martínez-Guzmán, Marco A.; Iñiguez-Gutierrez, Liliana; García-Orozco, Alejandra; Alvarado-Navarro, Anabell; Fafutis-Morris, Mary

    2017-01-01

    In addition to physical barriers, neutrophils are considered a part of the first line of immune defense. They can be found in the bloodstream, with a lifespan of 6–8 h, and in tissue, where they can last up to 7 days. The mechanisms that neutrophils utilize for host defense are phagocytosis, degranulation, cytokine production, and, the most recently described, neutrophil extracellular trap (NET) production. NETs are DNA structures released due to chromatin decondensation and spreading, and they thus occupy three to five times the volume of condensed chromatin. Several proteins adhere to NETs, including histones and over 30 components of primary and secondary granules, among them components with bactericidal activity such as elastase, myeloperoxidase, cathepsin G, lactoferrin, pentraxin 3, gelatinase, proteinase 3, LL37, peptidoglycan-binding proteins, and others with bactericidal activity able to destroy virulence factors. Three models for NETosis are known to date. (a) Suicidal NETosis, with a duration of 2–4 h, is the best described model. (b) In vital NETosis with nuclear DNA release, neutrophils release NETs without exhibiting loss of nuclear or plasma membrane within 5–60 min, and it is independent of reactive oxygen species (ROS) and the Raf/MERK/ERK pathway. (c) The final type is vital NETosis with release of mitochondrial DNA that is dependent on ROS and produced after stimuli with GM-CSF and lipopolysaccharide. Recent research has revealed neutrophils as more sophisticated immune cells that are able to precisely regulate their granular enzymes release by ion fluxes and can release immunomodulatory cytokines and chemokines that interact with various components of the immune system. Therefore, they can play a key role in autoimmunity and in autoinflammatory and metabolic diseases. In this review, we intend to show the two roles played by neutrophils: as a first line of defense against microorganisms and as a contributor to the pathogenesis of

  3. Chemokines: sirens of neutrophil recruitment-but is it just one song?

    PubMed

    McDonald, Braedon; Kubes, Paul

    2010-08-27

    Neutrophil trafficking to inflamed tissues requires the integration of multiple chemoattractant guidance signals. In this issue of Immunity, Chou et al. (2010) demonstrate that collaborative "cascades" of chemoattractant mediators control neutrophil recruitment to arthritic joints in mice.

  4. Dynamic Protonation Equilibrium of Solvated Acetic Acid

    SciTech Connect

    Gu, Wei; Frigato, Tomaso; Straatsma, TP; Helms, Volkhard H.

    2007-04-13

    For the first time, the dynamic protonation equilibrium between an amino acid side chain analogue and bulk water as well as the diffusion properties of the excess proton were successfully reproduced through unbiased computer simulations. During a 50 ns Q-HOP MD simulation, two different regimes of proton transfer were observed. Extended phases of frequent proton swapping between acetic acid and nearby water were separated by phases where the proton freely diffuses in the simulation box until it is captured again by acetic acid. The pKa of acetic acid was calculated around 3.0 based on the relative population of protonated and deprotonated states and the diffusion coefficient of excess proton was computed from the average mean squared displacement in the simulation. Both calculated values agree well with the experimental measurements.

  5. The physicochemical property characterization of agar acetate.

    PubMed

    Xia, Kai; Liu, Xin; Zhao, Jingkun; Zhang, Xiaodong

    2014-09-22

    A series of agar acetates with different degree of substitution (DS) were prepared, and their properties were determined and analyzed. The results showed that the gelling temperature, the gel melting temperature, the gel strength, the gel hardness, the gel fracturability, the gel springiness and the solution apparent viscosity of agar acetates all decreased except that their gel cohesiveness increased with the increase of DS. The variation process of agar molecules in solution from coil to helix could be also observed by measuring solution optical rotation in a lower concentration at which even the solution could not form a gel. The gel skeleton structures of agar acetates were of porous network structures, and the pores became smaller and denser with the increase of DS. After acetylation, the water holding capacity of the agar was improved, but its thermal stability was lowered.

  6. Fever and neutrophilic alveolitis caused by a vanadium based catalyst

    PubMed Central

    Vandenplas, O; Binard-Van, C; Gregoire, J; Brumagne, A; Larbanois, A

    2002-01-01

    Methods: The investigation included inhalation challenge with the suspected compound combined with monitoring of lung function tests and post-challenge bronchoalveolar lavage. Results: Exposure to the vanadium containing catalyst for 120 minutes resulted in a sustained decline in forced vital capacity and forced expiratory volume in one second, while the transfer factor for carbon monoxide did not change significantly. The subject developed fever and peripheral blood neutrophilia. Bronchoalveolar lavage performed 48 hours after the end of challenge exposure showed a marked increase in neutrophils (60% of total cell count). Conclusions: Exposure to vanadium can cause a metal fume fever-like syndrome associated with neutrophilic alveolitis. PMID:12409538

  7. Role of osteopontin in hepatic neutrophil infiltration during alcoholic steatohepatitis

    SciTech Connect

    Apte, Udayan M.; Banerjee, Atrayee; McRee, Rachel; Wellberg, Elizabeth; Ramaiah, Shashi K. . E-mail: sramaiah@cvm.tamu.edu

    2005-08-22

    Alcoholic liver disease (ALD) is a major complication of heavy alcohol (EtOH) drinking and is characterized by three progressive stages of pathology: steatosis, steatohepatitis, and fibrosis/cirrhosis. Alcoholic steatosis (AS) is the initial stage of ALD and consists of fat accumulation in the liver accompanied by minimal liver injury. AS is known to render the hepatocytes increasingly sensitive to toxicants such as bacterial endotoxin (LPS). Alcoholic steatohepatitis (ASH), the second and rate-limiting step in the progression of ALD, is characterized by hepatic fat accumulation, neutrophil infiltration, and neutrophil-mediated parenchymal injury. However, the pathogenesis of ASH is poorly defined. It has been theorized that the pathogenesis of ASH involves interaction of increased circulating levels of LPS with hepatocytes being rendered highly sensitive to LPS due to heavy EtOH consumption. We hypothesize that osteopontin (OPN), a matricellular protein (MCP), plays an important role in the hepatic neutrophil recruitment due to its enhanced expression during the early phase of ALD (AS and ASH). To study the role of OPN in the pathogenesis of ASH, we induced AS in male Sprague-Dawley rats by feeding EtOH-containing Lieber-DeCarli liquid diet for 6 weeks. AS rats experienced extensive fat accumulation and minimal liver injury. Moderate induction in OPN was observed in AS group. ASH was induced by feeding male Sprague-Dawley rats EtOH-containing Lieber-DeCarli liquid diet for 6 weeks followed by LPS injection. The ASH rats had substantial neutrophil infiltration, coagulative oncotic necrosis, and developed higher liver injury. Significant increases in the hepatic and circulating levels of OPN was observed in the ASH rats. Higher levels of the active, thrombin-cleaved form of OPN in the liver in ASH group correlated remarkably with hepatic neutrophil infiltration. Finally, correlative studies between OPN and hepatic neutrophil infiltration was corroborated in a simple

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

    PubMed

    Martinova, E A; Baranov, V I

    2014-01-01

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

  9. Microhydration of Neutral and Charged Acetic Acid.

    PubMed

    Krishnakumar, Parvathi; Maity, Dilip Kumar

    2017-01-19

    A systematic theoretical study has been carried out on the effect of sequential addition of water molecules to neutral and mono positively charged acetic acid molecules by applying first principle based electronic structure theory. Geometry, dipole moment, and polarizability of hydrated clusters of neutral and mono positively charged acetic acid of the type CH3COOH·nH2O (n = 1-8) and [CH3COOH·nH2O](+) (n = 1, 2) are calculated at the ωB97X-D/aug-cc-pVDZ level of theory. Free energies of formation of the hydrated acid clusters, at different temperatures and pressures are determined. Solvent stabilization energy and interaction energy are also calculated at the CCSD(T)/6-311++G(d,p) level of theory. It is observed that in the case of neutral acetic acid, proton transfer from the acid molecule to solvent water molecules does not occur even with eight water molecules and the acid molecule remains in the undissociated form. High-energy equilibrium structures showing dissociation of acetic acid are obtained in case of hexahydrated and larger hydrated clusters only. However, dissociation of mono positively charged acetic acid occurs with just two water molecules. Interestingly, it is noted that in the case of dissociation, calculated bond dipole moments of the dissociating bonds of acetic acid in microhydated clusters shows a characteristic feature. IR spectra of CH3COOH·nH2O (n = 1-8) and [CH3COOH·nH2O](+) (n = 1-3) clusters are simulated and compared with the available experimental data.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Adenosine (AD) decreases cobra venom factor (CVF)-induced lung injury

    SciTech Connect

    Bruner, L.H.; Till, G.O.; Ward, P.A.

    1986-03-01

    Systemic activation of complement following intravenous injection of CVF causes acute pulmonary microvascular injury. This injury is caused by release of neutrophil-derived oxygen radicals in the pulmonary microvasculature after complement activation. Phorbol myristate acetate (PMA)-stimulated neutrophils exposed to AD in vitro produce less O/sub 2/- than do neutrophils exposed to PMA alone. Thus, it was of interest to determine whether AD co-treatment would protect rats from injury due to CVF in vivo. Four groups of rats were treated with either CVF/AD, CVF/Saline (S), S/AD or S/S. AD or S vehicle was given 5 min prior to CVF or S vehicle. The rats were killed 30 min after CVF when pulmonary injury was assessed by measuring sequestration of /sup 125/I-labeled bovine serum albumin in the lungs. The lung injury in rats co-treated with CVF/AD was approximately 50% less than in rats receiving CVF/S. AD alone had no effects. These results indicate that AD decreases pulmonary damage due to CVF-induced systemic complement activation in vivo.

  13. Mechanism of inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922

    SciTech Connect

    Hoffman, M.D.; Wright, C.D.

    1986-03-05

    The allergic mediator release inhibitor CI-922 (3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo(3,2-b)indole-2-carboxamide) is a potent inhibitor of human neutrophil (PMN) respiratory and secretory responses in vitro. At concentrations from 1 to 100 micromolar, CI-922 inhibits activation of PMNs by agents which stimulate phospholipase C-dependent phosphoinositide hydrolysis to generate the second messengers inositol 1,4,5 trisphosphate and diacylglycerol, including: the plasma membrane receptor-specific ligands fMet-Leu-Phe and C5a; concanavalin A; and the guanine nucleotide regulatory protein-specific stimulus GTPgammaS. In contrast, CI-922 does not inhibit PMN responses to protein kinase C-specific stimuli such as phorbol myristate acetate (PMA) or sn-1,2-dioctanoyl-glycerol. CI-922 is also unable to inhibit the synergistic activation of PMNs by suboptimal concentrations of PMA and calcium ionophore A23187. These results suggest that CI-922 inhibits PMN activation at a site distal to signal transduction through the guanine nucleotide regulatory protein required for second messenger generation but proximal cophosphorylation reactions mediated by protein kinase C and calcium/calmodulin-dependent protein kinases.

  14. NEUTROPHIL DEPLETION ATTENUATES INTERLEUKIN-8 PRODUCTION IN MILD-OVERSTRETCHED VENTILATED NORMAL RABBIT LUNG

    EPA Science Inventory

    OBJECTIVE: Acute lung injury induced by lung overstretch is associated with neutrophil influx, but the pathogenic role of neutrophils in overstretch-induced lung injury remains unclear. DESIGN: To assess the contribution of neutrophils, we compared the effects of noninjurious lar...

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

    PubMed

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

    2012-03-01

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

  16. Commensal microbiota stimulate systemic neutrophil migration through induction of Serum amyloid A

    PubMed Central

    Kanther, Michelle; Tomkovich, Sarah; Sun, Xiaolun; Grosser, Melinda R.; Koo, Jaseol; Flynn, Edward J.; Jobin, Christian; Rawls, John F.

    2015-01-01

    Summary Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses. PMID:24373309

  17. Megestrol acetate in cachexia and anorexia

    PubMed Central

    Yeh, Shing-shing; Schuster, Michael W

    2006-01-01

    The aim is to review major clinical trials that have used megestrol acetate (MA) in the treatment of cachexia across several disease states. A review of general usage and potential side-effects are discussed. A theory that the newly approved nanocrystal formation of MA can better deliver this potent medication for treatment will also be reviewed. PMID:17722275

  18. 21 CFR 173.228 - Ethyl acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ethyl acetate. 173.228 Section 173.228 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Solvents, Lubricants, Release Agents and...

  19. Process for the 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 within a contact zone a mixture of ketene and acetaldehyde with an acid catalyst at about one bar pressure and between about 85.degree. and 200.degree. C. and removing the reaction products from the contact zone.

  20. Process for the preparation of vinyl acetate

    DOEpatents

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

    1998-02-17

    This invention pertains to the preparation of vinyl acetate by contacting within a contact zone a mixture of ketene and acetaldehyde with an acid catalyst at about one bar pressure and between about 85 and 200 C and removing the reaction products from the contact zone.

  1. [Hormonal desexing of boars with chlormadinone acetate].

    PubMed

    Busch, W; Hagelschuer, H; Gränz, G; Richter, G; Werner, K

    1979-01-01

    Chloromadinone acetate produces a dependable desexualising effect on boar by contant administration in feed rations of 30 mg per die over 70 days. Sexual odour thus can be widely eliminated. Other aspects studied in a group of 107 boars are body weight development, sexual behaviour, slaughter yield, and skin quality.

  2. Heat Bonding of Irradiated Ethylene Vinyl Acetate

    NASA Technical Reports Server (NTRS)

    Slack, D. H.

    1986-01-01

    Reliable method now available for joining parts of this difficult-tobond material. Heating fixture encircles ethylene vinyl acetate multiplesocket part, providing heat to it and to tubes inserted in it. Fixtures specially designed to match parts to be bonded. Tube-and-socket bonds made with this technique subjected to tensile tests. Bond strengths of 50 percent that of base material obtained consistently.

  3. 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 Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of...

  4. Corrosion of stainless steel during acetate production

    SciTech Connect

    Qi, J.S.; Lester, G.C.

    1996-07-01

    Corrosion of types 304, 304L, 316, and 316L stainless steel (SS) during the esterification of acetic acid and alcohol or glycol ether was investigated. The catalyst for this reaction, sulfuric acid or para-toluene sulfonic acid (PTSA), was shown to cause more corrosion on reactor equipment than CH{sub 3}COOH under the process conditions commonly practiced in industry. The corrosive action of the catalyst occurred only in the presence of water. Thus, for the batch processes, corrosion occurred mostly during the initial stage of esterification, where water produced by the reaction created an aqueous environment. After water was distilled off, the corrosion rate declined to a negligible value. The corrosion inhibitor copper sulfate, often used in industrial acetate processes, was found to work well for a low-temperature process (< 95 C) such as in production of butyl acetate, but it accelerated corrosion in the glycol ether acetate processes where temperatures were > 108 C. Process conditions that imparted low corrosion rates were determined.

  5. Advanced Colloids Experiment (ACE-T1)

    NASA Technical Reports Server (NTRS)

    Meyer, William V.; Sicker, Ron; Brown, Dan; Eustace, John

    2015-01-01

    Increment 45 - 46 Science Symposium presentation of Advanced Colloids Experiment (ACE-T1) to RPO. The purpose of this event is for Principal Investigators to present their science objectives, testing approach, and measurement methods to agency scientists, managers, and other investigators.

  6. 21 CFR 522.533 - Deslorelin acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Deslorelin acetate. 522.533 Section 522.533 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS IMPLANTATION OR INJECTABLE DOSAGE FORM NEW ANIMAL DRUGS §...

  7. 21 CFR 522.1073 - Gonadorelin acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Gonadorelin acetate. 522.1073 Section 522.1073 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS IMPLANTATION OR INJECTABLE DOSAGE FORM NEW ANIMAL DRUGS §...

  8. 21 CFR 522.1073 - Gonadorelin acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Gonadorelin acetate. 522.1073 Section 522.1073 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS IMPLANTATION OR INJECTABLE DOSAGE FORM NEW ANIMAL DRUGS §...

  9. Synthesis of Cellulose Acetate from Cotton Byproducts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton burr and cottonseed hull are relatively inexpensive cotton byproducts. In an effort to derive greater value out of these natural renewable materials, we have succeeded in converting part of them into cellulose acetate without prior chemical breakdown or physical separation of cellulose, ligni...

  10. Superoxide generation and cytotactic response of irradiated neutrophils

    SciTech Connect

    Eastlund, D.T.; Charbonneau, T.T.

    1988-07-01

    Irradiation of blood components has been used to prevent transfusion-related graft-versus-host disease (GVHD) in immunocompromised patients. This study was designed to determine the effect of irradiation on neutrophil aggregation, chemotaxis, and superoxide generation. Purified neutrophils were irradiated with a Cesium source at four doses ranging from 0 to 17,500 rads. Formyl-methionyl-leucyl-phenylalanine (FMLP) and zymosan-treated serum (ZTS) cytotaxin-induced chemotaxis and migration were determined in the agarose assay. Neutrophil aggregation to FMLP was determined by aggregometry. Superoxide generation and random migration were not affected by irradiation at doses up to 17,500 rads. When compared to nonirradiated controls, the chemotactic response to ZTS remained normal, with an insignificant decline from 174 +/- 31.0 to 150 +/- 42.3 (mean +/- SD) units. The chemotactic response to FMLP declined insignificantly, from 228 +/- 31.3 at 0 rad to 207 +/- 26.4 at 17,500 rads. The aggregation response to FMLP remained within the normal range but declined from 0.78 +/- 0.11 to 0.61 +/- 0.18. At the radiation doses currently used to reduce the risk of transfusion-related GVHD, neutrophil superoxide generation and chemotactic response remain essentially normal.

  11. TRPC6 regulates CXCR2-mediated chemotaxis of murine neutrophils.

    PubMed

    Lindemann, Otto; Umlauf, Daniel; Frank, Svetlana; Schimmelpfennig, Sandra; Bertrand, Jessica; Pap, Thomas; Hanley, Peter J; Fabian, Anke; Dietrich, Alexander; Schwab, Albrecht

    2013-06-01

    Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor-mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca(2+) influx but not Ca(2+) store release was attenuated in TRPC6(-/-) neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6(-/-) mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

  12. Immunomodulating action of low intensity millimeter waves on primed neutrophils.

    PubMed

    Safronova, Valentina G; Gabdoulkhakova, A G; Santalov, B F

    2002-12-01

    Comparative investigation of the susceptibility of intact and primed neutrophils of the NMRI strain mice to low intensity millimeter wave (mm wave) irradiation (41.95 GHz) was performed. The specific absorption rate was 0.45 W/kg. Isolated neutrophils were primed by a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) at a subthreshold concentration of 10 nM for 20 min, and then the cells were activated by 1 microM fMLP. Production of the reactive oxygen species (ROS) was estimated by the luminol dependent chemiluminescence technique. It was found that the preliminary mm wave irradiation of the resting cells at 20 degrees C did not act on the ROS production induced by the chemotactic peptide. The exposure of the primed cells results in a subsequent increase in the fMLP response. Therefore, the primed neutrophils are susceptible to the mm waves. Specific inhibitors of the protein kinases abolished the mm wave effect on the primed cells. The data indicate that protein kinases actively participate in transduction of the mm wave signal to effector molecules involved in neutrophil respiratory burst.

  13. Surface Acoustic Waves Enhance Neutrophil Killing of Bacteria

    PubMed Central

    Loike, John D.; Plitt, Anna; Kothari, Komal; Zumeris, Jona; Budhu, Sadna; Kavalus, Kaitlyn; Ray, Yonatan; Jacob, Harold

    2013-01-01

    Biofilms are structured communities of bacteria that play a major role in the pathogenicity of bacteria and are the leading cause of antibiotic resistant bacterial infections on indwelling catheters and medical prosthetic devices. Failure to resolve these biofilm infections may necessitate the surgical removal of the prosthetic device which can be debilitating and costly. Recent studies have shown that application of surface acoustic waves to catheter surfaces can reduce the incidence of infections by a mechanism that has not yet been clarified. We report here the effects of surface acoustic waves (SAW) on the capacity of human neutrophils to eradicate S. epidermidis bacteria in a planktonic state and within biofilms. Utilizing a novel fibrin gel system that mimics a tissue-like environment, we show that SAW, at an intensity of 0.3 mW/cm2, significantly enhances human neutrophil killing of S. epidermidis in a planktonic state and within biofilms by enhancing human neutrophil chemotaxis in response to chemoattractants. In addition, we show that the integrin CD18 plays a significant role in the killing enhancement observed in applying SAW. We propose from out data that this integrin may serve as mechanoreceptor for surface acoustic waves enhancing neutrophil chemotaxis and killing of bacteria. PMID:23936303

  14. ACTIVATED NEUTROPHILS INHIBIT PHAGOCYTOSIS BY HUMAN MONOCYTE CELLS IN VITRO

    EPA Science Inventory

    We have previously reported the correlation of decreased phagocytosis of opsonized zymosan by sputum monocytic cells with the increase in sputum neutrophils in volunteers 6h after inhalation of endotoxin (20,000 EU) (Alexis, et al. JACI, 2003;112:353). To define whether an intrin...

  15. Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis

    PubMed Central

    Jaeger, Baptiste N.; Donadieu, Jean; Cognet, Céline; Bernat, Claire; Ordoñez-Rueda, Diana; Barlogis, Vincent; Mahlaoui, Nizar; Fenis, Aurore; Narni-Mancinelli, Emilie; Beaupain, Blandine; Bellanné-Chantelot, Christine; Bajénoff, Marc; Malissen, Bernard; Malissen, Marie

    2012-01-01

    Natural killer (NK) cells are bone marrow (BM)–derived granular lymphocytes involved in immune defense against microbial infections and tumors. In an N-ethyl N-nitrosourea (ENU) mutagenesis strategy, we identified a mouse mutant with impaired NK cell reactivity both in vitro and in vivo. Dissection of this phenotype showed that mature neutrophils were required both in the BM and in the periphery for proper NK cell development. In mice lacking neutrophils, NK cells displayed hyperproliferation and poor survival and were blocked at an immature stage associated with hyporesponsiveness. The role of neutrophils as key regulators of NK cell functions was confirmed in patients with severe congenital neutropenia and autoimmune neutropenia. In addition to their direct antimicrobial activity, mature neutrophils are thus endowed with immunoregulatory functions that are conserved across species. These findings reveal novel types of cooperation between cells of the innate immune system and prompt examination of NK cell functional deficiency in patients suffering from neutropenia-associated diseases. PMID:22393124

  16. Cellular memory: Neutrophil orientation reverses during temporally decreasing chemoattractant concentrations

    PubMed Central

    Albrecht, Eric; Petty, Howard R.

    1998-01-01

    Cell directional orientation or shape polarization is the first cellular step in neutrophil locomotion. To better understand how chemoattractants interact with cells, we studied neutrophil polarization (or shape changes) during exposure to a temporally decreasing chemoattractant signal of N-formyl-methionyl-leucyl-phenylalanine (FMLP) in the absence of a spatial concentration gradient. To accomplish this objective, we used a manifold of differing FMLP concentrations attached to a stopped-flow microscope chamber. Spatial gradients of a fluorescent chemotactic peptide could not be detected in the chamber by using microfluorometry. When FMLP was injected at continually increasing concentrations at 10-s intervals, the shape and relative direction of the neutrophil persisted. However, when temporally decreasing FMLP concentrations were injected, ≈80% of the cells changed their direction with 44% of the total cells swinging about to 180° ± 15°. Most of these directional changes involved dissolution of both the lamellipodium and uropod and reformation of these structures 180° from their original positions. This research suggests that neutrophils reverse their morphological polarity when exposed to temporally decreasing ligand concentrations by “remembering” their ligand exposure history and relative direction. PMID:9560224

  17. Cyanate-mediated inhibition of neutrophil myeloperoxidase activity.

    PubMed Central

    Qian, M; Eaton, J W; Wolff, S P

    1997-01-01

    Cyanate (CNO-) forms spontaneously in solutions containing urea, and is present in urine and the body fluids of uraemic patients. We have explored the possibility that CNO- might be one of the unknown substances responsible for the reported impairment, by urine and uraemic plasma, of neutrophil oxidative metabolism (especially as measured by luminol-enhanced chemiluminescence). Luminol-enhanced chemiluminescence generated by human neutrophils derives predominantly from the activity of myeloperoxidase (MPO) which produces hypochlorous acid from H2O2 and Cl-. We hypothesized that CNO- (which resembles the 'pseudohalide' thiocyanate, an alternative substrate for MPO) might somehow interfere with the activity of MPO. In support of this, we find: (i) CNO- inhibits both peroxidative and halogenating activities of MPO and also inhibits the enzyme within intact human neutrophils; (ii) the inhibition is H2O2-dependent, irreversible, accompanied by covalent addition of [14C]CNO- (or a carbon-containing fragment thereof) to the enzyme; (iii) CNO- also inhibits Cl-/H2O2/MPO-mediated bacterial killing. Impairment of this arm of neutrophil bactericidal activity by CNO- formed from urea may be one factor in the risk of urinary-tract infection associated with urinary stasis and perhaps in the generalized increase in susceptibility to infection in uraemic patients. PMID:9337863

  18. Regulation of the autophagic machinery in human neutrophils.

    PubMed

    Mitroulis, Ioannis; Kourtzelis, Ioannis; Kambas, Konstantinos; Rafail, Stavros; Chrysanthopoulou, Akrivi; Speletas, Matthaios; Ritis, Konstantinos

    2010-05-01

    The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.

  19. Antiinflammatory benzimidazole derivative with inhibitory effects on neutrophil function.

    PubMed

    Lazer, E S; Farina, P R; Oliver, J T; Possanza, G J; Matteo, M R

    1987-08-01

    5-Methyl-2,2,2-trifluoroethylsulfonyl-1H-benzimidazole (BI-L-45 XX) inhibits both neutrophil enzyme release and chemotaxis in vitro and also inhibits chemotaxis in vivo. BI-L-45 XX has an IC50 between 16 microM and 25 microM in inhibiting lysosomal enzyme release from human peripheral blood neutrophils. In a Boyden chamber experiment, BI-L-45 XX inhibited migration in response to fMLP with an IC50 of 5 microM. When given orally to passively sensitized rats at doses of 0.1 to 1.0 mg/kg, it inhibited migration of neutrophils to the pleural cavity in response to an antigen (ovalbumin) challenge. BI-L-45 XX also shows activity in the developing adjuvant arthritis model, with an ED50 of 45 mg/kg, while exhibiting no significant inhibition of cyclooxygenase in a human platelet assay. This suggests the possibility that its antiinflammatory activity may be in part mediated by its effect on neutrophil function.

  20. Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions

    PubMed Central

    Sawant, Kirti V.; Poluri, Krishna Mohan; Dutta, Amit K.; Sepuru, Krishna Mohan; Troshkina, Anna; Garofalo, Roberto P.; Rajarathnam, Krishna

    2016-01-01

    The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury. PMID:27625115

  1. Granulocytic Spongiotic Papulovesiculosis (Neutrophilic Spongiosis): A Rare Entity

    PubMed Central

    Mendiratta, Vibhu; Sanke, Sarita; Ramchander; Nangia, Anita

    2017-01-01

    Neutrophilic spongiosis also known as granulocytic spongiotic papulovesiculosis (GSPV) is an uncommon disorder of uncertain classification. We report the case of a 45-year-old woman suffering from recurrent episodes of itchy, grouped papulovesicles over her body, histologically showing granulocytic spongiosis. The eruptions showed complete response to dapsone. PMID:28216731

  2. Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis

    PubMed Central

    Rada, Balázs

    2017-01-01

    Cystic fibrosis (CF) affects 70,000 patients worldwide. Morbidity and mortality in CF is largely caused by lung complications due to the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Cystic fibrosis airway inflammation is mediated by robust infiltration of polymorphonuclear neutrophil granulocytes (PMNs, neutrophils). Neutrophils are not capable of clearing lung infections and contribute to tissue damage by releasing their dangerous cargo. Pseudomonas aeruginosa is an opportunistic pathogen causing infections in immunocompromised individuals. P. aeruginosa is a main respiratory pathogen in CF infecting most patients. Although PMNs are key to attack and clear P. aeruginosa in immunocompetent individuals, PMNs fail to do so in CF. Understanding why neutrophils cannot clear P. aeruginosa in CF is essential to design novel therapies. This review provides an overview of the antimicrobial mechanisms by which PMNs attack and eliminate P. aeruginosa. It also summarizes current advances in our understanding of why PMNs are incapable of clearing P. aeruginosa and how this bacterium adapts to and resists PMN-mediated killing in the airways of CF patients chronically infected with P. aeruginosa. PMID:28282951

  3. Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

    PubMed

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

    2016-08-01

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

  4. Loss of Lung WWOX Expression Causes Neutrophilic Inflammation.

    PubMed

    Singla, Sunit; Chen, Jiwang; Sethuraman, Shruthi; Sysol, Justin R; Gampa, Amulya; Zhao, Shuangping; Machado, Roberto F

    2017-03-10

    The tumor suppressor, WWOX, exhibits regulatory interactions with an array of transcription factors and signaling molecules that are positioned at the well-known crossroads between inflammation and cancer. WWOX is also subject to downregulation by genotoxic environmental exposures, making it of potential interest to the study of lung pathobiology. Knockdown of lung WWOX expression in mice was observed to cause neutrophil influx, and accompanied by a corresponding vascular leak and inflammatory cytokine production. In cultured human alveolar epithelial cells, loss of WWOX expression resulted in increased c-Jun- and IL-8- dependent neutrophil chemotaxis towards cell monolayers. WWOX was observed to directly interact with c-Jun in these cells, and its absence resulted in increased nuclear translocation of c-Jun. Finally, inhibition of c-Jun activating kinase, JNK, abrogated the lung neutrophil influx observed during WWOX knockdown in mice. Altogether, these observations represent a novel mechanism of pulmonary neutrophil influx that is highly relevant to the pathobiology and potential treatment of a number of different lung inflammatory conditions.

  5. Coexistence of chronic neutrophilic leukemia with light chain myeloma.

    PubMed

    Cehreli, C; Undar, B; Akkoc, N; Onvural, B; Altungoz, O

    1994-01-01

    A 60-year-old woman who presented with weakness, night sweats, bone pain, easy bruising and weight loss was found to have ecchymoses and hepatosplenomegaly. Blood counts showed persistent neutrophilia of mature cell type with Döhle bodies and toxic granulation. Coexistence of chronic neutrophilic leukemia and multiple myeloma of kappa light chain type was documented by bone marrow examination and immunofixation.

  6. Acetal phosphatidic acids: novel platelet aggregating agents.

    PubMed

    Brammer, J P; Maguire, M H; Walaszek, E J; Wiley, R A

    1983-05-01

    1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 muM for human platelets and 0.25-0.5 muM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.3 PGAP-induced irreversible aggregation of [(14)C]-5-hydroxytryptamine ([(14)C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0+/-2.4% (s.e.) of the platelet [(14)C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [(14)C]-5-HT-labelled sheep platelets was dose-dependent.4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A(2) inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E(1) (PGE(1)); these agents abolished the second, but not the first phase of ADP-induced aggregation.5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 muM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 muM.6 It is concluded that the acetal phosphatidic acids

  7. Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis.

    PubMed Central

    Koch, A E; Kunkel, S L; Harlow, L A; Mazarakis, D D; Haines, G K; Burdick, M D; Pope, R M; Walz, A; Strieter, R M

    1994-01-01

    We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs

  8. Neutrophil Extracellular Traps in ANCA-Associated Vasculitis

    PubMed Central

    Söderberg, Daniel; Segelmark, Mårten

    2016-01-01

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

  9. Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

    PubMed Central

    Mariano, F.S.; Campanelli, A.P.; Nociti, F.H.; Mattos-Graner, R.O.; Gonçalves, R.B.

    2012-01-01

    Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens. PMID:22850872

  10. Bromelain treatment decreases neutrophil migration to sites of inflammation.

    PubMed

    Fitzhugh, David J; Shan, Siqing; Dewhirst, Mark W; Hale, Laura P

    2008-07-01

    Bromelain, a mixture of proteases derived from pineapple stem, has been reported to have therapeutic benefits in a variety of inflammatory diseases, including murine inflammatory bowel disease. The purpose of this work was to understand potential mechanisms for this anti-inflammatory activity. Exposure to bromelain in vitro has been shown to remove a number of cell surface molecules that are vital to leukocyte trafficking, including CD128a/CXCR1 and CD128b/CXCR2 that serve as receptors for the neutrophil chemoattractant IL-8 and its murine homologues. We hypothesized that specific proteolytic removal of CD128 molecules by bromelain would inhibit neutrophil migration to IL-8 and thus decrease acute responses to inflammatory stimuli. Using an in vitro chemotaxis assay, we demonstrated a 40% reduction in migration of bromelain- vs. sham-treated human neutrophils in response to rhIL-8. Migration to the bacterial peptide analog fMLP was unaffected, indicating that bromelain does not induce a global defect in leukocyte migration. In vivo bromelain treatment generated a 50-85% reduction in neutrophil migration in 3 different murine models of leukocyte migration into the inflamed peritoneal cavity. Intravital microscopy demonstrated that although in vivo bromelain treatment transiently decreased leukocyte rolling, its primary long-term effect was abrogation of firm adhesion of leukocytes to blood vessels at the site of inflammation. These changes in adhesion were correlated with rapid re-expression of the bromelain-sensitive CD62L/L-selectin molecules that mediate rolling following in vivo bromelain treatment and minimal re-expression of CD128 over the time period studied. Taken together, these studies demonstrate that bromelain can effectively decrease neutrophil migration to sites of acute inflammation and support the specific removal of the CD128 chemokine receptor as a potential mechanism of action.

  11. Peripheral blood neutrophil cytokine hyper-reactivity in chronic periodontitis.

    PubMed

    Ling, Martin R; Chapple, Iain L C; Matthews, John B

    2015-10-01

    Pro-inflammatory cytokine release (IL-8, IL-6, TNF-α, IL-1β) by peripheral blood neutrophils, isolated from periodontitis patients (before/after therapy) and matched controls, was determined after 18 h culture in the presence/absence of Escherichia coli LPS, opsonised Staphylococcus aureus, heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis. All cultures demonstrated differences in the amounts of each cytokine detected (P < 0.0001), with a clear release pattern (IL-8 > IL-6 > TNF-α = IL-1β). Median cytokine release from unstimulated patient neutrophils was consistently, but non-significantly, higher than from control cells. Stimulated cytokine release from untreated patient neutrophils was also consistently higher than from control cells. This hyper-reactivity was significant for all tested cytokines when data for all stimuli were combined (P < 0.016). In terms of individual stimuli, significant hyper-reactivity was detected with LPS (IL-8), F. nucleatum (IL-8, TNF-α), opsonised S. aureus (IL-8, TNF-α, IL-1β) and P. gingivalis (IL-8, IL-1β). Cytokine production by patient neutrophils did not reduce following successful non-surgical periodontal therapy and, except for responses to F. nucleatum, the cytokine hyper-reactivity detected pre-therapy was retained. These data demonstrate that chronic periodontitis is characterised by neutrophils that constitutively exhibit cytokine hyper-reactivity, the effects of which could modulate local and systemic inflammatory-immune responses and influence the risk and severity of periodontitis-associated systemic inflammatory diseases.

  12. Conjunctival Neutrophils Predict Progressive Scarring in Ocular Mucous Membrane Pemphigoid

    PubMed Central

    Williams, Geraint P.; Nightingale, Peter; Southworth, Sue; Denniston, Alastair K. O.; Tomlins, Paul J.; Turner, Stephen; Hamburger, John; Bowman, Simon J.; Curnow, S. John; Rauz, Saaeha

    2016-01-01

    Purpose Ocular mucous membrane pemphigoid (OcMMP) is a rare autoimmune disorder resulting in progressive conjunctival fibrosis and ocular surface failure leading to sight loss in up to 50%. This study was designed to optimize an ocular surface sampling technique for identification of novel biomarkers associated with disease activity and/or progressive fibrosis. Methods Fifty-seven patients with OcMMP underwent detailed examination of conjunctival inflammation and fibrosis using fornix depth measurement. Ocular surface impression cytology (OSIC) to sample superior bulbar conjunctiva combined with flow cytometry (OSIC-flow) profiled infiltrating leukocytes. Profiles were compared with healthy controls (HC) and disease controls (primary Sjögren's syndrome, pSS). Thirty-five OcMMP patients were followed every 3 months for 12 months. Results Overall neutrophils were elevated in OcMMP eyes when compared to pSS or HC (109 [18%] neutrophils/impression [NPI]; 2 [0.2%]; 6 [0.8%], respectively [P < 0.0001]) and in OcMMP patients with no visible inflammation when compared with HC (44.3 [7.9%]; 5.8 [0.8%]; P < 0.05). At 12 months follow-up, 53% of OcMMP eyes progressed, and this was associated with baseline conjunctival neutrophilia (P = 0.004). As a potential biomarker, a value of 44 NPI had sensitivity, specificity, and positive predictive values of 75%, 70%, and 73%, respectively. Notably, eyes with no visible inflammation and raised conjunctival neutrophils were more likely to progress and have a greater degree of conjunctival shrinkage compared to those without raised neutrophils. Conclusions These data suggest that OSIC-flow cytometric analyses may facilitate repeated patient sampling. Neutrophils may act as a biomarker for monitoring disease activity, progressive fibrosis, and response to therapy in OcMMP even when the eye appears clinically uninflamed. PMID:27760272

  13. Bromelain Treatment Decreases Neutrophil Migration to Sites of Inflammation

    PubMed Central

    Fitzhugh, David J.; Shan, Siqing; Dewhirst, Mark W.; Hale, Laura P.

    2008-01-01

    Bromelain, a mixture of proteases derived from pineapple stem, has been reported to have therapeutic benefits in a variety of inflammatory diseases, including murine inflammatory bowel disease. The purpose of this work was to understand potential mechanisms for this anti-inflammatory activity. Exposure to bromelain in vitro has been shown to remove a number of cell surface molecules that are vital to leukocyte trafficking, including CD128a/CXCR1 and CD128b/CXCR2 that serve as receptors for the neutrophil chemoattractant IL-8 and its murine homologues. We hypothesized that specific proteolytic removal of CD128 molecules by bromelain would inhibit neutrophil migration to IL-8 and thus decrease acute responses to inflammatory stimuli. Using an in vitro chemotaxis assay, we demonstrated a 40% reduction in migration of bromelain- vs. sham-treated human neutrophils in response to rhIL-8. Migration to the bacterial peptide analog fMLP was unaffected, indicating that bromelain does not induce a global defect in leukocyte migration. In vivo bromelain treatment generated a 50 – 85% reduction in neutrophil migration in 3 different murine models of leukocyte migration into the inflamed peritoneal cavity. Intravital microscopy demonstrated that although in vivo bromelain treatment transiently decreased leukocyte rolling, its primary long-term effect was abrogation of firm adhesion of leukocytes to blood vessels at the site of inflammation. These changes in adhesion were correlated with rapid re-expression of the bromelain-sensitive CD62L/L-selectin molecules that mediate rolling following in vivo bromelain treatment and minimal re-expression of CD128 over the time period studied. Taken together, these studies demonstrate that bromelain can effectively decrease neutrophil migration to sites of acute inflammation and support the specific removal of the CD128 chemokine receptor as a potential mechanism of action. PMID:18482869

  14. Interaction between arsenic trioxide (ATO) and human neutrophils.

    PubMed

    Binet, François; Chiasson, Sonia; Girard, Denis

    2011-05-01

    The cytotoxic effect of arsenic trioxide (ATO) is known to be mediated by its ability to induce cell apoptosis in a variety of cells, including neutrophils. More recently, we demonstrated that ATO induced several parameters involved in endoplasmic reticulum (ER) stress-induced neutrophil apoptosis but that caspase-4 was not involved. The aim of this study was to better understand how neutrophils are activated by ATO and to further demonstrate that ATO is an ER stressor. Human neutrophils were isolated from healthy blood donors and incubated in vitro in the presence or absence of ATO and several parameters were investigated. We found that ATO induced the expression of the proapoptotic GADD153 protein, a key player involved in ER stress-induced apoptosis, activated nuclear nuclear factor κB (NF-κB) DNA binding activities, and increased prostaglandine E2 (PGE2) production. Using an antibody array approach, we found that ATO increased the production of several cytokines, with interleukin 8 (IL-8) being the predominant one. We confirmed that ATO increased the production of IL-8 by enzyme-linked-immunosorbent assay (ELISA). Treatment with a caspase-4 inhibitor did not inhibit IL-8 production. The results of the present study further support the notion that ATO is an ER stressor and that, although its toxic effect is mediated by induction of apoptosis, this chemical also induced, in parallel, NF-κB activation, the production of PGE2 and several cytokines probably involved in other cell functions. Also, we conclude that the production of IL-8 is not induced by a caspase-4-dependent mechanism, suggesting that ATO-induced caspase-4 activation is involved in other as yet unidentified functions in human neutrophils.

  15. Phenyl Acetate Preparation from Phenol and Acetic Acid: Reassessment of a Common Textbook Misconception.

    ERIC Educational Resources Information Center

    Hocking, M. B.

    1980-01-01

    Reassesses a common textbook misconception that "...phenols cannot be esterified directly." Results of experiments are discussed and data tables provided of an effective method for the direct preparation of phenyl acetate. (CS)

  16. The microwave spectrum of n-hexyl acetate and structural aspects of n-alkyl acetates

    NASA Astrophysics Data System (ADS)

    Attig, T.; Kannengießer, R.; Kleiner, I.; Stahl, W.

    2014-04-01

    The microwave spectrum of n-hexyl acetate was recorded in the range of 10-13.5 GHz using the Aachen MB-FTMW spectrometer. The rotational constants of the most abundant conformer were determined to be A = 3.3591100(32) GHz, B = 0.39596553(53) GHz, and C = 0.36999804(31) GHz. Quantum chemical calculations for specific conformers were carried out at the MP2/6-311++G(d,p) level. The programs XIAM and BELGI were used to analyze the internal rotation of the acetyl methyl group. The observed conformer of n-hexyl acetate was compared to the lowest energy conformers of n-butyl acetate and n-pentyl acetate.

  17. Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow

    PubMed Central

    Devi, Sapna; Wang, Yilin; Chew, Weng Keong; Lima, Ronald; A-González, Noelia; Mattar, Citra N.Z.; Chong, Shu Zhen; Schlitzer, Andreas; Bakocevic, Nadja; Chew, Samantha; Keeble, Jo L.; Goh, Chi Ching; Li, Jackson L.Y.; Evrard, Maximilien; Malleret, Benoit; Larbi, Anis; Renia, Laurent; Haniffa, Muzlifah; Tan, Suet Mien; Chan, Jerry K.Y.; Balabanian, Karl; Nagasawa, Takashi; Bachelerie, Françoise; Hidalgo, Andrés; Ginhoux, Florent; Kubes, Paul

    2013-01-01

    Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4–CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4–CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients. PMID:24081949

  18. Assessment of the Developmental Toxicity of Propylene Glycol Monomethyl Ether Acetate (PM Acetate) in Rats

    DTIC Science & Technology

    1989-12-01

    Lyiql §hIi r --- I . ISTRACT (Continue on reverse if Aocessary ntify by block number) his study evaluated tfte pot-,,i I maternal, embryotoxic and...RATS DECEMBER 1989 1. PURPOSE. We performed this study to evaluate the potential maternal, embryotoxic and teratogenic parameters of PM Acetate in...We performed this study to evaluate the potential maternal, embryotoxic and teratogenic parameters of PM Acetate in Sprague-Dawley rats following

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

    PubMed

    Bajrami, Besnik; Zhu, Haiyan; Kwak, Hyun-Jeong; Mondal, Subhanjan; Hou, Qingming; Geng, Guangfeng; Karatepe, Kutay; Zhang, Yu C; Nombela-Arrieta, César; Park, Shin-Young; Loison, Fabien; Sakai, Jiro; Xu, Yuanfu; Silberstein, Leslie E; Luo, Hongbo R

    2016-09-19

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

  20. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus.

    PubMed

    Wardini, Amanda B; Guimarães-Costa, Anderson B; Nascimento, Michelle T C; Nadaes, Natalia R; Danelli, Maria G M; Mazur, Carlos; Benjamim, Claudia F; Saraiva, Elvira M; Pinto-da-Silva, Lucia H

    2010-01-01

    Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats compared with FeLV(+) symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV(+) symptomatic cats were higher than those in unstimulated neutrophils from FeLV(-) and FeLV(+) asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV(+) cats and that this feature could be considered a marker of disease progression in FeLV infection.

  1. Lipopolysaccharide: a p38 MAPK-dependent disrupter of neutrophil chemotaxis.

    PubMed

    Khan, Adil I; Heit, Bryan; Andonegui, Graciela; Colarusso, Pina; Kubes, Paul

    2005-01-01

    In sepsis, and in models of sepsis including endotoxemia, impaired neutrophil recruitment and chemotaxis have been reported. The inability of the endotoxemic neutrophil to chemotax could be attributed to the fact that intracellular signaling via LPS overrides signals from endogenous chemokines or, alternatively, that sequestration of neutrophils into lungs prevents access to peripheral tissues. Using both in vitro and in vivo chemotaxis assays the authors established that neutrophils from healthy mice chemotaxed in vivo toward MIP-2, whereas endotoxemic neutrophils did not. Since LPS activates leukocytes via the p38 MAPK pathway, SKF86002, a p38 MAPK inhibitor, was given to endotoxemic animals. SKF86002 significantly reversed the LPS-induced impairment in emigration of endotoxic neutrophils in response to MIP-2. Neutrophil chemotaxis in vitro was also impaired by LPS, via a p38 MAPK-dependent pathway, and this impairment could be reversed via p38 MAPK inhibition. Although neutrophil numbers dropped in the circulation and trapped in lungs during endotoxemia, SKF86002 did not reverse these parameters, demonstrating that p38 MAPK inhibition did not release trapped neutrophils from the lungs. In conclusion, the data suggest that the impaired emigration and chemotaxis of neutrophils at peripheral sites during endotoxemia may be partially due to a p38 MAPK-mediated inhibition of neutrophil responses to endogenous chemokines.

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

    PubMed

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

    2009-10-01

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

  3. ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

    PubMed

    Woodfin, Abigail; Beyrau, Martina; Voisin, Mathieu-Benoit; Ma, Bin; Whiteford, James R; Hordijk, Peter L; Hogg, Nancy; Nourshargh, Sussan

    2016-02-18

    Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

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

    PubMed Central

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

    2016-01-01

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

  5. Partial correction of neutrophil dysfunction by oral galactose therapy in glycogen storage disease type Ib.

    PubMed

    Letkemann, Rudolf; Wittkowski, Helmut; Antonopoulos, Aristotelis; Podskabi, Teodor; Haslam, Stuart M; Föll, Dirk; Dell, Anne; Marquardt, Thorsten

    2017-03-01

    Glycogen storage disease type Ib (GSD-Ib) is characterized by impaired glucose homeostasis, neutropenia and neutrophil dysfunction. Mass spectrometric glycomic profiling of GSD-Ib neutrophils showed severely truncated N-glycans, lacking galactose. Experiments indicated the hypoglycosylation of the electron transporting subunit of NADPH oxidase, which is crucial for the defense against bacterial infections. In phosphoglucomutase 1 (PGM1) deficiency, an inherited disorder with an enzymatic defect just one metabolic step ahead, hypogalactosylation can be successfully treated by dietary galactose. We hypothesized the same pathomechanism in GSD-Ib and started a therapeutic trial with oral galactose and uridine. The aim was to improve neutrophil dysfunction through the correction of hypoglycosylation in neutrophils. The GSD-Ib patient was treated for 29weeks. Monitoring included glycomics analysis of the patient's neutrophils and neutrophil function tests including respiratory burst activity, phagocytosis and migration. Although no substantial restoration of neutrophil glycosylation was found, there was partial improvement of respiratory burst activity.

  6. Sex Hormones Coordinate Neutrophil Immunity in the Vagina by Controlling Chemokine Gradients.

    PubMed

    Lasarte, Sandra; Samaniego, Rafael; Salinas-Muñoz, Laura; Guia-Gonzalez, Mauriel A; Weiss, Linnea A; Mercader, Enrique; Ceballos-García, Elena; Navarro-González, Teresa; Moreno-Ochoa, Laura; Perez-Millan, Federico; Pion, Marjorie; Sanchez-Mateos, Paloma; Hidalgo, Andres; Muñoz-Fernandez, Maria A; Relloso, Miguel

    2016-02-01

    Estradiol-based contraceptives and hormonal replacement therapy predispose women to Candida albicans infections. Moreover, during the ovulatory phase (high estradiol), neutrophil numbers decrease in the vaginal lumen and increase during the luteal phase (high progesterone). Vaginal secretions contain chemokines that drive neutrophil migration into the lumen. However, their expression during the ovarian cycle or in response to hormonal treatments are controversial and their role in vaginal defense remains unknown.To investigate the transepithelial migration of neutrophils, we used adoptive transfer of Cxcr2(-/-) neutrophils and chemokine immunofluorescence quantitative analysis in response to C. albicans vaginal infection in the presence of hormones.Our data show that the Cxcl1/Cxcr2 axis drives neutrophil transepithelial migration into the vagina. Progesterone promotes the Cxcl1 gradient to favor neutrophil migration. Estradiol disrupts the Cxcl1 gradient and favors neutrophil arrest in the vaginal stroma; as a result, the vagina becomes more vulnerable to pathogens.

  7. Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

    SciTech Connect

    Ahluwalia, Jatinder

    2008-10-31

    Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I{sub e}. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 {mu}M), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

  8. An overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration

    PubMed Central

    Selders, Gretchen S.; Fetz, Allison E.; Radic, Marko Z.; Bowlin, Gary L.

    2017-01-01

    Despite considerable recent progress in defining neutrophil functions and behaviors in tissue repair, much remains to be determined with regards to its overall role in the tissue integration of biomaterials. This article provides an overview of the neutrophil’s numerous, important roles in both inflammation and resolution, and subsequently, their role in biomaterial integration. Neutrophils function in three primary capacities: generation of oxidative bursts, release of granules and formation of neutrophil extracellular traps (NETs); these combined functions enable neutrophil involvement in inflammation, macrophage recruitment, M2 macrophage differentiation, resolution of inflammation, angiogenesis, tumor formation and immune system activation. Neutrophils exhibit great flexibility to adjust to the prevalent microenvironmental conditions in the tissue; thus, the biomaterial composition and fabrication will potentially influence neutrophil behavior following confrontation. This review serves to highlight the neutrophil’s plasticity, reiterating that neutrophils are not just simple suicidal killers, but the true maestros of resolution and regeneration. PMID:28149530

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

    PubMed Central

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

    2015-01-01

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

  10. Differential uptake of grepafloxacin by human circulating blood neutrophils and those exudated into tissues.

    PubMed

    Niwa, M; Hotta, K; Kanamori, Y; Matsuno, H; Kozawa, O; Hirota, M; Uematsu, T

    2001-09-28

    The uptake of the antimicrobial quinolone agent, grepafloxacin, both by human circulating blood neutrophils and by those exudated into tissues, was evaluated in vitro by comparing the intracellular drug concentrations. In circulating blood neutrophils, the uptake of grepafloxacin was rapid and saturable at 37 degrees C. The uptake of grepafloxacin into circulating blood neutrophils was reduced by lowering the environmental temperature or by the presence of metabolic inhibitors, suggesting the involvement of an active transport mechanism. Furthermore, the uptake of grepafloxacin by tissue (salivary) neutrophils was also partially temperature-dependent and was significantly greater than that by circulating blood neutrophils, i.e. exudation of neutrophils into tissue results in a markedly enhanced transport mechanism for grepafloxacin. This phenomenon may be related to the higher defense activity against infection seen in exudated tissue neutrophils.

  11. Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils

    PubMed Central

    Funchal, Giselle A.; Jaeger, Natália; Czepielewski, Rafael S.; Machado, Mileni S.; Muraro, Stéfanie P.; Stein, Renato T.; Bonorino, Cristina B. C.; Porto, Bárbara N.

    2015-01-01

    Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis. PMID:25856628

  12. Viscometric study of chitosan solutions in acetic acid/sodium acetate and acetic acid/sodium chloride.

    PubMed

    Costa, Cristiane N; Teixeira, Viviane G; Delpech, Marcia C; Souza, Josefa Virginia S; Costa, Marcos A S

    2015-11-20

    A viscometric study was carried out at 25°C to assess the physical-chemical behavior in solution and the mean viscometric molar mass (M¯v) of chitosan solutions with different deacetylation degrees, in two solvent mixtures: medium 1-acetic acid 0.3mol/L and sodium acetate 0.2mol/L; and medium 2-acetic acid 0.1mol/L and sodium chloride 0.2mol/L. Different equations were employed, by graphical extrapolation, to calculate the intrinsic viscosities [η] and the viscometric constants, to reveal the solvent's quality: Huggins (H), Kraemer (K) and Schulz-Blaschke (SB). For single-point determination, the equations used were SB, Solomon-Ciuta (SC) and Deb-Chanterjee (DC), resulting in a faster form of analysis. The values of ̄M¯v were calculated by applying the equation of Mark-Houwink-Sakurada. The SB and SC equations were most suitable for single-point determination of [η] and ̄M¯v and the Schulz-Blachke constant (kSB), equal to 0.28, already utilized for various systems, can also be employed to analyze chitosan solutions under the conditions studied.

  13. Nasal pungency, odor, and eye irritation thresholds for homologous acetates.

    PubMed

    Cometto-Muñiz, J E; Cain, W S

    1991-08-01

    We measured detection thresholds for nasal pungency (in anosmics), odor (in normosmics) and eye irritation employing a homologous series of acetates: methyl through octyl acetate, decyl and dodecyl acetate. All anosmics reliably detected the series up to heptyl acetate. Only the anosmics without smell since birth (congenital) reliably detected octyl acetate, and only one congenital anosmic detected decyl and dodecyl acetate. Anosmics who lost smell from head trauma proved to be selectively less sensitive. As expected, odor thresholds lay well below pungency thresholds. Eye irritation thresholds for selected acetates came close to nasal pungency thresholds. All three types of thresholds decreased logarithmically with carbon chain length, as previously seen with homologous alcohols and as seen in narcotic and toxic phenomena. Results imply that nasal pungency for these stimuli rests upon a physical, rather than chemical, interaction with susceptible mucosal structures. When expressed as thermodynamic activity, nasal pungency thresholds remain remarkably constant within and across the homologous series of acetates and alcohols.

  14. Donor dependent, interferon-γ induced HLA-DR expression on human neutrophils in vivo

    PubMed Central

    REINISCH, W; LICHTENBERGER, C; STEGER, G; TILLINGER, W; SCHEINER, O; GANGL, A; MAURER, D; WILLHEIM, M

    2003-01-01

    Neutrophils are effector cells of innate immune responses. Stimulated by interferon-γ (IFN-γ) to express HLA-DR, neutrophils acquire accessory cell functions for superantigen-mediated T cell activation. In vitro HLA-DR induction on neutrophils varies in a functionally relevant way as levels of MHC class II expression and magnitude of neutrophil induced T cell responses are correlated functions. The aim of this study was to assess whether IFN-γ induces HLA-DR on human neutrophils in a donor dependent fashion in vivo and to define regulatory events operative in MHC class II expression of neutrophils. In vivo administration of rhIFN-γ in 55 patients with renal cell carcinoma resulted in a varying increase of HLA-DR on neutrophils. By setting a cut-off for response at>10% HLA-DR positive neutrophils, HLA-DR responders (51%) were as frequent as nonresponders (49%). In vivo kinetic studies revealed a peak expression of HLA-DR on neutrophils 48 h after rhIFN-γ application, while nonresponders remained HLA-DR negative over a 72-h period. In vitro IFN-γ stimulated neutrophils recapitulated the response profiles observed in vivo. No differences in IFN-γ dependent CD64 and invariant chain expression, and IFN-γ serum levels were observed among the response subgroups. HLA-DR mRNA was detected in neutrophils from rhIFN-γ treated responders and nonresponders, HLA-DR protein solely in lysates of responder neutrophils. IFN-γ stimulated HLA-DR expression on neutrophils is subject to donor dependent variations in vivo, which result from rather post-transcriptional than transcriptional regulation. Due to their abundance in inflammatory reactions heterogeneous HLA-DR expression by neutrophils could determine the outcome of superantigen-driven diseases. PMID:12930377

  15. Expression of Acetate Permease-like (apl) Genes in Subsurface Communities of Geobacter Species Under Fluctuating Acetate Concentrations

    SciTech Connect

    Elifantz, H; N'Guessan, A L; Mouser, Paula; Williams, Kenneth H; Wilkins, Michael J; Risso, Carla; Holmes, Dawn; Long, Philip E; Lovley, Derek R

    2010-09-01

    The addition of acetate to uranium-contaminated aquifers in order to stimulate the growth and activity of Geobacter species that reduce uranium is a promising in situ bioremediation option. Optimizing this bioremediation strategy requires that sufficient acetate be added to promote Geobacter species growth. We hypothesized that under acetate-limiting conditions, subsurface Geobacter species would increase the expression of either putative acetate symporters genes (aplI and aplII). Acetate was added to a uranium-contaminated aquifer (Rifle, CO) in two continuous amendments separated by 5 days of groundwater flush to create changing acetate concentrations. While the expression of aplI in monitoring well D04 (high acetate) weakly correlated with the acetate concentration over time, the transcript levels for this gene were relatively constant in well D08 (low acetate). At the lowest acetate concentrations during the groundwater flush, the transcript levels of aplII were the highest. The expression of aplII decreased 2–10-fold upon acetate reintroduction. However, the overall instability of acetate concentrations throughout the experiment could not support a robust conclusion regarding the role of apl genes in response to acetate limitation under field conditions, in contrast to previous chemostat studies, suggesting that the function of a microbial community cannot be inferred based on lab experiments alone.

  16. Expression of acetate permease-like (apl) genes in subsurface communities of Geobacter species under fluctuating acetate concentrations

    SciTech Connect

    Elifantz, H.; N'Guessan, L.A.; Mouser, P.J.; Williams, K H.; Wilkins, M J.; Risso, C.; Holmes, D.E.; Long, P.E.; Lovley, D.R.

    2010-03-01

    The addition of acetate to uranium-contaminated aquifers in order to stimulate the growth and activity of Geobacter species that reduce uranium is a promising in situ bioremediation option. Optimizing this bioremediation strategy requires that sufficient acetate be added to promote Geobacter species growth. We hypothesized that under acetate-limiting conditions, subsurface Geobacter species would increase the expression of either putative acetate symporters genes (aplI and aplII). Acetate was added to a uranium-contaminated aquifer (Rifle, CO) in two continuous amendments separated by 5 days of groundwater flush to create changing acetate concentrations. While the expression of aplI in monitoring well D04 (high acetate) weakly correlated with the acetate concentration over time, the transcript levels for this gene were relatively constant in well D08 (low acetate). At the lowest acetate concentrations during the groundwater flush, the transcript levels of aplII were the highest. The expression of aplII decreased 2-10-fold upon acetate reintroduction. However, the overall instability of acetate concentrations throughout the experiment could not support a robust conclusion regarding the role of apl genes in response to acetate limitation under field conditions, in contrast to previous chemostat studies, suggesting that the function of a microbial community cannot be inferred based on lab experiments alone.

  17. Separating acetic acid from furol (furfural) by electrodialysis method

    SciTech Connect

    Guan, S.F.; Li, C.S. Ye, S.T.; Shen, S.Y.; Wang, Y.T.; Yu, S.H.

    1981-01-01

    Furfural production by hydrolysis of fibrous plant materials is accompanied by formation of acetic acid in amounts depending on the material used. The amount of acetic formed in the hydrolysis of the fruit shell of oil-tea camellia (Camellia oleosa) (an oilseed-bearing tree) is equal to the amount of furfural. The acetic acid can be separated from the furfural and concentrated to 10% by electrodialysis. A smaller amount of furfural is separated with acetic acid.

  18. Warm-up exercise suppresses platelet-eosinophil/neutrophil aggregation and platelet-promoted release of eosinophil/neutrophil oxidant products enhanced by severe exercise in men.

    PubMed

    Wang, Jong-Shyan; Yen, Hsiang-Ling; Yang, Chuen-Mao

    2006-03-01

    Heterotypic platelet-eosinophil/neutrophil aggregation and subsequent release of eosinophil/neutrophil oxidant products contribute to pathogenesis of conditions such as asthma and inflammatory bowel diseases. This study investigates whether warmup exercise (WUE) affects platelet-eosinophil/neutrophil interaction mediated by high-intensity exercise (HIE). Twenty-three healthy sedentary men performed on three occasions light-intensity exercise (LIE, 40%VO(2 max) for 40 min) and HIE (80%VO(2 max) for 40 min) with and without WUE (40%VO(2 max) for 20 min). Before and immediately after exercise, platelet-eosinophil and platelet-neutrophil aggregation (PEA and PNA), reactive oxygen species production of eosinophils and neutrophils (EROS and NROS) enhanced by platelets, and adhesion molecule expression on platelets, eosinophils, and neutrophils were measured. The results of this study demonstrated that HIE enhanced PEA, PNA, and platelet-induced EROS and NROS, was accompanied by increased expressions of Mac-1 on eosinophils and neutrophils and P-selectin on platelets at 5 dyne/cm(2) of shear stress, 100 microg/ml lipopolysaccharide, and 1 microM N-formylmethionyl- leucyl-phenylalanine treatments, whereas the enhancement of HIE on platelet-eosinophil/neutrophil interaction was suppressed by WUE. Conversely, LIE significantly reduced PEA and PNA, suppressed platelet-induced EROS and NROS, and down-regulated eosinophil/neutrophil Mac-1 and platelet P-selectin expressions under various stimuli and shear flow conditions. Moreover, these effects were more pronounced in platelet interaction with eosinophils than with neutrophils. It is concluded that HIE enhances hetero-aggregation, adhesion molecules expressions, and subsequent oxidative bursts mediated by platelets and eosinophils/neutrophils, this effect diminishes after WUE. However, LIE minimizes the risk of thromboinflammation.

  19. Leuprolide acetate and central retinal vein occlusion.

    PubMed

    Federici, Thomas J

    2007-01-01

    A 63-year-old man suffered a central retinal vein occlusion 2 months after he began taking leuprolide acetate for prostate cancer. Despite control for possible systemic hypertension (126/90 mm Hg) and mild hypercholesterolemia (total cholesterol level =246 mg/dL [range: 16 to 200 mg/dL], high-density lipoprotein level =67 mg/dL [range: 40 to 59 mg/dL], and low-density lipoprotein level =144 mg/dL [range: 0 to 130 mg/dL]), progression of the venous occlusive disease occurred. Leuprolide acetate, which is associated with thromboembolic events and diffuse intravascular coagulation, may be implicated in central retinal vein occlusion.

  20. Acetic acid vapor levels associated with facial prosthetics

    SciTech Connect

    McElroy, T.H.; Guerra, O.N.; Lee, S.A.

    1985-01-01

    The use of Silastic Medical Adhesive Type A in the fabrication of facial prostheses may cause health hazards to the patient and the operator because of acetic acid emissions. Caution must be exercised to remove acetic acid vapors from the air and unliberated acetic acid from material applied directly to the skin.

  1. 21 CFR 582.5933 - Vitamin A acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Vitamin A acetate. 582.5933 Section 582.5933 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5933 Vitamin A acetate. (a) Product. Vitamin A acetate. (b) Conditions of use....

  2. 21 CFR 582.5933 - Vitamin A acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Vitamin A acetate. 582.5933 Section 582.5933 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5933 Vitamin A acetate. (a) Product. Vitamin A acetate. (b) Conditions of use....

  3. 21 CFR 582.5933 - Vitamin A acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Vitamin A acetate. 582.5933 Section 582.5933 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5933 Vitamin A acetate. (a) Product. Vitamin A acetate. (b) Conditions of use....

  4. 21 CFR 582.5933 - Vitamin A acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Vitamin A acetate. 582.5933 Section 582.5933 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5933 Vitamin A acetate. (a) Product. Vitamin A acetate. (b) Conditions of use....

  5. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Ethyl alcohol containing ethyl acetate. 584.200... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100...

  6. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Ethyl alcohol containing ethyl acetate. 584.200... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100...

  7. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Ethyl alcohol containing ethyl acetate. 584.200... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100...

  8. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Ethyl alcohol containing ethyl acetate. 584.200... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100...

  9. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ethyl alcohol containing ethyl acetate. 584.200... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100...

  10. Acetate concentrations and oxidation in salt marsh sediments

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Acetate concentrations and rates of acetate oxidation and sulfate reduction were measured in S. alterniflora sediments in New Hampshire and Massachusetts. Pore water extracted from cores by squeezing or centrifugation contained in greater than 0.1 mM acetate and, in some instances, greater than 1.0 mM. Pore water sampled nondestructively contained much less acetate, often less than 0.01 mM. Acetate was associated with roots, and concentrations varied with changes in plant physiology. Acetate turnover was very low whether whole core or slurry incubations were used. Radiotracers injected directly into soils yielded rates of sulfate reduction and acetate oxidation not significantly different from core incubation techniques. Regardless of incubation method, acetate oxidation did not account for a substantial percentage of sulfate reduction. These results differ markedly from data for unvegetated coastal sediments where acetate levels are low, oxidation rate constants are high, and acetate oxication rates greatly exceed rates of sulfate reduction. The discrepancy between rates of acetate oxidation and sulfate reduction in these marsh soils may be due either to the utilization of substrates other than acetate by sulfate reducers or artifacts associated with measurements of organic utilization by rhizosphere bacteria. Care must be taken when interpreting data from salt marsh sediments since the release of material from roots during coring may affect the concentrations of certain compounds as well as influencing results obtained when sediment incubations are employed.

  11. 21 CFR 582.5933 - Vitamin A acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Vitamin A acetate. 582.5933 Section 582.5933 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5933 Vitamin A acetate. (a) Product. Vitamin A acetate. (b) Conditions of use....

  12. 21 CFR 177.1350 - Ethylene-vinyl acetate copolymers.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ethylene-vinyl acetate copolymers. 177.1350... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1350 Ethylene-vinyl acetate copolymers. Ethylene-vinyl acetate copolymers may be safely used as articles or components of...

  13. 21 CFR 177.1350 - Ethylene-vinyl acetate copolymers.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ethylene-vinyl acetate copolymers. 177.1350 Section... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1350 Ethylene-vinyl acetate copolymers. Ethylene-vinyl acetate copolymers may be safely used as articles or components of...

  14. 21 CFR 177.1350 - Ethylene-vinyl acetate copolymers.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ethylene-vinyl acetate copolymers. 177.1350... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1350 Ethylene-vinyl acetate copolymers. Ethylene-vinyl acetate copolymers may be safely used as articles or components of...

  15. 21 CFR 177.1350 - Ethylene-vinyl acetate copolymers.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ethylene-vinyl acetate copolymers. 177.1350... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1350 Ethylene-vinyl acetate copolymers. Ethylene-vinyl acetate copolymers may be safely used as articles or components of...

  16. Kinetics of Ethyl Acetate Synthesis Catalyzed by Acidic Resins

    ERIC Educational Resources Information Center

    Antunes, Bruno M.; Cardoso, Simao P.; Silva, Carlos M.; Portugal, Ines

    2011-01-01

    A low-cost experiment to carry out the second-order reversible reaction of acetic acid esterification with ethanol to produce ethyl acetate is presented to illustrate concepts of kinetics and reactor modeling. The reaction is performed in a batch reactor, and the acetic acid concentration is measured by acid-base titration versus time. The…

  17. 21 CFR 522.2477 - Trenbolone acetate and estradiol.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... milligrams (mg) trenbolone acetate and 24 mg estradiol (one implant consisting of 6 pellets, each pellet containing 20 mg trenbolone acetate and 4 mg estradiol) per implant dose. (B) 120 mg trenbolone acetate and 24 mg estradiol (one implant consisting of 7 pellets, each of 6 pellets containing 20 mg...

  18. 21 CFR 522.2477 - Trenbolone acetate and estradiol.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... milligrams (mg) trenbolone acetate and 24 mg estradiol (one implant consisting of 6 pellets, each pellet containing 20 mg trenbolone acetate and 4 mg estradiol) per implant dose. (B) 120 mg trenbolone acetate and 24 mg estradiol (one implant consisting of 7 pellets, each of 6 pellets containing 20 mg...

  19. Activated neutrophils injure the isolated, perfused rat liver by an oxygen radical-dependent mechanism.

    PubMed Central

    Dahm, L. J.; Schultze, A. E.; Roth, R. A.

    1991-01-01

    Under certain circumstances, segmented neutrophils (PMNs) injure extrahepatic tissue by releasing toxic oxygen species and degradative enzymes. The authors used an isolated, perfused rat liver preparation to determine whether PMNs might injure the liver. Livers from fasted rats were perfused with Krebs-Ringer bicarbonate buffer (pH 7.4) containing 3% bovine serum albumin (BSA) in a recirculating system. Rat peritoneal PMNs (4 x 10(8] or vehicle (Hank's balanced salt solution [HBSS], pH 7.35) were added, and liver injury was assessed 90 minutes later by release of alanine aminotransferase (ALT) into the perfusion medium and histopathologic analysis of liver sections. Perfusion of livers receiving only HBSS for 90 minutes resulted in a small increase in ALT activity in the perfusion medium but did not significantly alter histologic features of liver sections. Addition of unstimulated PMNs did not increase further the ALT activity and, with the exception of vascular neutrophilia, did not significantly change the histomorphology compared with controls. When PMNs activated with a combination of phorbol myristate acetate (PMA, 31 ng/ml) and lithocholate (100 mumol/l [micromolar]) were added to the perfusion system, however, livers released greater amounts of ALT than those perfused with PMA, lithocholate, and HBSS. Activated PMNs caused a transient reduction in flow of perfusion medium that lasted approximately 5 to 15 minutes. Liver sections had multifocal to coalescing foci of moderate to severe, acute hepatocellular necrosis associated with the areas of intense sinusoidal neutrophilia. In addition a second type of lesion was observed and was characterized by triangular foci of necrosis located adjacent to periportal regions of sinusoids or portal veins containing neutrophilic thrombi. These lesions were void of PMNs and were consistent with infarcts. A combination of superoxide dismutase and catalase added to the perfusion medium (500 U/ml each) prevented the

  20. Ultrasound-assisted dyeing of cellulose acetate.

    PubMed

    Udrescu, C; Ferrero, F; Periolatto, M

    2014-07-01

    The possibility of reducing the use of auxiliaries in conventional cellulose acetate dyeing with Disperse Red 50 using ultrasound technique was studied as an alternative to the standard procedure. Dyeing of cellulose acetate yarn was carried out by using either mechanical agitation alone, with and without auxiliaries, or coupling mechanical and ultrasound agitation in the bath where the temperature range was maintained between 60 and 80 °C. The best results of dyeing kinetics were obtained with ultrasound coupled with mechanical agitation without auxiliaries (90% of bath exhaustion value at 80 °C). Hence the corresponding half dyeing times, absorption rate constants according to Cegarra-Puente modified equation and ultrasound efficiency were calculated confirming the synergic effect of sonication on the dyeing kinetics. Moreover the apparent activation energies were also evaluated and the positive effect of ultrasound added to mechanical agitation was evidenced by the lower value (48 kJ/mol) in comparison with 112 and 169 kJ/mol for mechanical stirring alone with auxiliaries and without, respectively. Finally, the fastness tests gave good values for samples dyed with ultrasound technique even without auxiliaries. Moreover color measurements on dyed yarns showed that the color yield obtained by ultrasound-assisted dyeing at 80 °C of cellulose acetate without using additional chemicals into the dye bath reached the same value yielded by mechanical agitation, but with remarkably shorter time.

  1. The neutrophil-to-lymphocyte ratio: a narrative review

    PubMed Central

    Faria, Sara Socorro; Fernandes, Paulo César; Silva, Marcelo José Barbosa; Lima, Vladmir C; Fontes, Wagner; Freitas-Junior, Ruffo; Eterovic, Agda Karina; Forget, Patrice

    2016-01-01

    Cellular-mediated inflammatory response, lymphocytes, neutrophils, and monocytes are increasingly being recognised as having an important role in tumorigenesis and carcinogenesis. In this context, studies have suggested that the neutrophil-to-lymphocyte ratio (NLR) can be used as an independent prognostic factor in a variety of cancers. Particularly in breast cancer, several studies have shown that a high NLR is associated with shorter survival. Because the NLR can be easily determined from the full blood count, it could potentially provide a simple and inexpensive test cancer prognosis. This review addresses the possibilities and limitations of using the NLR as a clinical tool for risk stratification helpful for individual treatment of breast cancer patients. The potential underlying phenomena and some perspectives are discussed. PMID:28105073

  2. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy

    PubMed Central

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer. PMID:27618112

  3. The pore-forming toxin listeriolysin O is degraded by neutrophil metalloproteinase-8 and fails to mediate Listeria monocytogenes intracellular survival in neutrophils.

    PubMed

    Arnett, Eusondia; Vadia, Stephen; Nackerman, Colleen C; Oghumu, Steve; Satoskar, Abhay R; McLeish, Kenneth R; Uriarte, Silvia M; Seveau, Stephanie

    2014-01-01

    The pore-forming toxin listeriolysin O (LLO) is a major virulence factor secreted by the facultative intracellular pathogen Listeria monocytogenes. This toxin facilitates L. monocytogenes intracellular survival in macrophages and diverse nonphagocytic cells by disrupting the internalization vesicle, releasing the bacterium into its replicative niche, the cytosol. Neutrophils are innate immune cells that play an important role in the control of infections, yet it was unknown if LLO could confer a survival advantage to L. monocytogenes in neutrophils. We report that LLO can enhance the phagocytic efficiency of human neutrophils and is unable to protect L. monocytogenes from intracellular killing. To explain the absence of L. monocytogenes survival in neutrophils, we hypothesized that neutrophil degranulation leads to the release of LLO-neutralizing molecules in the forming phagosome. In support of this, L. monocytogenes is a potent inducer of neutrophil degranulation, since its virulence factors, such as LLO, facilitate granule exocytosis. Within the first few minutes of interaction with L. monocytogenes, granules can fuse with the plasma membrane at the bacterial interaction site before closure of the phagosome. Furthermore, granule products directly degrade LLO, irreversibly inhibiting its activity. The matrix metalloproteinase-8, stored in secondary granules, was identified as an endoprotease that degrades LLO, and blocking neutrophil proteases increased L. monocytogenes intracellular survival. In conclusion, we propose that LLO degradation by matrix metalloproteinase-8 during phagocytosis protects neutrophil membranes from perforation and contributes to maintaining L. monocytogenes in a bactericidal phagosome from which it cannot escape.

  4. Phase-field approach to chemotactic driving of neutrophil morphodynamics

    NASA Astrophysics Data System (ADS)

    Najem, Sara; Grant, Martin

    2013-09-01

    To simulate the motion of neutrophils and their morphodynamics in response to chemical cues, we construct a model based on the phase-field method utilizing a description with a free-energy functional and associated dynamics which captures the basic features of the phenomenon. We additionally incorporate spatial sensing by introducing an auxiliary field which depicts the polymerization of the region of the cell facing the highest concentration of the chemical attractant.

  5. Comprehensive multiplexed protein quantitation delineates eosinophilic and neutrophilic experimental asthma

    PubMed Central

    2014-01-01

    Background Improvements in asthma diagnosis and management require deeper understanding of the heterogeneity of the complex airway inflammation. We hypothesise that differences in the two major inflammatory phenotypes of asthma; eosinophilic and neutrophilic asthma, will be reflected in the lung protein expression profile of murine asthma models and can be delineated using proteomics of bronchoalveolar lavage (BAL). Methods BAL from mice challenged with ovalbumin (OVA/OVA) alone (standard model of asthma, here considered eosinophilic) or OVA in combination with endotoxin (OVA/LPS, model of neutrophilic asthma) was analysed using liquid chromatography coupled to high resolution mass spectrometry, and compared with steroid-treated animals and healthy controls. In addition, conventional inflammatory markers were analysed using multiplexed ELISA (Bio-Plex™ assay). Multivariate statistics was performed on integrative proteomic fingerprints using principal component analysis. Proteomic data were complemented with lung mechanics and BAL cell counts. Results Several of the analysed proteins displayed significant differences between the controls and either or both of the two models reflecting eosinophilic and neutrophilic asthma. Most of the proteins found with mass spectrometry analysis displayed a considerable increase in neutrophilic asthma compared with the other groups. Conversely, the larger number of the inflammatory markers analysed with Bio-Plex™ analysis were found to be increased in the eosinophilic model. In addition, major inflammation markers were correlated to peripheral airway closure, while commonly used asthma biomarkers only reflect central inflammation. Conclusion Our data suggest that the commercial markers we are currently relying on to diagnose asthma subtypes are not giving us comprehensive or specific enough information. The analysed protein profiles allowed to discriminate the two models and may add useful information for characterization of

  6. Adipocytes and Neutrophils Give a Helping Hand to Pancreatic Cancers.

    PubMed

    Bronte, Vincenzo; Tortora, Giampaolo

    2016-08-01

    Obesity-induced inflammation can build up a confined microenvironment in pancreatic adenocarcinoma that is associated with increased desmoplasia, neutrophil recruitment, reduced delivery of chemotherapeutic drugs, and immune evasion. Targeting molecular pathways empowering this circuit might represent a necessary measure to reach clinical efficacy for combination therapies in patients with excess body weight. Cancer Discov; 6(8); 821-3. ©2016 AACR.See related article by Incio et al., p. 852.

  7. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  8. Central Role of Conventional Dendritic Cells in Regulation of Bone Marrow Release and Survival of Neutrophils

    PubMed Central

    Jiao, Jingjing; Dragomir, Ana-Cristina; Kocabayoglu, Peri; Rahman, Adeeb H.; Chow, Andrew; Hashimoto, Daigo; Leboeuf, Marylene; Kraus, Thomas; Moran, Thomas; Carrasco-Avino, Gonzalo; Friedman, Scott L.; Merad, Miriam; Aloman, Costica

    2014-01-01

    Neutrophils are the most abundant cell type in the immune system and play an important role in the innate immune response. Using a diverse range of mouse models with either defective DC development or conditional DC depletion, we provide in vivo evidence indicating that conventional dendritic cells (cDC) play an important role in the regulation of neutrophil homeostasis. Flk2, Flt3L and Batf3 knockout mice, which have defects in DC development, have increased numbers of liver neutrophils in the steady state. Conversely, neutrophil frequency is reduced in DC-specific PTEN knockout mice, which have an expansion of CD8+ and CD103+ DCs. In chimeric CD11c-DTR mice, cDC depletion results in a systemic increase of neutrophils in peripheral organs in the absence of histological inflammation or an increase in pro-inflammatory cytokines. This effect is also present in splenectomized chimeric CD11c-DTR mice and is absent in chimeric mice with 50% normal bone marrow. In chimeric CD11c-DTR mice, DT treatment results in enhanced neutrophil trafficking from the bone marrow into circulation and increased neutrophil recruitment. Moreover, there is an increased expression of chemokines/cytokines involved in neutrophil homeostasis and reduced neutrophil apoptosis. These data underscore the role of the DC pool in regulating the neutrophil compartment in non-lymphoid organs. PMID:24591364

  9. Neutrophils contact to plasma membrane of keratinocytes including desmosomal structures in canine pemphigus foliaceus.

    PubMed

    Yabuzoe, Atsushi; Nishifuji, Koji; Sekiguchi, Maiko; Shimizu, Atsushi; Momoi, Yasuyuki; Ishiko, Akira; Iwasaki, Toshiroh

    2008-08-01

    Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that affects certain mammals including dogs. In canine PF, neutrophils are infiltrated intensely into pustular lesions including acantholytic cells, although neutrophilic infiltration is not characterized in human PF. The roles of the neutrophils in the cutaneous lesions of canine PF have not yet been understood. The purpose of this study was to characterize the ultrastructural features underlying the acantholysis with pustule formation in canine PF. Four dogs diagnosed as PF on the basis of clinical signs, histopathological findings, and direct and indirect immunofluorescence examinations were performed. Electron microscopy revealed that the acantholytic cells were adjacent to multiple neutrophils in the pustules. At the contact points between neutrophils and acantholytic keratinocytes, half-desmosomes of acantholytic keratinocytes with intact attachment plaques were observed within invaginations of neutrophils. Furthermore, on the surface of acantholytic cells in the pustules, neutrophil granules seemed to be secreted to the surface of acantholytic cells and to degenerate the half-desmosome structures. Neutrophils were also observed within the epidermis adjacent to the pustule. At the intercellular gap between two dissociated keratinocytes, neutrophils inserted its pseudopodia into the gap between the two half-desmosomes of keratinocytes. These findings taken together suggested that, at least in the areas where we analyzed ultrastructurally, neutrophils contact desmosomal structures and seem to play some parts in separation of keratinocytes and degeneration of split-desmosomes in pustules of dogs with PF.

  10. Technical Advance: Changes in neutrophil migration patterns upon contact with platelets in a microfluidic assay.

    PubMed

    Frydman, Galit H; Le, Anna; Ellett, Felix; Jorgensen, Julianne; Fox, James G; Tompkins, Ronald G; Irimia, Daniel

    2017-03-01

    Neutrophils are traditionally regarded as the "first responders" of the immune system. However, recent observations revealed that platelets often respond earlier to recruit and activate neutrophils within sites of injury and inflammation. Currently, platelet-neutrophil interactions are studied by intravital microscopy. Although such studies provide exceptional, physiologic in vivo data, they are also laborious and have low throughput. To accelerate platelet-neutrophil interaction studies, we have developed and optimized an ex vivo microfluidic platform with which the interactions between platelets and moving neutrophils are measured at single-cell level in precise conditions and with high throughput. With the use of this new assay, we have evaluated changes in neutrophil motility upon direct contact with platelets. Motility changes include longer distances traveled, frequent changes in direction, and faster neutrophil velocities compared with a standard motility response to chemoattractant fMLP. We also found that the neutrophil-platelet direct interactions are transient and mediated by CD62P-CD162 interactions, localized predominantly at the uropod of moving neutrophils. This "crawling," oscillatory neutrophil behavior upon platelet contact is consistent with previous in vivo studies and validates the use of this new test for the exploration of this interactive relationship.

  11. Neutrophil recruitment by allergens contribute to allergic sensitization and allergic inflammation

    PubMed Central

    Hosoki, Koa; Boldogh, Istvan; Sur, Sanjiv

    2016-01-01

    Purpose of review To discuss the presence and role of neutrophils in asthma and allergic diseases, and outline importance of pollen and cat dander-induced innate neutrophil recruitment in induction of allergic sensitization and allergic inflammation. Recent findings Uncontrolled asthma is associated with elevated numbers of neutrophils, and levels of neutrophil-attracting chemokine IL-8 and IL-17 in BAL fluids. These parameters negatively correlate with lung function. Pollen allergens and cat dander recruit neutrophils to the airways in a TLR4, MD2 and CXCR2-dependent manner. Repeated recruitment of activated neutrophils by these allergens facilitates allergic sensitization and airway inflammation. Inhibition of neutrophil recruitment with CXCR2 inhibitor, disruption of TLR4, or siRNA against MD2 also inhibits allergic inflammation. The molecular mechanisms by which neutrophils shift the inflammatory response of the airways to inhaled allergens to an allergic phenotype is an area of active research. Summary Recent studies have revealed that neutrophil recruitment is important in development of allergic sensitization and inflammation. Inhibition of neutrophils recruitment may be strategy to control allergic inflammation. PMID:26694038

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

    PubMed

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

    2015-08-01

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

  13. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis

    PubMed Central

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C.; Wilson, Mary E.; Carvalho, Edgar M.; Bacellar, Olívia

    2016-01-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects. PMID:27167379

  14. Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus

    PubMed Central

    Finsterbusch, Michaela; Hall, Pam; Li, Anqi; Devi, Sapna; Westhorpe, Clare L. V.; Kitching, A. Richard

    2016-01-01

    Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclea