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Sample records for neutrophil activity improves

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

    PubMed

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

    2014-08-01

    Therapy with monoclonal antibodies to tumor necrosis factor (TNF)-α and the interleukin (IL)-12/23 p40 subunit has significantly improved the clinical outcome of patients with psoriasis. These antibodies inhibit the effects of the target cytokines and thus the major concern during their use is the induction of excessive immunosuppression. Recent studies evaluating the long-term efficacy and safety of biologic therapy in psoriasis have shown no significant appearance of serious adverse effects including infections and malignancies. However, the immunological consequence and the mechanism by which the blockade of a single cytokine by biologics can successfully control the activity of psoriasis remain unclear. In the current study, we investigated the effect of biologic therapy on cytokine production of various lymphocytes and on the activity of monocytes and neutrophils in psoriatic patients. Neutrophils, monocytes and T cells were purified from heparinized peripheral venous blood by Ficoll density gradient centrifugation, and γ-interferon, TNF-α and IL-17 production from lymphocytes was measured by flow cytometer. The activation maker of neutrophils and the activated subsets of monocytes were also analyzed. Biologic therapy induced no significant changes in the cytokine production by lymphocytes from the skin and gut-homing T cells. However, neutrophil activity and the ratio of activated monocyte population increased in severely psoriatic patients were normalized in psoriatic patients receiving biologic therapy. The present study showed that biologic therapy ameliorates clinical symptoms and controls the immune response in patients with psoriasis. PMID:25099154

  2. Insulin Treatment Directly Restores Neutrophil Phagocytosis and Bactericidal Activity in Diabetic Mice and Thereby Improves Surgical Site Staphylococcus aureus Infection

    PubMed Central

    Yano, Hidekazu; Fujino, Keiichi; Nakashima, Masahiro; Yamamoto, Yoritsuna; Miyazaki, Hiromi; Hamada, Koji; Ono, Satoshi; Iwaya, Keiichi; Saitoh, Daizoh; Seki, Shuhji; Tanaka, Yuji

    2012-01-01

    Bacterial infections, including surgical site infections (SSI), are a common and serious complication of diabetes. Staphylococcus aureus, which is eliminated mainly by neutrophils, is a major cause of SSI in diabetic patients. However, the precise mechanisms by which diabetes predisposes to staphylococcal infection are not fully elucidated. The effect of insulin on this infection is also not well understood. We therefore investigated the effect of insulin treatment on SSI and neutrophil function in diabetic mice. S. aureus was inoculated into the abdominal muscle in diabetic db/db and high-fat-diet (HFD)-fed mice with or without insulin treatment. Although the diabetic db/db mice developed SSI, insulin treatment ameliorated the infection. db/db mice had neutrophil dysfunction, such as decreased phagocytosis, superoxide production, and killing activity of S. aureus; however, insulin treatment restored these functions. Ex vivo treatment (coincubation) of neutrophils with insulin and euglycemic control by phlorizin suggest that insulin may directly activate neutrophil phagocytic and bactericidal activity independently of its euglycemic effect. However, insulin may indirectly restore superoxide production by neutrophils through its euglycemic effect. HFD-fed mice with mild hyperglycemia also developed more severe SSI by S. aureus than control mice and had impaired neutrophil phagocytic and bactericidal activity, which was improved by insulin treatment. Unlike db/db mice, in HFD mice, superoxide production was increased in neutrophils and subsequently suppressed by insulin treatment. Glycemic control by insulin also normalized the neutrophil superoxide-producing capability in HFD mice. Thus, insulin may restore neutrophil phagocytosis and bactericidal activity, thereby ameliorating SSI. PMID:23027538

  3. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    SciTech Connect

    Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  4. Mechanotransduction in neutrophil activation and deactivation.

    PubMed

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

    2015-11-01

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

  5. NET amyloidogenic backbone in human activated neutrophils.

    PubMed

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

    2016-03-01

    Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role. PMID:26462606

  6. Activation of bovine neutrophils by Brucella spp.

    PubMed

    Keleher, Lauren L; Skyberg, Jerod A

    2016-09-01

    Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. PMID:27436438

  7. Exercise, training and neutrophil microbicidal activity.

    PubMed

    Smith, J A; Telford, R D; Mason, I B; Weidemann, M J

    1990-06-01

    The concentration in human plasma of putative neutrophil-"priming" cytokines like endogenous pyrogens is known to increase significantly in response to moderate exercise (11). This is characteristic of an acute-phase response. The ability of blood neutrophils isolated from both trained and untrained human subjects (n = 11, 9) to produce microbicidal reactive oxygen species was determined using luminol-enhanced chemiluminescence both before and after one hour of aerobic exercise at 60% VO2max. Irrespective of training and stimulus concentration, exercise nearly always caused significant "priming" of the capacity of neutrophils to produce H2O2 and HOCl upon stimulation with opsonized zymosan (P less than 0.01); however, compared to their untrained counterparts, the activity of cells isolated from trained individuals was depressed about 50% at unit stimulus concentration, both before and after exercise (P less than 0.075), whilst remaining unaltered at saturating concentrations. Although neutrophil oxygenation activity is only one parameter that contributes to immunological status, regular episodes of moderate exercise may increase resistance to infection by priming the "killing capacity" of neutrophils. In contrast, prolonged periods of intensive training may lead to increased susceptibility to common infections by diminishing this activity. PMID:2115507

  8. Pneumolysin activates neutrophil extracellular trap formation.

    PubMed

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

    2016-06-01

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

  9. 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. PMID:17086359

  10. [Neutrophil activation by sea hydrobiont biopolymers].

    PubMed

    Zaporozhets, T S

    2003-01-01

    Biopolymers of sea hydrobionts such as mytilan, alpha-1,4;1,6-D-glycan isolated from the muntle of the mussel Crenomytilus grayanus; translam, beta-1,3;1,6-D-glucan isolated from the seaweed Laminaria cichorioides; fucoidan, a sulfated polysccharide isolated from the algae Fucus evanescens; zosterin, a pectin isolated from sea grass of the family Zosteraceae were comparatively studied. The mechanisms of the phagocyte activation were investigated and the dose-dependent ability of the biopolymers to increase in vitro adhesion of the intact cells and to restore the neutrophil functions at cyclophosphamide-induced immunodepression was detected. The neutrophil activation by mytilan, zosterin and fucoidan linked with the adhesion potentiation was shown to be associated with their ability to increase the number of the adhesion receptors and in particular CD116b on the cell surface. The lower potential of the neutrophils preincubated in vitro with high doses of translam beta-glucan could be due to blockade of the beta-glucan receptors participating in the complex multicomponent adhesion process. The use of the biopolymers of the sea hydrobionts of the glycobiological nature for modulation of the immunity processes provided rather convenient in vivo management of intracellular processes through direct and competing carbohydrate specific interactions of the modifiers with the membrane receptors and formation of active and inactive lectin-glycoligand and carbohydrate-carbohydrate complexes. PMID:15002173

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

    PubMed

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

    1997-01-01

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

  12. Neutrophil depletion after subarachnoid hemorrhage improves memory via NMDA receptors.

    PubMed

    Provencio, Jose Javier; Swank, Valerie; Lu, Haiyan; Brunet, Sylvain; Baltan, Selva; Khapre, Rohini V; Seerapu, Himabindu; Kokiko-Cochran, Olga N; Lamb, Bruce T; Ransohoff, Richard M

    2016-05-01

    Cognitive deficits after aneurysmal subarachnoid hemorrhage (SAH) are common and disabling. Patients who experience delayed deterioration associated with vasospasm are likely to have cognitive deficits, particularly problems with executive function, verbal and spatial memory. Here, we report neurophysiological and pathological mechanisms underlying behavioral deficits in a murine model of SAH. On tests of spatial memory, animals with SAH performed worse than sham animals in the first week and one month after SAH suggesting a prolonged injury. Between three and six days after experimental hemorrhage, mice demonstrated loss of late long-term potentiation (L-LTP) due to dysfunction of the NMDA receptor. Suppression of innate immune cell activation prevents delayed vasospasm after murine SAH. We therefore explored the role of neutrophil-mediated innate inflammation on memory deficits after SAH. Depletion of neutrophils three days after SAH mitigates tissue inflammation, reverses cerebral vasoconstriction in the middle cerebral artery, and rescues L-LTP dysfunction at day 6. Spatial memory deficits in both the short and long-term are improved and associated with a shift of NMDA receptor subunit composition toward a memory sparing phenotype. This work supports further investigating suppression of innate immunity after SAH as a target for preventative therapies in SAH. PMID:26872422

  13. 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...

  14. 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. PMID:24909063

  15. Blocking neutrophil integrin activation prevents ischemia–reperfusion injury

    PubMed Central

    Yago, Tadayuki; Petrich, Brian G.; Zhang, Nan; Liu, Zhenghui; Shao, Bojing; Ginsberg, Mark H.

    2015-01-01

    Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia–reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin’s capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia–reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin–mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin–mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia–reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable. PMID:26169939

  16. G-CSF improves murine G6PC3-deficient neutrophil function by modulating apoptosis and energy homeostasis

    PubMed Central

    Jun, Hyun Sik; Lee, Young Mok; Song, Ki Duk; Mansfield, Brian C.

    2011-01-01

    G6PC3 (or glucose-6-phosphatase-β) deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced endoplasmic reticulum (ER) stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. Here we show that murine G6pc3−/− neutrophils undergoing ER stress activate protein kinase-like ER kinase and phosphatidylinositol 3,4,5-trisphosphate/Akt signaling pathways, and that neutrophil apoptosis is mediated in part by the intrinsic mitochondrial pathway. In G6PC3-deficient patients, granulocyte colony-stimulating factor (G-CSF) improves neutropenia, but its impact on neutrophil apoptosis and dysfunction is unknown. We now show that G-CSF delays neutrophil apoptosis in vitro by modulating apoptotic mediators. However, G6pc3−/− neutrophils in culture exhibit accelerated apoptosis compared with wild-type neutrophils both in the presence or absence of G-CSF. Limiting glucose (0.6mM) accelerates apoptosis but is more pronounced for wild-type neutrophils, leading to similar survival profiles for both neutrophil populations. In vivo G-CSF therapy completely corrects neutropenia and normalizes levels of p-Akt, phosphatidylinositol 3,4,5-trisphosphate, and active caspase-3. Neutrophils from in vivo G-CSF–treated G6pc3−/− mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and adenosine-5′-triphosphate, leading to improved functionality. Together, the results strongly suggest that G-CSF improves G6pc3−/− neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis. PMID:21292774

  17. Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration.

    PubMed

    Ramachandran, Mohanraj; Jin, Chuan; Yu, Di; Eriksson, Fredrik; Essand, Magnus

    2014-09-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. Both HP-NAP protein and oncolytic viruses encoding HP-NAP have been suggested as immunotherapeutic anticancer agents and adjuvants for vaccination but with little known about its mode of action to activate adaptive immunity. Dendritic cells (DCs) are key players in bridging innate and adaptive immune responses, and in this study we aim to evaluate the effect of HP-NAP on DC maturation, migration, and induction of adaptive immune response. Maturation markers CD83, CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and relatively low IL-10 secretion, and migrated toward CCL19. Ag-specific T cells were efficiently expanded by Ag-presenting HP-NAP-activated DCs, which is an important property of functionally mature DCs. Furthermore, intradermal injections of HP-NAP-encoding adenovirus in C57BL/6 mice enhanced resident DC migration to draining lymph nodes, which was verified by imaging lymph nodes by two-photon microscopy and by phenotyping migrating cells by flow cytometry. In conclusion, therapeutic effects of HP-NAP are mediated by maturation of DCs and subsequent activation of Ag-specific T cells in addition to provoking innate immunity. PMID:25049358

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

    PubMed

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

    1993-06-15

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

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

    PubMed Central

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

    2016-01-01

    Neutrophils play a critical role in the initiation and maintenance of intestinal inflammation. However, conventional neutrophil-targeted therapies can impair normal host defense. Tanshinone IIA has been recently revealed to act directly on neutrophils. Hence, we aimed at investigating whether Tanshinone IIA can protect against experimental colitis through modulation of neutrophils. We induced colitis in C57BL/6 mice by giving 3% dextran sulfate sodium (DSS) orally, and meanwhile, we treated mice daily with Tanshinone IIA intraperitoneally. The severity of colitis was evaluated by calculating disease activity index (DAI) and histological parameters. Neutrophil infiltration and activation in the colons of mice were measured. Moreover, whether Tanshinone IIA has direct effects on neutrophil migration and activation was determined in vitro. Our data showed that Tanshinone IIA significantly ameliorated the severity of DSS-induced colitis in mice, evidenced by the reduced DAI and improved colonic inflammation. In addition, Tanshinone IIA decreased neutrophil infiltration of intestinal mucosa and activation and reduced colonic inflammatory cytokines in DSS-treated mice. Furthermore, Tanshinone IIA was demonstrated to significantly suppress neutrophil migration and activation. These results provide compelling evidence that Tanshinone IIA has a therapeutic potential for alleviating inflammatory colitis in mice, which is possibly mediated by the immunomodulation of neutrophils. PMID:26881040

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

    PubMed

    Ren, Xiaoming; Ding, Yun; Lu, Naihao

    2016-03-15

    Oxidative stress is usually considered as an important factor to the pathogenesis of various diseases. Peroxynitrite (ONOO(-)) and hypochlorite (OCl(-)) are formed in immune cells as a part of the innate host defense system, but excessive reactive oxygen species generation can cause progressive inflammation and tissue damage. It has been proven that through mediating nitric oxide (NO) homeostasis, inorganic nitrite (NO2(-)) shows organ-protective effects on oxidative stress and inflammation. However, the effects of NO2(-) on the function of immune cells were still not clear. The potential role of NO2(-) in modulating ONOO(-) and OCl(-) generation in neutrophil cells was investigated in this study. As an immune cell activator, lipopolysaccharide (LPS) increased both ONOO(-) and OCl(-) production in neutrophils, which was significantly attenuated by NO2(-). NO2(-) reduced superoxide (O2(·-)) generation via a NO-dependent mechanism and increased NO formation in activated neutrophils, suggesting a crucial role of O2(·-) in NO2(-)-mediated reduction of ONOO(-). Moreover, the reduced effect of NO2(-) on OCl(-) production was attributed to that NO2(-) reduced H2O2 production in activated neutrophils without influencing the release of myeloperoxidase (MPO), thus limiting OCl(-) production by MPO/H2O2 system. Therefore, NO2(-) attenuates ONOO(-) and OCl(-) formation in activated neutrophils, opening a new direction to modulate the inflammatory response. PMID:26854590

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

    PubMed

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

    2014-05-15

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

  2. Dimethylfumarate Impairs Neutrophil Functions.

    PubMed

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

    2016-01-01

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

  3. Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils.

    PubMed

    Hebeda, Cristina Bichels; Pinedo, Fernanda Júdice; Bolonheis, Simone Marques; Ferreira, Zulma F; Muscará, Marcelo Nicolas; Teixeira, Simone Aparecida; Farsky, Sandra Helena Poliselli

    2012-11-01

    Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 μM; 2 h) and subsequently incubated with LPS (5 μg/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or β(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor κB (NF-κB) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure. PMID:22717997

  4. Effects of MCI-186 upon neutrophil-derived active oxygens.

    PubMed

    Sumitomo, K; Shishido, N; Aizawa, H; Hasebe, N; Kikuchi, K; Nakamura, M

    2007-01-01

    Reactions of 3-methyl-1-phenyl-2-pyrazoline-5-one (MCI-186) with hypochlorous acid and superoxide were analysed by spectrophotometry and mass spectrometry. The results were applied to the neutrophil system to evaluate the scavenging activity of neutrophil-derived active oxygen species by MCI-186. MCI-186 reacted rapidly with hypochlorous acid (1 x 10(6) M(-1)s(-1)) to form a chlorinated intermediate, followed by a slow conversion to a new spectrum. MCI-186 consumed 3 moles of hypochlorous acid and did not react with superoxide. The newly synthesized fluorescence probes, 2-[6-(4'-amino)-phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) and 2-[6-(4'-hydroxy)phenoxy-3H-anthen-3-on-9-yl]benzoic acid (HPF) successfully detected neutrophil-derived active oxygens (Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003; 278: 3170-3175). The rate constants for the reaction of hypochlorous acid with MCI-186 and fluorescence probes was in the order of MCI-186 > APF > HPF. Fluorescence due to the oxidation of APF and HPF was observed with the stimulated neutrophils. The result that the intensity from APF oxidation was higher than that from HPF oxidation is compatible with reports that APF selectively reacts with hypochlorous acid. Fluorescence due to oxidation of both APF and HPF decreased when the reactions were carried out in the presence of a fluorescence probe and MCI-186 in a dose-dependent manner. These results indicate that MCI-186 effectively scavenges neutrophil-derived hypochlorous acid and other active oxygens. PMID:17705989

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

    PubMed

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

    2016-04-01

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

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

    SciTech Connect

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

    1987-05-01

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

  7. Activation of human neutrophils by mycobacterial phenolic glycolipids

    PubMed Central

    Fäldt, J; Dahlgren, C; Karlsson, A; Ahmed, A M S; Minnikin, D E; Ridell, M

    1999-01-01

    The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells. PMID:10540187

  8. Association of microparticles and neutrophil activation with decompression sickness.

    PubMed

    Thom, Stephen R; Bennett, Michael; Banham, Neil D; Chin, Walter; Blake, Denise F; Rosen, Anders; Pollock, Neal W; Madden, Dennis; Barak, Otto; Marroni, Alessandro; Balestra, Costantino; Germonpre, Peter; Pieri, Massimo; Cialoni, Danilo; Le, Phi-Nga Jeannie; Logue, Christopher; Lambert, David; Hardy, Kevin R; Sward, Douglas; Yang, Ming; Bhopale, Veena B; Dujic, Zeljko

    2015-09-01

    Decompression sickness (DCS) is a systemic disorder, assumed due to gas bubbles, but additional factors are likely to play a role. Circulating microparticles (MPs)--vesicular structures with diameters of 0.1-1.0 μm--have been implicated, but data in human divers have been lacking. We hypothesized that the number of blood-borne, Annexin V-positive MPs and neutrophil activation, assessed as surface MPO staining, would differ between self-contained underwater breathing-apparatus divers suffering from DCS vs. asymptomatic divers. Blood was analyzed from 280 divers who had been exposed to maximum depths from 7 to 105 meters; 185 were control/asymptomatic divers, and 90 were diagnosed with DCS. Elevations of MPs and neutrophil activation occurred in all divers but normalized within 24 h in those who were asymptomatic. MPs, bearing the following proteins: CD66b, CD41, CD31, CD142, CD235, and von Willebrand factor, were between 2.4- and 11.7-fold higher in blood from divers with DCS vs. asymptomatic divers, matched for time of sample acquisition, maximum diving depth, and breathing gas. Multiple logistic regression analysis documented significant associations (P < 0.001) between DCS and MPs and for neutrophil MPO staining. Effect estimates were not altered by gender, body mass index, use of nonsteroidal anti-inflammatory agents, or emergency oxygen treatment and were modestly influenced by divers' age, choice of breathing gas during diving, maximum diving depth, and whether repetitive diving had been performed. There were no significant associations between DCS and number of MPs without surface proteins listed above. We conclude that MP production and neutrophil activation exhibit strong associations with DCS. PMID:26139218

  9. Antioxidant, antimicrobial and neutrophil-modulating activities of herb extracts.

    PubMed

    Denev, Petko; Kratchanova, Maria; Ciz, Milan; Lojek, Antonin; Vasicek, Ondrej; Blazheva, Denitsa; Nedelcheva, Plamena; Vojtek, Libor; Hyrsl, Pavel

    2014-01-01

    The present study provides a comprehensive data on the antioxidant, antimicrobial and neutrophil-modulating activities of extracts from six medicinal plants--blackberry (Rubus fruticosus) leaves, chokeberry (Aronia melanocarpa) leaves, hawthorn (Crataegus monogyna) leaves, lady's mantle (Alchemilla glabra) aerial parts, meadowsweet (Filipendula ulmaria) aerial parts and raspberry (Rubus idaeus) leaves. In order to analyze the antioxidant activity of the herbs, several methods (ORAC, TRAP, HORAC and inhibition of lipid peroxidation) were used. Blackberry leaves and meadowsweet extracts revealed the highest antioxidant activities via all methods. All extracts studied blocked almost completely the opsonized zymosan particle-activated ROS production by neutrophils from human whole blood. On the other hand, the effect of extracts on phorbol myristate acetate-activated ROS production was much milder and even nonsignificant in the case of chokeberry leaves. This latter result suggests that extracts (apart from their antioxidative activity) interfere with the signaling cascade of phagocyte activation upstream of the protein kinase C activation. The antimicrobial activity of the investigated extracts against 11 human pathogens was investigated using three different methods. Meadowsweet and blackberry leaves extracts had the highest antimicrobial effect and the lowest minimal inhibiting concentrations (MICs) against the microorganisms tested. PMID:24945135

  10. ISOLATION OF MOUSE NEUTROPHILS

    PubMed Central

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

    2015-01-01

    Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT 19.6. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments. PMID:26237011

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

    PubMed Central

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

    2007-01-01

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

  12. Activated neutrophils disrupt endothelial monolayer integrity by an oxygen radical-independent mechanism

    SciTech Connect

    Harlan, J.M.; Schwartz, B.R.; Reidy, M.A.; Schwartz, S.M.; Ochs, H.D.; Harker, L.A.

    1985-02-01

    The effect of activated neutrophils on endothelial monolayer integrity in vitro has been measured by assessing the capacity of endothelial monolayers on polycarbonate filters to exclude /sup 125/I-albumin. Although formylmethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils failed to induce /sup 51/Cr-release or detachment after 4 hours of incubation with endothelial monolayers cultured in polystyrene wells, FMLP-activated neutrophils produced a marked increase in the passage of /sup 125/I-albumin across bovine aortic or pulmonary artery endothelial monolayers on polycarbonate filters. This effect was evident as early as 30 minutes following the addition of FMLP-activated neutrophils to the monolayer and reached 180% over control values at 2 hours (p . 0.001). Light and transmission electron microscopic examination of the polycarbonate filters exposed to FMLP-activated neutrophils revealed focal disruption of the endothelial monolayers. Chronic granulomatous disease neutrophils produced similar disruption of the endothelial monolayer at 2 hours. Moreover, catalase and superoxide dismutase failed to reduce significantly the neutrophil-mediated increase in /sup 125/I-albumin passage at 2 hours. Cell-free postsecretory supernatants of FMLP-activated neutrophils, leukotriene C4, and platelet activating factor did not induce a significant increase in /sup 125/I-albumin passage across the endothelial monolayers. Of note, FMLP-activated neutrophils from a patient with a congenital abnormality of neutrophil adhesion and chemotaxis did not induce disruption of the monolayer or increase /sup 125/I-albumin passage.

  13. FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

    PubMed Central

    Worthen, G S; Avdi, N; Buhl, A M; Suzuki, N; Johnson, G L

    1994-01-01

    Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants. Images PMID:8040337

  14. Nanoparticle Targeting of Neutrophils for Improved Cancer Immunotherapy

    PubMed Central

    Chu, Dafeng; Zhao, Qi; Yu, Jian; Zhang, Faya; Zhang, Hui; Wang, Zhenjia

    2016-01-01

    Cancer immunotherapy using tumor specific monoclonal antibodies (mAbs) presents a novel approach for cancer treatment. A monoclonal antibody TA99 specific for gp75 antigen of melanoma, initiates neutrophil recruitment in tumor responsible for cancer therapy. Here we report a strategy for hijacking neutrophils in vivo using nanoparticles (NPs) to deliver therapeutics into tumor. In a mouse model of melanoma, we showed that systemically delivered albumin NPs increased in tumor when TA99 antibody was injected; and the nanoparticle tumor accumulation was mediated by neutrophils. After the administration of pyropheophorbide-a (Ppa) loaded albumin NPs and TA99, photodynamic therapy significantly suppressed the tumor growth and increased mouse survival compared with treatment with the NPs or TA99. The study reveals a new avenue to treat cancer by nanoparticle hitchhiking of immune systems to enhance delivery of therapeutics into tumor sites. PMID:26989887

  15. 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

  16. NFκB Is Persistently Activated in Continuously Stimulated Human Neutrophils

    PubMed Central

    Miskolci, Veronika; Rollins, Janet; Vu, Hai Yen; Ghosh, Chandra C; Davidson, Dennis; Vancurova, Ivana

    2007-01-01

    Increased activation of the transcription factor NFκB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFκB activation in human neutrophils, activation of NFκB in persistently stimulated neutrophils has not been previously studied. Because it is the persistent NFκB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFκB activity. The goal of this study was to analyze the NFκB activation and expression of IκB and NFκB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNFα) induces, in addition to the early activation at 30–60 min, a previously unrecognized late phase of NFκB activation. In LPS-stimulated neutrophils, this NFκB activity typically had a biphasic character, whereas TNFα-stimulated neutrophils exhibited a continuous NFκB activity peaking around 9 h after stimulation. In contrast to the early NFκB activation that inversely correlates to the nuclear levels of IκBα, however, in continuously stimulated neutrophils, NFκB is persistently activated despite considerable levels of IκBα present in the nucleus. Our data suggest that NFκB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFκB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators. PMID:17592547

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

    PubMed Central

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

    2011-01-01

    Helicobacter pylori (H. pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. Within this work we present the implication of C-terminal region of H. pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evidence that the C-terminal region of H. pylori activating protein is indispensable for neutrophil adhesion to endothelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins derived from chios mastic gum, the natural resin of the plant Pistacia lentiscus var. Chia inhibit neutrophil activation in vitro. PMID:21677824

  18. Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins.

    PubMed Central

    Mak, P; Wójcik, K; Thogersen, I B; Dubin, A

    1996-01-01

    Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3. PMID:8890190

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

    PubMed

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

    2016-04-01

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

  20. Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.

    PubMed

    Garratt, Luke W; Sutanto, Erika N; Ling, Kak-Ming; Looi, Kevin; Iosifidis, Thomas; Martinovich, Kelly M; Shaw, Nicole C; Kicic-Starcevich, Elizabeth; Knight, Darryl A; Ranganathan, Sarath; Stick, Stephen M; Kicic, Anthony

    2015-08-01

    Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis.Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis.A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis.Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease. PMID:25929954

  1. Neutrophil-mediated damage to human vascular endothelium. Role of cytokine activation.

    PubMed Central

    Westlin, W. F.; Gimbrone, M. A.

    1993-01-01

    Cytokine activation of cultured human vascular endothelial cells renders them hyperadhesive for blood leukocytes. Co-incubation of freshly isolated, unstimulated human blood neutrophils with confluent cytokine-activated human endothelial monolayers for 90 minutes results in extensive endothelial detachment and destruction of monolayer integrity. In contrast, unactivated endothelial monolayers remain intact. Using this in vitro model, we have explored the neutrophil-effector mechanisms involved in this injury. Coincubation in the presence of a serine protease inhibitor (phenylmethylsulfonyl fluoride) or specific elastase inhibitors (Ala-Ala-Pro-Val-chloromethyl ketone or alpha-1-protease inhibitor) markedly diminished injury. In contrast, scavengers or inhibitors of oxygen-derived free radicals (superoxide dismutase, catalase, mannitol, or sodium azide) were not protective. Purified human neutrophil elastase mimicked the effect of the neutrophils suggesting a key role for elastase in the neutrophil-mediated injury in this model. Interfering with direct neutrophil-endothelial cell contact by interposing a microporous barrier insert prevented endothelial cell detachment. Furthermore, this neutrophil-mediated detachment could be inhibited with interleukin-8, an action correlated with a decrease in neutrophil adhesion to activated endothelial monolayers. By defining the role of endothelial activation in neutrophil-mediated injury, this in vitro model may provide useful insights into potential therapeutic interventions designed to prevent disruption of the endothelial barrier function. Images Figure 1 Figure 6 PMID:8424450

  2. Application of intracellular alkaline phosphatase activity measurement in detection of neutrophil adherence in vitro.

    PubMed

    Bednarska, Katarzyna; Klink, Magdalena; Sulowska, Zofia

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (10(4)-10(6)). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro. PMID:17047286

  3. The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists.

    PubMed

    Chen, Kaiwen W; Bezbradica, Jelena S; Groß, Christina J; Wall, Adam A; Sweet, Matthew J; Stow, Jennifer L; Schroder, Kate

    2016-04-01

    Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL-1β production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL-1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum-induced IL-1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response. PMID:27062120

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

    PubMed

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

    2016-06-01

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

  5. Neutrophils in cancer.

    PubMed

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

    2016-09-01

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

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

    PubMed Central

    Köffel, René; Meshcheryakova, Anastasia; Warszawska, Joanna; Hennig, Annika; Wagner, Karin; Jörgl, Almut; Gubi, Daniela; Moser, Doris; Hladik, Anastasiya; Hoffmann, Ulrike; Fischer, Michael B.; van den Berg, Wim; Koenders, Marije; Scheinecker, Clemens; Gesslbauer, Bernhard; Knapp, Sylvia

    2014-01-01

    During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly “transdifferentiate” into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal–induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor–dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF–pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo–isolated G-CSF–mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils. PMID:25214442

  7. Comparative evaluation of assays for the measurement of bovine neutrophil oxidative burst activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). The objectives of the current stu...

  8. Modelling c-Abl Signalling in Activated Neutrophils: the Anti-inflammatory Effect of Seliciclib.

    PubMed

    Jackson, Robert C; Radivoyevitch, Tomas

    2013-03-01

    When mammalian tissues are infected by bacteria or fungi, inflammatory cytokines are released that cause circulating neutrophils to invade the infected tissue. The cytosolic tyrosine kinase, c-Abl, in these tissue neutrophils is activated by TNFα. c-Abl then phosphorylates STAT transcription factors, which results in production of the antiapoptotic protein Mcl-1. The normally short-lived tissue neutrophils are then unable to enter apoptosis. c-Abl also causes release of reactive oxygen species (ROS) from the mitochondria of the activated neutrophils. These ROS, and ROS generated by NADPH oxidase, are bactericidal agents of the innate immune system. In some inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), the invading neutrophils become permanently activated, and the resulting ROS overproduction causes severe tissue damage. The cyclin-dependent kinase inhibitor, seliciclib, blocks transcription through inhibition of cdk9. This results in a relatively rapid decline of antiapoptotic Mcl-1 transcripts in activated neutrophils, an increase in neutrophil apoptosis, and less ROS leakage and oxidative damage. We present here a model of neutrophil kinetics that simulates the principal pathways of c-Abl signalling and use it to explore possible treatment options for inflammatory lung disease. PMID:24765523

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-07-01

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

  11. Neutrophil ageing is regulated by the microbiome.

    PubMed

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

    2015-09-24

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

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

    PubMed Central

    Murcia, Ruby Yoana; Vargas, Amandine; Lavoie, Jean-Pierre

    2016-01-01

    Background Glucocorticoids (GCs) are the most effective drugs for the treatment of human asthma. However, a subgroup of asthmatic patients with neutrophilic airway inflammation is insensitive to GCs. Interleukin-17 (IL-17), a cytokine upregulated in the airways of a subset of human asthmatic patients, contributes to the recruitment of neutrophils and induces a glucocorticoid resistance in human airway epithelial cells. We hypothesized that IL-17 similarly activates neutrophils and contributes to their persistence in the asthmatic airways in spite of glucocorticoid therapy. Objective To determine whether IL-17 directly activates neutrophils and whether this response is attenuated by GCs. Methods Neutrophils were isolated from the blood of horses and incubated in the presence of recombinant equine IL-17, LPS and dexamethasone. mRNA and protein expression of IL-17 receptors (IL-17RA/IL-17RC) were assessed by qPCR and immunoblot, respectively. Pro-inflammatory cytokine expression, cell viability and apoptosis were determined by qPCR, Trypan Blue exclusion test, and flow cytometry, respectively. Results Equine neutrophils express both IL-17RA and IL-17RC at the mRNA and protein levels. Neutrophil stimulation with IL-17 increases the mRNA expression of IL-8, which is not attenuated by dexamethasone (p = 0.409). Also, neutrophil viability is significantly increased (p<0.0001) by IL-17 in the presence of LPS when compared to LPS alone. Flow cytometry and light microscopy revealed that LPS-induced apoptosis is decreased by IL-17 (p = 0.02 and p = 0.006 respectively). Conclusion These results indicate that IL-17 directly activates equine neutrophils at 24 hours, and that the expression of IL-8 thus induced is not attenuated by GCs. Additionally, IL-17 increases neutrophil viability and decreases apoptosis. These findings suggest an important role of IL-17 in pulmonary persistence of neutrophils in the asthmatic airways. PMID:27138006

  13. Effects of CXC chemokines on neutrophil activation and sequestration in hepatic vasculature.

    PubMed

    Bajt, M L; Farhood, A; Jaeschke, H

    2001-11-01

    The initiating step of neutrophil-induced cytotoxicity in the liver is the recruitment of these phagocytes into sinusoids. The aim of our study was to compare the efficacy of systemic exposure with individual inflammatory mediators on neutrophil activation and sequestration in the hepatic vasculature of C3Heb/FeJ mice as assessed by flow cytometry and histochemistry, respectively. The CXC chemokine macrophage inflammatory protein-2 (MIP-2; 20 microg/kg) induced a time-dependent upregulation of Mac-1 (318% at 4 h) and shedding of L-selectin (41% at 4 h). MIP-2 treatment caused a temporary increase of sinusoidal neutrophil accumulation at 0.5 h [97 +/- 6 polymorphonuclear leukocytes (PMN)/50 high-power fields (HPF)], which declined to baseline (8 +/- 2) at 4 h. The CXC chemokine KC was largely ineffective in activating neutrophils or recruiting them into the liver. Cytokines (tumor necrosis factor-alpha and interleukin-1alpha) and cobra venom factor substantially increased Mac-1 expression and L-selectin shedding on neutrophils and caused stable sinusoidal neutrophil accumulation (170-220 PMN/50 HPF). Only cytokines induced venular neutrophil margination. Thus CXC chemokines in circulation are less effective than cytokines or complement in activation of neutrophils and their recruitment into the hepatic vasculature in vivo. PMID:11668027

  14. Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia.

    PubMed

    McDonald, Braedon; Jenne, Craig N; Zhuo, Lisheng; Kimata, Koji; Kubes, Paul

    2013-12-01

    A key pathological feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4(-/-)Tie2(tlr4)), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan-associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver. PMID:24113769

  15. Effect of acute exercise on some haematological parameters and neutrophil functions in active and inactive subjects.

    PubMed

    Benoni, G; Bellavite, P; Adami, A; Chirumbolo, S; Lippi, G; Brocco, G; Cuzzolin, L

    1995-01-01

    In this work we studied the possible effects of acute exercise on some haematological parameters and on some functions of neutrophils in seven active and six inactive subjects. Physical exercise (10 min on a cycle ergometer at a heart rate of 150 beats.min-1) induced a significant increase in total leucocyte, lymphocyte and neutrophil concentrations in active subjects; serum iron and ferritin concentrations were lower in active compared to inactive subjects. Cellular adhesion, bactericidal activity and superoxide anion production did not change after exercise, while we also observed some differences between active and inactive subjects before exercise. In particular, the neutrophils from active subjects showed a significantly higher percentage of adhesion, higher bactericidal activity and lower superoxide anion production. In conclusion, the training induced changes in some neutrophil functions, while acute exercise influenced, overall, leucocyte concentrations. PMID:7768243

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

    PubMed Central

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

    2016-01-01

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

  17. Intravenous immunoglobulins modulate neutrophil activation and vascular injury through FcγRIII and SHP-1

    PubMed Central

    Jang, Jung-Eun; Hidalgo, Andrés; Frenette, Paul S.

    2012-01-01

    Rationale Intravascular neutrophil recruitment and activation are a key pathogenic factor that contributes to vascular injury. Intravenous immunoglobulin (IVIG) has been shown to have a beneficial effect in systemic inflammatory disorders; however, the mechanisms underlying IVIG’s inhibitory effect on neutrophil recruitment and activation are not understood. Objective We studied the mechanisms by which IVIG exerts protection from neutrophil-mediated acute vascular injury. Methods and Results We examined neutrophil behavior in response to IVIG in vivo using real time intravital microscopy. We found that an antibody that blocks both FcγRIII and its inhibitory receptor counterpart, FcγRIIB, abrogated the inhibitory effect of IVIG on leukocyte recruitment and heterotypic RBC interactions with adherent leukocytes in wild-type mice. In the context of sickle cell disease, the blockade of both FcγRIIB and III abrogated the protective effect of IVIG on acute vaso-occlusive crisis caused by neutrophil recruitment and activation. Analysis of FcγRIIB- and FcγRIII-deficient mice revealed the predominant expression of FcγRIII on circulating neutrophils. FcγRIII mediated IVIG-triggered inhibition of leukocyte recruitment, circulating RBC capture, and enhanced Mac-1 activity, whereas FcγRIIB was dispensable. In addition, FcγRIII-induced IVIG anti-inflammatory activity in neutrophils was mediated by recruitment of Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1). Indeed, the protective effect of IVIG on leukocyte recruitment and activation was abrogated in SHP-1-mutant mice. Conclusions FcγRIII, a classical activating receptor, has an unexpected inhibitory role on neutrophil adhesion and activation via recruitment of SHP-1 in response to IVIG. Our results identify SHP-1 as a therapeutic target in neutrophil-mediated vascular injury. PMID:22415018

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

    SciTech Connect

    Williams, C. David; Bajt, Mary Lynn; Sharpe, Matthew R.; McGill, Mitchell R.; Farhood, Anwar; Jaeschke, Hartmut

    2014-03-01

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

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

    PubMed Central

    Fliss, H; Weissbach, H; Brot, N

    1983-01-01

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

  20. Mechanism of neutrophil activation and toxicity elicited by engineered nanomaterials.

    PubMed

    Johnston, Helinor; Brown, David M; Kanase, Nilesh; Euston, Matthew; Gaiser, Birgit K; Robb, Calum T; Dyrynda, Elisabeth; Rossi, Adriano G; Brown, Euan R; Stone, Vicki

    2015-08-01

    The effects of nanomaterials (NMs) on biological systems, especially their ability to stimulate inflammatory responses requires urgent investigation. We evaluated the response of the human differentiated HL60 neutrophil-like cell line to NMs. It was hypothesised that NM physico-chemical characteristics would influence cell responsiveness by altering intracellular Ca2+ concentration [Ca2+]i and reactive oxygen species production. Cells were exposed (1.95-125 μg/ml, 24 h) to silver (Ag), zinc oxide (ZnO), titanium dioxide (TiO2), multi-walled carbon nanotubes (MWCNTs) or ultrafine carbon black (ufCB) and cytotoxicity assessed (alamar blue assay). Relatively low (TiO2, MWCNTs, ufCB) or high (Ag, ZnO) cytotoxicity NMs were identified. Sub-lethal impacts of NMs on cell function were investigated for selected NMs only, namely TiO2, Ag and ufCB. Only Ag stimulated cell activation. Within minutes, Ag stimulated an increase in [Ca2+]i (in Fura-2 loaded cells), and a prominent inward ion current (assessed by electrophysiology). Within 2-4 h, Ag increased superoxide anion release and stimulated cytokine production (MCP-1, IL-8) that was diminished by Ca2+ inhibitors or trolox. Light microscopy demonstrated that cells had an activated phenotype. In conclusion NM toxicity was ranked; Ag>ufCB>TiO2, and the battery of tests used provided insight into the mechanism of action of NM toxicity to guide future testing strategies. PMID:25962642

  1. Obesity is Associated with More Activated Neutrophils in African American Male Youth

    PubMed Central

    Xu, Xiaojing; Su, Shaoyong; Wang, Xin; Barnes, Vernon; De Miguel, Carmen; Ownby, Dennis; Pollock, Jennifer; Snieder, Harold; Chen, Weiqin; Wang, Xiaoling

    2014-01-01

    Background There is emerging evidence suggesting the role of peripheral blood leukocytes in the pathogenesis of obesity and related diseases. However, few studies have taken a genome wide approach to investigating gene expression profiles in peripheral leukocytes between obese and lean individuals with the consideration of obesity related shifts in leukocyte types. Method We conducted this study in 95 African Americans of both genders (age 14-20, 46 lean and 49 obese). Complete blood count with differential test (CBC) was performed in whole blood. Genome wide gene expression analysis was obtained using Illumina HumanHT-12 V4 Beadchip with RNA extracted from peripheral leukocytes. Out of the 95 participants, 64 had neutrophils stored. The validation study was based on Real-time polymerase chain reaction with RNA extracted from purified neutrophils. Results CBC test suggested that in males, obesity was associated with increased neutrophil percentage (p=0.03). Genome wide gene expression analysis showed that in males, the majority of the most differentially expressed genes were related to neutrophil activation. Validation of the gene expression levels of ELANE (neutrophil elastase) and MPO (myeloperoxidase) in purified neutrophils demonstrated that the expression of these two genes – important biomarkers of neutrophils activation – were significantly elevated in obese males (p=0.01 and p=0.02, respectively). Conclusion The identification of increased neutrophil percentage and activation in obese African American males suggests that neutrophils play an essential role in the pathogenesis of obesity related disease. Further functional and mechanistic studies on neutrophils may contribute to the development of novel intervention strategies reducing the burden associated with obesity-related health problems. PMID:25388404

  2. A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

    PubMed Central

    García-García, Erick; Uribe-Querol, Eileen; Rosales, Carlos

    2013-01-01

    Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types. PMID:23603868

  3. Inflammatory response, neutrophil activation, and free radical production after acute myocardial infarction: effect of thrombolytic treatment.

    PubMed Central

    Bell, D; Jackson, M; Nicoll, J J; Millar, A; Dawes, J; Muir, A L

    1990-01-01

    Activated neutrophils releasing proteolytic enzymes and oxygen free radicals have been implicated in extending myocardial injury after myocardial infarction. Neutrophil elastase was used as a marker of neutrophil activation and the non-peroxide diene conjugate of linoleic acid was used as an indicator of free radical activity in 32 patients after acute myocardial infarction; 17 were treated by intravenous thrombolysis. Patients with acute myocardial infarction had higher plasma concentrations of neutrophil elastase and the non-peroxide diene conjugated isomer of linoleic acid than normal volunteers or patients with stable ischaemic heart disease. Patients treated by thrombolysis had an early peak of neutrophil elastase at eight hours while those who had not been treated by thrombolysis showed a later peak 40 hours after infarction. The plasma concentration of non-peroxide conjugated diene of linoleic acid was highest 16 hours after the infarction irrespective of treatment by thrombolysis. Quantitative imaging with single photon emission tomography showed decreased uptake of indium-111 labelled neutrophils in the infarcted myocardium (as judged from technetium-99m pyrophosphate) in those who had received thrombolysis, suggesting a decreased inflammatory response. The results indicate increased neutrophil activation and free radical production after myocardial infarction; they also suggest that thrombolysis does not amplify the inflammatory response and may indeed suppress it. Images PMID:2317413

  4. DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis.

    PubMed

    Kunisaki, Yuya; Nishikimi, Akihiko; Tanaka, Yoshihiko; Takii, Ryosuke; Noda, Mayuko; Inayoshi, Ayumi; Watanabe, Ken-ichi; Sanematsu, Fumiyuki; Sasazuki, Takehiko; Sasaki, Takehiko; Fukui, Yoshinori

    2006-08-28

    Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation. PMID:16943182

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

    PubMed

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

    2015-06-01

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

  6. Transformation of lupus-inducing drugs to cytotoxic products by activated neutrophils.

    PubMed

    Jiang, X; Khursigara, G; Rubin, R L

    1994-11-01

    Drug-induced lupus is a serious side effect of certain medications, but the chemical features that confer this property and the underlying pathogenesis are puzzling. Prototypes of all six therapeutic classes of lupus-inducing drugs were highly cytotoxic only in the presence of activated neutrophils. Removal of extracellular hydrogen peroxide before, but not after, exposure of the drug to activated neutrophils prevented cytotoxicity. Neutrophil-dependent cytotoxicity required the enzymatic action of myeloperoxidase, resulting in the chemical transformation of the drug to a reactive product. The capacity of drugs to serve as myeloperoxidase substrates in vitro was associated with the ability to induce lupus in vivo. PMID:7973636

  7. Systemic neutrophil activation in a mouse model of ischemic stroke and reperfusion.

    PubMed

    Morrison, Helena; McKee, Dana; Ritter, Leslie

    2011-04-01

    As a natural response to injury and disease, neutrophils activate, adhere to the microvasculature, migrate into brain tissue, and release toxic substances such as reactive oxygen species and proteases. This neutrophil response occurs when blood flow is returned to brain tissue (reperfusion) after ischemic stroke. Thus, the presence of activated systemic neutrophils increases the potential for tissue injury during reperfusion after ischemic stroke. Although experiments in rat models suggest that activated neutrophils play a pivotal role in cerebral ischemia reperfusion injury, little is known about systemic neutrophil activation during reperfusion following ischemic stroke in a mouse model. The purpose of this study was to characterize systemic leukocyte responses and neutrophil CD11b expression 15-min and 24-hr post-reperfusion in a mouse model of ischemic stroke. The intraluminal filament method of transient middle cerebral artery occlusion (tMCAO) with reperfusion or a sham procedure was performed in male C57Bl/6 mice. Automated leukocyte counts and manual white blood cell (WBC) differential counts were measured. Flow cytometry was used to assess systemic neutrophil surface CD11b expression. The data suggest that the damaging potential of systemic neutrophil activation begins as early as 15 min and remains evident at 24 hr after the initiation of reperfusion. In addition, because transgenic mouse models, bred on a C57Bl/6 background, are increasingly used to elucidate single mechanisms of reperfusion injury after ischemic stroke, findings from this study are foundational for future investigations examining the damaging potential of neutrophil responses post-reperfusion after ischemic stroke in genetically altered mouse models within this background strain. PMID:21044968

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

    PubMed

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

    2015-06-25

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

  9. Detection of Bidirectional Signaling During Integrin Activation and Neutrophil Adhesion

    PubMed Central

    Altman, Stuart M.; Dixit, Neha; Simon, Scott I.

    2014-01-01

    Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype. PMID:24504956

  10. N-Formyl peptides drive mitochondrial damage associated molecular pattern induced neutrophil activation through ERK1/2 and P38 MAP kinase signalling pathways.

    PubMed

    Hazeldine, Jon; Hampson, Peter; Opoku, Francis Adusei; Foster, Mark; Lord, Janet M

    2015-01-01

    that MAPK activation is required for mtDAMP-induced neutrophil activation and function. Our findings demonstrate that signalling through FPR-1 and activation of p38 and ERK1/2 MAPKs are key events in mtDAMP-induced neutrophil activation. Gaining an understanding of the signalling pathways involved in mtDAMP-induced neutrophil activation may assist in the development of future therapeutic strategies aimed at targeting the SIRS response to improve the outcome of the hospitalised trauma patient. Reducing the severity of the inflammatory response may realise substantial benefits for the severely injured trauma patient. PMID:25817163

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

    PubMed

    Williams, C David; Bajt, Mary Lynn; Sharpe, Matthew R; McGill, Mitchell R; Farhood, Anwar; Jaeschke, Hartmut

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: >800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91(phox)⁻/⁻ mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. PMID:24440789

  12. Neutrophil Activation During Acetaminophen Hepatotoxicity and Repair in Mice and Humans

    PubMed Central

    Williams, C. David; Bajt, Mary Lynn; Sharpe, Matthew R.; McGill, Mitchell R.; Farhood, Anwar; Jaeschke, Hartmut

    2014-01-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by release of cellular contents from necrotic hepatocytes into systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution is controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: >800U/L) had serial blood draws during the injury and recovery phases for determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91phox-/- mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. PMID:24440789

  13. IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

    PubMed

    Liu, Rebecca; Lauridsen, Holly M; Amezquita, Robert A; Pierce, Richard W; Jane-Wit, Dan; Fang, Caodi; Pellowe, Amanda S; Kirkiles-Smith, Nancy C; Gonzalez, Anjelica L; Pober, Jordan S

    2016-09-15

    A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space. PMID:27534549

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

    PubMed

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

    2016-02-01

    Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1β and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 μg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 μg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation. PMID:26433579

  15. Myeloperoxidase activity and the oxidized proteins in blood neutrophils of patients with pneumonia.

    PubMed

    Muravlyova, Larissa; Molotov-Luchanskiy, Vilen; Bakirova, Ryszhan; Klyuyev, Dmitriy; Demidchik, Ludmila; Kolesnikova, Yevgeniya

    2014-10-01

    The main purpose of our investigation was to study myeloperoxidase activity and concentration of oxidized proteins in blood neutrophils of patients with ambulant pneumonia and secondary pneumonia which has arisen on a background of chronic obstructive pulmonary disease (COPD). Patients were divided into 2 groups. 17 patients with ambulant pneumonia moderate severity and respiratory insufficiency of grade 2 were included in the 1-st group. 20 COPD patients with secondary pneumonia moderate severity and with respiratory insufficiency of grade 2 were included in the 2-nd group. The control group consisted of 15 healthy subjects. The reactive protein carbonyl derivates, advanced oxidation protein products (AOPP) and myeloperoxidase activity were detected in neutrophils. In neutrophils of 1-st group patients the augmentation of reactive protein carbonyl derivates was observed in comparison with healthy ones. In neutrophils of 2-nd group patients the slight decrease of reactive protein carbonyl derivates was observed in comparison with healthy ones (by 17%). In neutrophils of 2-nd group patients the significant increasing AOPP in comparison with healthy ones (p <0.01) and 1 group patients (p <0.05) was fixed. Myeloperoxidase activity was higher in neutrophils of 1-th group patients in comparison with healthy ones. In neutrophils of 2-nd group patients myeloperoxidase activity was higher in comparison with the same of 1 group patients (by 67%, p <0.05). Our results showed the different direction of oxidized proteins formation neutrophils of patients with primary and secondary pneumonia. Besides that the varied degree of myeloperoxidase activity was fixed. Our results require more detailed understanding because they can reflect peculiar mechanisms of pneumonia development and determine the characteristics of their progression. PMID:26461373

  16. 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...

  17. Neutrophil depletion in the early inflammatory phase delayed cutaneous wound healing in older rats: improvements due to the use of un-denatured camel whey protein

    PubMed Central

    2014-01-01

    Background While it is known that advanced age alters the recruitment of neutrophils during wound healing, thereby delaying the wound healing process, little is known about prolonged wound healing in advanced ages. Thus, we investigated the correlation of neutrophil recruitment with healing events, and the impact of whey protein (WP) on neutrophil activation. Methods The animals were allocated into wounded young group, wounded older group and wounded older rats with daily treatment of WP at a dose of 100 mg/kg of body weight. Results Our results pointed to a marked deficiency in the number of neutrophils in the wounds of older rats, which was accompanied with impairment of the healing process. In the group of older rats, phagocytic activity, as tested by fluorescence microscopy, declined throughout the first 24 hours after wounding. Both the neutrophil number and the phagocytic activity recovered in older rats which received WP supplementation. Interestingly, WP was found to significantly up-regulate the MIP-1α and CINC-1 mRNA expression in old rats. On the other hand, the wound size in older rats was significantly higher than that in younger ones. Blood angiogenesis was also significantly delayed in the older group as opposed to the young rats. WP, however, was found to return these indices to normal levels in the older rats. Proliferation and epidermal migration of the keratinocytes and the collagen deposition were also returned to the normal rates. Conclusions This data confirms the critical role of neutrophil recruitment in the early inflammatory phase of wound healing in older rats. In addition, WP protein was used to improve neutrophil function in older rats, healing events returned to a more normal profile. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2100966986117779. PMID:24593823

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

    PubMed

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

    2009-06-10

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

  19. Cefoperazone prevents the inactivation of alpha(1)-antitrypsin by activated neutrophils.

    PubMed

    Dallegri, F; Dapino, P; Arduino, N; Bertolotto, M; Ottonello, L

    1999-09-01

    At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor alpha(1)-antitrypsin. In the present study, cefoperazone prevented alpha(1)-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with alpha(1)-antitrypsin without affecting the cells' ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with alpha(1)-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation. PMID:10471586

  20. Cefoperazone Prevents the Inactivation of α1-Antitrypsin by Activated Neutrophils

    PubMed Central

    Dallegri, Franco; Dapino, Patrizia; Arduino, Nicoletta; Bertolotto, Maria; Ottonello, Luciano

    1999-01-01

    At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor α1-antitrypsin. In the present study, cefoperazone prevented α1-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with α1-antitrypsin without affecting the cells’ ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with α1-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation. PMID:10471586

  1. Activated human valvular interstitial cells sustain interleukin-17 production to recruit neutrophils in infective endocarditis.

    PubMed

    Yeh, Chiou-Yueh; Shun, Chia-Tung; Kuo, Yu-Min; Jung, Chiau-Jing; Hsieh, Song-Chou; Chiu, Yen-Ling; Chen, Jeng-Wei; Hsu, Ron-Bin; Yang, Chia-Ju; Chia, Jean-San

    2015-06-01

    The mechanisms that underlie valvular inflammation in streptococcus-induced infective endocarditis (IE) remain unclear. We previously demonstrated that streptococcal glucosyltransferases (GTFs) can activate human heart valvular interstitial cells (VIC) to secrete interleukin-6 (IL-6), a cytokine involved in T helper 17 (Th17) cell differentiation. Here, we tested the hypothesis that activated VIC can enhance neutrophil infiltration through sustained IL-17 production, leading to valvular damage. To monitor cytokine and chemokine production, leukocyte recruitment, and the induction or expansion of CD4(+) CD45RA(-) CD25(-) CCR6(+) Th17 cells, primary human VIC were cultured in vitro and activated by GTFs. Serum cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA), and neutrophils and Th17 cells were detected by immunohistochemistry in infected valves from patients with IE. The expression of IL-21, IL-23, IL-17, and retinoic acid receptor-related orphan receptor C (Rorc) was upregulated in GTF-activated VIC, which may enhance the proliferation of memory Th17 cells in an IL-6-dependent manner. Many chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), were upregulated in GTF-activated VIC, which might recruit neutrophils and CD4(+) T cells. Moreover, CXCL1 production in VIC was induced in a dose-dependent manner by IL-17 to enhance neutrophil chemotaxis. CXCL1-expressing VIC and infiltrating neutrophils could be detected in infected valves, and serum concentrations of IL-17, IL-21, and IL-23 were increased in patients with IE compared to healthy donors. Furthermore, elevated serum IL-21 levels have been significantly associated with severe valvular damage, including rupture of chordae tendineae, in IE patients. Our findings suggest that VIC are activated by bacterial modulins to recruit neutrophils and that such activities might be further enhanced by the production of Th17-associated cytokines. Together, these factors can amplify

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

    PubMed

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

    2016-08-01

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

  3. Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist

    PubMed Central

    Macleod, Tom; Doble, Rosella; McGonagle, Dennis; Wasson, Christopher W.; Alase, Adewonuola; Stacey, Martin; Wittmann, Miriam

    2016-01-01

    The interleukin-36 receptor antagonist (IL-36Ra) which regulates IL-36α, -β and -γ is linked to psoriatic inflammation, especially loss-of-function mutations in pustular psoriasis subtypes. As observed with other IL-1 superfamily proteins, the IL-36 members require N-terminal cleavage for full biological activity but the mechanisms of IL-36Ra activation remain poorly defined. Using different blood leukocyte and skin resident cell preparations, and recombinant proteins, we have identified that neutrophil elastase, but not other neutrophil derived proteases, cleaves IL-36Ra into its highly active antagonistic form. The activity of this processed form of IL-36Ra was confirmed in human primary dermal fibroblasts and keratinocytes and in skin equivalents. A significant dose dependent reduction of IL-36γ induced IL-8 and chemokine ligand 20 (CCL20) levels were detected following addition of the cleaved IL-36Ra compared to full length IL-36Ra. By activating IL-36Ra, the neutrophil derived protease can inhibit IL-36 induced chemokine production, including IL-8 and CCL20, and reduce further inflammatory cell infiltration. These findings strongly indicate neutrophil elastase to be a key enzyme in the biological function of IL-36Ra and that neutrophils can play a regulatory role in psoriatic inflammation with regard to balancing the pro-inflammatory activity of IL-36. PMID:27101808

  4. Enhanced neutrophil activity is associated with shorter time to tumor progression in glioblastoma patients

    PubMed Central

    Rahbar, Afsar; Cederarv, Madeleine; Wolmer-Solberg, Nina; Tammik, Charlotte; Stragliotto, Giuseppe; Peredo, Inti; Fornara, Olesja; Xu, Xinling; Dzabic, Mensur; Taher, Chato; Skarman, Petra; Söderberg-Nauclér, Cecilia

    2016-01-01

    ABSTRACT Glioblastoma multiforme (GBM) is a highly malignant tumor with a poor outcome that is often positive for human cytomegalovirus (HCMV). GBM patients often have excessive numbers of neutrophils and macrophages near and within the tumor. Here, we characterized the cytokine patterns in the blood of GBM patients with and without Valganciclovir treatment. Furthermore, we determined whether neutrophil activation is related to HCMV status and patient outcome. Blood samples for analyses of cytokines and growth factors were collected from 42 GBM patients at the time of diagnosis (n = 42) and at weeks 12 and 24 after surgery. Blood neutrophils of 28 GBM patients were examined for CD11b expression. The levels of pro- and anti-inflammatory cytokines and chemokines—including interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-β1, interferon-γ, interferon-α, tumor necrosis factor α, and monocyte chemoattractant protein (MCP)-1were analyzed with a bead-based flow cytometry assay. During the first six months after surgery, neutrophil activity was increased in 12 patients and was unchanged or decreased in 16. Patients with increased neutrophil activity had enhanced IL-12p70, high grade HCMV and a shorter time to tumor progression (TTP) than patients without or decreased neutrophil activity (median TTP; 5.4 vs. 12 months, 95% confidence interval; 1.6–10 vs. 0.1–0.6, hazard ratio = 3 vs. 0.4, p = 0.004). The levels of IL-12p70 were significantly decreased in Valganciclovir treated patients (n = 22, T 12W vs. T 24W, p = 0.03). In conclusion, our findings suggest that neutrophil activation is an early sign of tumor progression in GBM patients. PMID:27057448

  5. Regulation of neutrophil apoptosis by modulation of PKB/Akt activation.

    PubMed

    Rane, Madhavi J; Klein, Jon B

    2009-01-01

    The serine/threonine kinase, Akt, also known as PKB (Protein Kinase B) is one important signal transduction pathway that mediates the delay of neutrophil apoptosis caused by inflammatory mediators. Proteins controlled by the PKB/Akt pathway have been reported to prevent or reverse apoptotic-signaling pathways and regulate cell survival. In this review we discuss the role of PKB/Akt activation in the regulation of neutrophil activation during inflammation, and the importance of resolving the inflammatory response by inhibiting PKB/Akt activation and neutrophil survival. Furthermore, we introduce the concept of a dynamic Akt signal complex that is altered when an extracellular signal is initiated such that changes in protein-protein interactions within the Akt signal complex regulates Akt activity and cell survival. Various substrates of PKB/Akt as well as positive and negative regulators of PKB/Akt activation are discussed which in turn inhibit or enhance cellular survival. PMID:19273208

  6. Activation of NLRP3 inflammasome in human neutrophils by Helicobacter pylori infection.

    PubMed

    Pérez-Figueroa, Erandi; Torres, Javier; Sánchez-Zauco, Norma; Contreras-Ramos, Alejandra; Alvarez-Arellano, Lourdes; Maldonado-Bernal, Carmen

    2016-02-01

    TLRs and NLRs participate in the immune system recognition of Helicobacter pylori. However, little is known about the mechanisms leading to inflammasome activation by H. pylori and if NLRs in neutrophils are involved in the process. We studied how NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome components are involved in IL-1β maturation in human neutrophils in response to the infection and if they are dependent on T4SS (type IV secretion system) and TLRs. Human neutrophils were cultured and infected with the 26695 or the VirD4- H. pylori strains; the IL-1β concentration was analyzed by ELISA, and we also evaluated the activation of TLRs 2 and 4. The infection of neutrophils with both strains of H. pylori induced production of IL-1β and expression of the NLRP3 inflammasome components such as apoptosis-associated speck-like protein with CARD domain and NLRP3 protein. The infection also increased the activity of caspase-1, which is required for the maturation of IL-1β. Our study shows, for the first time, that H. pylori infection induces the expression and activation of components of NLRP3 inflammasomes in human neutrophils and that the activation is independent of a functional T4SS and TLR2 and TLR4. PMID:26610398

  7. Diminished nitric oxide generation from neutrophils suppresses platelet activation in chronic renal failure.

    PubMed

    Abrantes, Daniele C; Brunini, Tatiana M C; Matsuura, Cristiane; Mury, Wanda Vianna; Corrêa, Carolina R; Santos, Sérgio F; Ormonde do Carmo, Monique B O; Mendes-Ribeiro, Antônio Cláudio

    2015-03-01

    Chronic renal failure (CRF) is a complex clinical condition associated with accelerated atherosclerosis and thrombosis leading to cardiovascular events. The aim of this study was to investigate in detail the NO pathway in neutrophils obtained from hemodialysis patients and its association with platelet function and oxidative status. Fifteen CRF patients on hemodialysis and fifteen controls were included in this study. Laboratory and experimental evaluations were performed after hemodialysis in CRF patients. We evaluated L-[³H] arginine transport, NO synthase (NOS) activity, amino acid concentration in neutrophils, and expressions of NOS isoforms and p47(phox) by western blotting. Platelet aggregation was analyzed in the presence or absence of neutrophils. Oxidative status was measured through glutathione peroxidase, catalase activities, protein oxidation, lipid peroxidation, and DNA/RNA oxidation in serum. Basal NOS activity (pmol/10⁶ cells/min) was impaired in CRF patients on hemodialysis (0.33 ± 0.17) compared to controls (0.65 ± 0.12), whereas the expression of NOS isoforms remained unaltered. L-Arginine transport into neutrophils was similar in CRF patients on hemodialysis and controls. In addition, intracellular concentration of L-arginine was increased fourfold in the patient group. Systemic oxidative stress markers were not affected by CRF. On the other hand, NADPH oxidase subunit p47(phox) in neutrophils was overexpressed in CRF. In the presence of neutrophils, there was a reduction time-dependent in platelet aggregation in both groups with no difference between them. This data suggest that reduced basal generation of NO by neutrophils in CRF patients on hemodialysis occurs independently of L-arginine bioavailability and is able to suppress platelet activation. PMID:25524601

  8. Role of Transient Receptor Potential Vanilloid 4 in Neutrophil Activation and Acute Lung Injury.

    PubMed

    Yin, Jun; Michalick, Laura; Tang, Christine; Tabuchi, Arata; Goldenberg, Neil; Dan, Qinghong; Awwad, Khader; Wang, Liming; Erfinanda, Lasti; Nouailles, Geraldine; Witzenrath, Martin; Vogelzang, Alexis; Lv, Lu; Lee, Warren L; Zhang, Haibo; Rotstein, Ori; Kapus, Andras; Szaszi, Katalin; Fleming, Ingrid; Liedtke, Wolfgang B; Kuppe, Hermann; Kuebler, Wolfgang M

    2016-03-01

    The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI. PMID:26222277

  9. Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism.

    PubMed

    Antoine, Francis; Girard, Denis

    2015-01-01

    Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis. PMID:24926560

  10. Noninvasive In Vivo Quantification of Neutrophil Elastase Activity in Acute Experimental Mouse Lung Injury

    PubMed Central

    Kossodo, Sylvie; Zhang, Jun; Groves, Kevin; Cuneo, Garry J.; Handy, Emma; Morin, Jeff; Delaney, Jeannine; Yared, Wael; Rajopadhye, Milind; Peterson, Jeffrey D.

    2011-01-01

    We developed a neutrophil elastase-specific near-infrared fluorescence imaging agent, which, combined with fluorescence molecular tomographic imaging, allowed us to detect and quantify neutrophil elastase activity in vivo, in real time, and noninvasively in an acute model of lung injury (ALI). Significantly higher fluorescent signal was quantified in mice with LPS/fMLP-induced ALI as compared to healthy controls, correlating with increases in the number of bronchoalveolar lavage cells, neutrophils, and elastase activity. The agent was significantly activated ex vivo in lung sections from ALI but not from control mice, and this activation was ablated by the specific inhibitor sivelestat. Treatment with the specific inhibitor sivelestat significantly reduced lung signal in mice with ALI. These results underscore the unique ability of fluorescence molecular imaging to quantify specific molecular processes in vivo, crucial for understanding the mechanisms underlying disease progression and for assessing and monitoring novel pharmacological interventions. PMID:21941648

  11. Wortmannin and 1-butanol block activation of a novel family of protein kinases in neutrophils.

    PubMed

    Ding, J; Badwey, J A

    1994-07-11

    Neutrophils contain four uncharacterized protein kinases with molecular masses of ca. 69, 63, 49 and 40 kDa that are rapidly activated upon stimulation of these cells with the chemoattractant fMet-Leu-Phe [Ding, J. and Badwey, J.A. (1993) J. Biol. Chem. 268, 17326-17333]. We now report that wortmannin and 1-butanol block activation of all four of these kinases. These reagents are known to inhibit superoxide generation in neutrophils stimulated with this agonist. Wortmannin inhibits phosphatidylinositol 3-kinase and blocks activation of phospholipase D, whereas 1-butanol can reduce the generation of phosphatidate in cells by serving as a substrate for phospholipase D. These data suggest that phosphatidylinositol 3-kinase and phospholipase D may be involved in the activation of several novel protein kinases in neutrophils and that one or more of these kinases is/are involved in superoxide release. PMID:8034030

  12. Activity determination, kinetic analyses and isoenzyme identification of gamma glutamyltransferase in human neutrophils.

    PubMed

    Sener, Azize; Yardimci, Turay

    2005-05-31

    Gamma-glutamyltransferase (GGT, EC 2.3.2.2) which hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH concentration. GGT is a membrane enzyme present in leukocytes and platelets. Its activity has also been observed in human neutrophils. In this study, GGT was purified from Triton X-100 solubilized neutrophils and its kinetic parameters were determined. For kinetic analyses of transpeptidation reaction, gamma-glutamyl p-nitroanilide was used as the substrate and glycylglycine as the acceptor. Apparent K(m) values were determined as 1.8 mM for gamma-glutamyl p-nitroanilide and 16.9 mM for glycylglycine. The optimum pH of GGT activity was 8.2 and the optimum temperature was 37 degrees C. It had thermal stability with 58 % relative activity at 56 degrees C for 30 min incubation. L-serine, in the presence of borate, was detected as the competitive inhibitor. Bromcresol green inhibited neutrophil GGT activity as a noncompetitive inhibitor. The neutrophils seem to contain only the isoenzyme that is present in platelets. We characterized the kinetic properties and compared the type of the isoenzyme of neutrophil GGT with platelet GGT via polyacrylamide gel electrophoresis (PAGE) under a standard set of conditions. PMID:15943911

  13. Time profile of oxidative stress and neutrophil activation in ovine acute lung injury and sepsis.

    PubMed

    Lange, Matthias; Szabo, Csaba; Traber, Daniel L; Horvath, Eszter; Hamahata, Atsumori; Nakano, Yoshimitsu; Traber, Lillian D; Cox, Robert A; Schmalstieg, Frank C; Herndon, David N; Enkhbaatar, Perenlei

    2012-05-01

    The formation of oxidative stress in the lung and activation of neutrophils are major determinants in the development of respiratory failure after acute lung injury and sepsis. However, the time changes of these pathogenic factors have not been sufficiently described. Twenty-four chronically instrumented sheep were subjected to cotton smoke inhalation injury and instillation of live Pseudomonas aeruginosa into both lungs. The sheep were euthanized at 4, 8, 12, 18, and 24 h after injury. Additional sheep received sham injury and were euthanized after 24 h. Pulmonary function was assessed by determination of oxygenation index and pulmonary shunt fraction. In addition, lung tissue was harvested at the respective time points for the measurement of malondialdehyde, interleukin 6, poly(ADP ribose), myeloperoxidase, and alveolar polymorphonuclear neutrophil score. The injury induced severe respiratory failure that was associated with an early increase in lipid peroxidation and interleukin 6 expression. The injury further led to an increase in poly(ADP ribose) activity that reached its peak at 12 h after injury and declined afterward. In addition, progressive increases in markers of neutrophil accumulation in the lung were observed. The peak of neutrophil accumulation in the lung was associated with a severe depletion of circulating neutrophils. The results from our model may enhance the understanding of the pathophysiological alterations after acute lung injury and sepsis and thus be useful in exploring therapeutic interventions directed at modifying the expression or activation of inflammatory mediators. PMID:22266977

  14. Effects of Neutrophils on Cefazolin Activity and Penicillin-Binding Proteins in Staphylococcus aureus Abscesses

    PubMed Central

    Bamberger, David M.; Herndon, Betty L.; Fitch, Jeffrey; Florkowski, Aaron; Parkhurst, Vera

    2002-01-01

    Bacteria survive within abscesses despite antimicrobial therapy, usually necessitating drainage. Our previous work showed that bacterial killing is diminished within the neutrophils of animals with abscesses. To further assess the role of neutrophils in Staphylococcus aureus survival and the poor activities of β-lactams in abscesses, tissue cage abscess-bearing rats were given polymorphonuclear leukocyte (PMN)-depleting antibody prior to and several times following inoculation of the tissue cages with S. aureus. Cefazolin (300 mg/kg of body weight/day) was administered to all animals in appropriately divided doses. After 7 days of antimicrobial therapy, the 17 animals that received anti-PMN serum had significantly fewer abscess neutrophils than the 18 controls and fewer abscess bacteria (5.55 versus 3.79 log10 CFU/ml [P = 0.04]) than the 18 controls. The data were consistent with the premise that cefazolin is more effective in abscesses depleted of neutrophils. To investigate further, S. aureus was incubated with rat peritoneal neutrophils; and bacterial cell membrane proteins were isolated, labeled with biotinylated ampicillin, separated by electrophoresis, blotted onto nitrocellulose, and stained for biotin reactivity. PBP 2 expression was consistently and significantly decreased after a brief, nonkilling PMN exposure. These experiments showed that PMN depletion enhanced the activity of cefazolin in the abscess milieu. Furthermore, altered bacterial cell wall cefazolin targets may be the mechanism by which the PMN diminishes antimicrobial activity, suggesting the importance of the staphylococcus-PMN interaction in the outcome of established infections. PMID:12183241

  15. Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

    PubMed Central

    Tavares-Murta, B. M.; Cunha, F. Q.; Dias-Baruffi, M.; Roque-Barreira, M. C.

    1996-01-01

    In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal−) and bound (D-gal+) fractions, with MNCF being found in the D-gal+ fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal− fraction. Furthermore, the incubation of the D-gal− fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+ activity. Overall, these results suggest that the D-gal− inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal+ fraction. PMID:18475709

  16. Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.

    PubMed

    Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, Jürgen; Panasenko, Oleg M

    2014-03-01

    Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the β subunit of β2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524

  17. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    SciTech Connect

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. The physiological role and pharmacological potential of nitric oxide in neutrophil activation.

    PubMed

    Armstrong, R

    2001-08-01

    There is contention over whether human neutrophils produce physiologically significant levels of nitric oxide (NO) during inflammatory reactions. Nevertheless, regardless of its cell source, NO does exert regulatory effects on neutrophil function. Depending on experimental conditions, NO can either inhibit or enhance neutrophil activation, in both cases probably acting through cyclic GMP. The explanation for these apparently contradictory findings may be that the effect depends upon the concentration of NO: low concentrations of NO being stimulatory and high concentrations inhibitory. Nitrite, produced at high concentrations from NO during inflammation, can react with neutrophil myeloperoxidase-derived hypochlorous acid (HOCl) to form the active oxidant nitryl chloride, a species capable of nitrating tyrosine and tyrosyl residues on proteins. Whether nitryl chloride acts to limit or amplify the oxidant effects of myeloperoxidase is not yet clear, although formation of nitrotyrosine has been linked with nitration of phagocytosed bacteria. Clearly, a better understanding of the inflammatory effects of NO on neutrophils is needed before the therapeutic potential of NO donors or inhibitors in inflammation can be realised. PMID:11515815

  20. Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

    PubMed Central

    Karlsson, Anna

    1999-01-01

    Wheat germ agglutinin (WGA), a lectin with specificity for N-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures. PMID:10377127

  1. Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

    PubMed

    Zimmermann, Maili; Aguilera, Francisco Bianchetto; Castellucci, Monica; Rossato, Marzia; Costa, Sara; Lunardi, Claudio; Ostuni, Renato; Girolomoni, Giampiero; Natoli, Gioacchino; Bazzoni, Flavia; Tamassia, Nicola; Cassatella, Marco A

    2015-01-01

    Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPβ recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells. PMID:25616107

  2. Stimulation of Fas signaling down-regulates activity of neutrophils from major trauma patients with SIRS.

    PubMed

    Paunel-Görgülü, Adnana; Lögters, Tim; Flohé, Sascha; Cinatl, Jindrich; Altrichter, Jens; Windolf, Joachim; Scholz, Martin

    2011-03-01

    Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34±1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling. PMID:20832139

  3. 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

  4. IFNα enhances the production of IL-6 by human neutrophils activated via TLR8

    PubMed Central

    Zimmermann, Maili; Arruda-Silva, Fabio; Bianchetto-Aguilera, Francisco; Finotti, Giulia; Calzetti, Federica; Scapini, Patrizia; Lunardi, Claudio; Cassatella, Marco A.; Tamassia, Nicola

    2016-01-01

    Recently, we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. In this study, we demonstrate that IFNα, a cytokine that modulates the early innate immune responses toward viral and bacterial infections, potently enhances the production of IL-6 in neutrophils stimulated with R848, a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848, but, rather, it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine, in an autocrine manner, leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPβ transcription factor to the IL-6 promoter. Moreover, we show that neutrophils from SLE patients with active disease state, hence displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether, data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils, which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases. PMID:26790609

  5. 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.

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

    PubMed Central

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

    1994-01-01

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

  7. Neutrophil ageing is regulated by the microbiome

    PubMed Central

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

    2015-01-01

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

  8. Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4.

    PubMed

    Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J; Lin, S Jack; Kirchhofer, Daniel; Salvesen, Guy S; Drag, Marcin

    2015-01-01

    Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs. PMID:26172376

  9. Evaluation of NAD(P)-Dependent Dehydrogenase Activities in Neutrophilic Granulocytes by the Bioluminescent Method.

    PubMed

    Savchenko, A A

    2015-09-01

    Bioluminescent method for measurements of the neutrophilic NAD(P)-dependent dehydrogenases (lactate dehydrogenase, NAD-dependent malate dehydrogenase, NADP-dependent decarboxylating malate dehydrogenase, NAD-dependent isocitrate dehydrogenase, and glucose- 6-phosphate dehydrogenase) is developed. The sensitivity of the method allows minimization of the volume of biological material for measurements to 104 neutrophils per analysis. The method is tried in patients with diffuse purulent peritonitis. Low levels of NADPH synthesis enzymes and high levels of enzymes determining the substrate flow by the Krebs cycle found in these patients can lead to attenuation of functional activity of cells. PMID:26468025

  10. An Infection-enhanced Oncolytic Adenovirus Secreting H. pylori Neutrophil-activating Protein with Therapeutic Effects on Neuroendocrine Tumors

    PubMed Central

    Ramachandran, Mohanraj; Yu, Di; Wanders, Alkwin; Essand, Magnus; Eriksson, Fredrik

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. HP-NAP can mediate antitumor effects by recruiting neutrophils and inducing Th1-type differentiation in the tumor microenvironment. It therefore holds strong potential as a therapeutic gene. Here, we armed a replication-selective, infection-enhanced adenovirus with secretory HP-NAP, Ad5PTDf35-[Δ24-sNAP], and evaluated its therapeutic efficacy against neuroendocrine tumors. We observed that it could specifically infect and eradicate a wide range of tumor cells lines from different origin in vitro. Insertion of secretory HP-NAP did not affect the stability or replicative capacity of the virus and infected tumor cells could efficiently secrete HP-NAP. Intratumoral administration of the virus in nude mice xenografted with neuroendocrine tumors improved median survival. Evidence of biological HP-NAP activity was observed 24 hours after treatment with neutrophil infiltration in tumors and an increase of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and MIP2-α in the systemic circulation. Furthermore, evidence of Th1-type immune polarization was observed as a result of increase in IL-12/23 p40 cytokine concentrations 72 hours postvirus administration. Our observations suggest that HP-NAP can serve as a potent immunomodulator in promoting antitumor immune response in the tumor microenvironment and enhance the therapeutic effect of oncolytic adenovirus. PMID:23817216

  11. Statin treatment is associated with reduction in serum levels of receptor activator of NF-κB ligand and neutrophil activation in patients with severe carotid stenosis.

    PubMed

    Lenglet, Sébastien; Quercioli, Alessandra; Fabre, Mathias; Galan, Katia; Pelli, Graziano; Nencioni, Alessio; Bauer, Inga; Pende, Aldo; Python, Magaly; Bertolotto, Maria; Spinella, Giovanni; Pane, Bianca; Palombo, Domenico; Dallegri, Franco; Mach, François; Vuilleumier, Nicolas; Montecucco, Fabrizio

    2014-01-01

    Systemic and intraplaque biomarkers have been widely investigated in clinical cohorts as promising surrogate parameters of cardiovascular vulnerability. In this pilot study, we investigated if systemic and intraplaque levels of calcification biomarkers were affected by treatment with a statin in a cohort of patients with severe carotid stenosis and being asymptomatic for ischemic stroke. Patients on statin therapy had reduced serum osteopontin (OPN), RANKL/osteoprotegerin (OPG) ratio, and MMP-9/pro-MMP-9 activity as compared to untreated patients. Statin-treated patients exhibited increased levels of collagen and reduced neutrophil infiltration in downstream portions of carotid plaques as compared to untreated controls. In upstream plaque portions, OPG content was increased in statin-treated patients as compared to controls. Other histological parameters (such as lipid, macrophage, smooth muscle cell, and MMP-9 content) as well as RANKL, RANK, and OPG mRNA levels did not differ between the two patient groups. Serum RANKL/OPG ratio positively correlated with serum levels of neutrophilic products, intraplaque neutrophil, and MMP-9 content within downstream portions of carotid plaques. In conclusion, statin treatment was associated with improvement in serum RANKL levels and reduced neutrophil activity both systemically and in atherosclerotic plaques. PMID:24648660

  12. Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils.

    PubMed

    Tateno, N; Matsumoto, N; Motowaki, T; Suzuki, K; Aratani, Y

    2013-05-01

    Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H2O2 treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H2O2 due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan. PMID:23438680

  13. Neutrophil oxidative burst activates ATM to regulate cytokine production and apoptosis

    PubMed Central

    Harbort, C. J.; Soeiro-Pereira, Paulo Vitor; von Bernuth, Horst; Kaindl, Angela M.; Costa-Carvalho, Beatriz Tavares; Condino-Neto, Antonio; Reichenbach, Janine; Roesler, Joachim; Zychlinsky, Arturo

    2015-01-01

    Neutrophils play an essential role in the initial stages of inflammation by balancing pro- and antiinflammatory signals. Among these signals are the production of proinflammatory cytokines and the timely initiation of antiinflammatory cell death via constitutive apoptosis. Here we identify ataxia-telangiectasia mutated (ATM) kinase as a modulator of these neutrophil functions. Ataxia-telangiectasia (AT) is a pleiotropic multisystem disorder caused by mutations in the gene-encoding ATM, a master regulator of the DNA damage response. In addition to progressive neurodegeneration and high rates of cancer, AT patients have numerous symptoms that can be linked to chronic inflammation. We report that neutrophils isolated from patients with AT overproduce proinflammatory cytokines and have a prolonged lifespan compared with healthy controls. This effect is partly mediated by increases in activation of p38 MAP kinase. Furthermore, we show that the oxidative burst, catalyzed by nicotinamide adenine dinucleotide phosphate oxidase, can activate ATM in neutrophils. Finally, activation of ATM and DNA damage signaling suppress cytokine production and can abrogate the overproduction of IL-8 in ROS-deficient cells. This reveals a novel mechanism for the regulation of cytokine production and apoptosis, establishing DNA damage as a downstream mediator of immune regulation by reactive oxygen species. We propose that deficiencies in the DNA damage response, like deficiencies in the oxidative burst seen in chronic granulomatous disease, could lead to pathologic inflammation. PMID:26491069

  14. Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

    PubMed Central

    Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

    2016-01-01

    Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

  15. Neutrophil oxidative burst activates ATM to regulate cytokine production and apoptosis.

    PubMed

    Harbort, C J; Soeiro-Pereira, Paulo Vitor; von Bernuth, Horst; Kaindl, Angela M; Costa-Carvalho, Beatriz Tavares; Condino-Neto, Antonio; Reichenbach, Janine; Roesler, Joachim; Zychlinsky, Arturo; Amulic, Borko

    2015-12-24

    Neutrophils play an essential role in the initial stages of inflammation by balancing pro- and antiinflammatory signals. Among these signals are the production of proinflammatory cytokines and the timely initiation of antiinflammatory cell death via constitutive apoptosis. Here we identify ataxia-telangiectasia mutated (ATM) kinase as a modulator of these neutrophil functions. Ataxia-telangiectasia (AT) is a pleiotropic multisystem disorder caused by mutations in the gene-encoding ATM, a master regulator of the DNA damage response. In addition to progressive neurodegeneration and high rates of cancer, AT patients have numerous symptoms that can be linked to chronic inflammation. We report that neutrophils isolated from patients with AT overproduce proinflammatory cytokines and have a prolonged lifespan compared with healthy controls. This effect is partly mediated by increases in activation of p38 MAP kinase. Furthermore, we show that the oxidative burst, catalyzed by nicotinamide adenine dinucleotide phosphate oxidase, can activate ATM in neutrophils. Finally, activation of ATM and DNA damage signaling suppress cytokine production and can abrogate the overproduction of IL-8 in ROS-deficient cells. This reveals a novel mechanism for the regulation of cytokine production and apoptosis, establishing DNA damage as a downstream mediator of immune regulation by reactive oxygen species. We propose that deficiencies in the DNA damage response, like deficiencies in the oxidative burst seen in chronic granulomatous disease, could lead to pathologic inflammation. PMID:26491069

  16. 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.

  17. The anti-inflammatory drug nimesulide inhibits neutrophil adherence to and migration across monolayers of cytokine-activated endothelial cells.

    PubMed

    Dapino, P; Ottonello, L; Dallegri, F

    1994-01-01

    Neutrophil migration through the microvascular endothelium represents a fundamental event for the cell accumulation at sites of tissue injury. Owing to their capacity to modify the structural and functional characteristics of endothelial cells, inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) play a pivotal role in directing circulating neutrophils away from the bloodstream to the interstitial tissue. In order to study neutrophil transendothelial migration, human umbilical vein endothelial cells were grown to confluence on the polycarbonate filter of two-compartment migration chambers. Pretreatment of the endothelial cell monolayers with TNF alpha for 4 h resulted in rapid migration of approximately 50% of subsequently added neutrophils across the layers. In contrast, < 10% of added neutrophils penetrated untreated endothelial monolayers. Using TNF alpha-treated endothelium, neutrophil transmigration was inhibited by the methane sulfonanilide anti-inflammatory drug nimesulide. Moreover, neutrophil adherence to TNF alpha-treated endothelial monolayers, cultured in microtiter wells, was markedly reduced by nimesulide. A linear correlation between the drug-dependent inhibition of neutrophil transmigration and neutrophil adherence was found. Finally, nimesulide did not interfere with the TNF alpha ability to convert resting endothelium into a pro-adhesive and pro-locomotory cell layer. The data suggest that nimesulide reduces neutrophil transendothelial migration primarily by limiting the cell anchorage to the TNF alpha-activated endothelium. Therefore, the drug has the potential to down-regulate neutrophil extravasation and, in turn, the burden of neutrophil oxidants and proteases leading to tissue injury at sites of inflammation. PMID:7824814

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

    PubMed

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

    2016-04-01

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

  19. Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

    PubMed

    Levine, Adam P; Duchen, Michael R; de Villiers, Simon; Rich, Peter R; Segal, Anthony W

    2015-01-01

    The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH. PMID:25885273

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

    PubMed

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

    2011-06-10

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

  1. Effects of Docosahexaenoic Supplementation and In Vitro Vitamin C on the Oxidative and Inflammatory Neutrophil Response to Activation

    PubMed Central

    Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Tur, Josep Antoni; Pons, Antoni

    2015-01-01

    We studied the effects of diet supplementation with docosahexaenoic (DHA) and in vitro vitamin C (VitC) at physiological concentrations on oxidative and inflammatory neutrophil response to phorbol myristate acetate (PMA). Fifteen male footballers ingested a beverage enriched with DHA or a placebo for 8 weeks in a randomized double-blind study. Neutrophils were isolated from blood samples collected in basal conditions at the end of nutritional intervention. Neutrophils were cultured for 2 hours at 37°C in (a) control media, (b) media with PMA, and (c) media with PMA + VitC. PMA induces neutrophil degranulation with increased extracellular myeloperoxidase and catalase activities, nitric oxide production, expression of the inflammatory genes cyclooxygenase-2, nuclear factor κβ, interleukin 8 and tumor necrosis factor α, and interleukin 6 production. DHA diet supplementation boosts the exit of CAT from neutrophils but moderates the degranulation of myeloperoxidase granules induced by PMA. VitC facilitates azurophilic degranulation of neutrophils and increases gene expression of myeloperoxidase induced by PMA. VitC and DHA diet supplementation prevent PMA effects on inflammatory gene expression, although together they do not produce additional effects. DHA diet supplementation enhances antioxidant defences and anti-inflammatory neutrophil response to in vitro PMA activation. VitC facilitates neutrophil degranulation but prevents an inflammatory response to PMA. PMID:25960826

  2. Pharmacological Beta-Adrenergic Receptor Activation Attenuates Neutrophil Recruitment by a Mechanism Dependent on Nicotinic Receptor and the Spleen.

    PubMed

    Silva, Rangel L; Castanheira, Fernanda V; Figueiredo, Jozi G; Bassi, Gabriel S; Ferreira, Sérgio H; Cunha, Fernando Q; Cunha, Thiago M; Kanashiro, Alexandre

    2016-08-01

    The aim of this study was to identify the effect of beta-adrenergic receptor activation on neutrophil migration in experimental peritonitis elucidating the neuroimmune components involved such as nicotinic receptors and the spleen. Mice pre-treated with mecamylamine (nicotinic antagonist) and propranolol (beta-adrenergic antagonist) or splenectomized animals were treated with isoproterenol (beta-adrenergic agonist) prior to intraperitoneal injection of carrageenan. After 4 h, the infiltrating neutrophils and the local cytokine/chemokine levels were evaluated in the peritoneal lavage. The effect of isoproterenol on neutrophil chemotaxis was investigated in a Boyden chamber. Isoproterenol inhibited neutrophil trafficking, reducing the cytokine/chemokine release and neutrophil chemotaxis. Surprisingly, the isoproterenol effect on neutrophil migration was totally reverted by splenectomy and mecamylamine pre-treatment. In contrast, the inhibitory effect of nicotine on neutrophil migration was abrogated only by splenectomy but not by propranolol pre-treatment. Collectively, our data show that beta-adrenergic receptor activation regulates the acute neutrophil recruitment via splenic nicotinic receptor. PMID:27262431

  3. Role of activated neutrophils in chest trauma-induced septic acute lung injury.

    PubMed

    Perl, Mario; Hohmann, Christoph; Denk, Stephanie; Kellermann, Philipp; Lu, Dapeng; Braumüller, Sonja; Bachem, Max G; Thomas, Jörg; Knöferl, Markus W; Ayala, Alfred; Gebhard, Florian; Huber-Lang, Markus S

    2012-07-01

    More than 50% of severely injured patients have chest trauma. Second insults frequently result in acute lung injury (ALI), with sepsis being the main underlying condition. We aimed to develop a standardized, reproducible, and clinically relevant double-hit mouse model of ALI induced by chest trauma and polymicrobial sepsis and to investigate the pathophysiologic role of activated neutrophils. Lung contusion was applied to C57Bl/6 mice via a focused blast wave. Twenty-four hours later, sepsis was induced by cecal ligation and puncture. For polymorphonuclear leukocyte (PMN) depletion, animals received intravenous injections of PMN-depleting antibody. In response to blunt chest trauma followed by sepsis as well as after sepsis alone, a significant local and systemic inflammatory response with increased cytokine/chemokine levels in lung and plasma was observed. In contrast, lung apoptosis was markedly elevated only after a double hit. Intra-alveolar neutrophils and total bronchoalveolar lavage protein concentrations were markedly increased following isolated chest trauma or the combined insult, but not after sepsis alone. Lung myeloperoxidase activity was enhanced only in response to the double hit accompanied by histological disruption of the alveolar architecture, lung congestion, and marked cellular infiltrates. Neutrophil depletion significantly diminished lung interleukin 1β and interleukin 6 concentrations and reduced the degree of septic ALI. Here we have established a novel and highly reproducible mouse model of chest trauma-induced septic ALI characterizing a clinical relevant double-hit scenario. In particular, the depletion of neutrophils substantially mitigated the extent of lung injury, indicating a pathomechanistic role for neutrophils in chest trauma-induced septic ALI. PMID:22552016

  4. Antithrombin Attenuates Vascular Leakage via Inhibiting Neutrophil Activation in Acute Lung Injury

    PubMed Central

    Rehberg, Sebastian; Yamamoto, Yusuke; Sousse, Linda E.; Jonkam, Collette; Zhu, Yong; Traber, Lillian D.; Cox, Robert A.; Prough, Donald S.; Traber, Daniel L.; Enkhbaatar, Perenlei

    2014-01-01

    Objective To test the hypothesis that restoration of antithrombin plasma concentrations attenuates vascular leakage by inhibiting neutrophil activation through syndecan-4 receptor inhibition in an established ovine model of acute lung injury. Design Randomized controlled laboratory experiment. Setting University animal research facility. Subjects Eighteen chronically instrumented sheep. Interventions Following combined burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn; 4 × 12 breaths of cold cotton smoke), chronically instrumented sheep were randomly assigned to receive an IV infusion of 6 IU/kg/hr recombinant human antithrombin III or normal saline (n = 6 each) during the 48-hour study period. In addition, six sham animals (not injured, continuous infusion of vehicle) were used to obtain reference values for histological and immunohistochemical analyses. Measurements and Main Results Compared to control animals, recombinant human antithrombin III reduced the number of neutrophils per hour in the pulmonary lymph (p < 0.01 at 24 and 48 hr), alveolar neutrophil infiltration (p = 0.04), and pulmonary myeloperoxidase activity (p = 0.026). Flow cytometric analysis revealed a significant reduction of syndecan-4-positive neutrophils (p = 0.002 vs control at 24 hr). Treatment with recombinant human antithrombin III resulted in a reduction of pulmonary nitrosative stress (p = 0.002), airway obstruction (bronchi: p = 0.001, bronchioli: p = 0.013), parenchymal edema (p = 0.044), and lung bloodless wet-to-dry-weight ratio (p = 0.015). Clinically, recombinant human antithrombin III attenuated the increased pulmonary transvascular fluid flux (12–48 hr: p ≤ 0.001 vs control each) and the deteriorated pulmonary gas exchange (12–48 hr: p < 0.05 vs control each) without increasing the risk of bleeding. Conclusions The present study provides evidence for the interaction between antithrombin and neutrophils in vivo, its pathophysiological

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-24

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

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

    PubMed Central

    Jandl, Katharina; Stacher, Elvira; Bálint, Zoltán; Sturm, Eva Maria; Maric, Jovana; Peinhaupt, Miriam; Luschnig, Petra; Aringer, Ida; Fauland, Alexander; Konya, Viktoria; Dahlen, Sven-Erik; Wheelock, Craig E.; Kratky, Dagmar; Olschewski, Andrea; Marsche, Gunther; Schuligoi, Rufina; Heinemann, Akos

    2016-01-01

    Background Prostaglandin (PG) D2 is an early-phase mediator in inflammation, but its action and the roles of the 2 D-type prostanoid receptors (DPs) DP1 and DP2 (also called chemoattractant receptor–homologous molecule expressed on TH2 cells) in regulating macrophages have not been elucidated to date. Objective We investigated the role of PGD2 receptors on primary human macrophages, as well as primary murine lung macrophages, and their ability to influence neutrophil action in vitro and in vivo. Methods In vitro studies, including migration, Ca2+ flux, and cytokine secretion, were conducted with primary human monocyte-derived macrophages and neutrophils and freshly isolated murine alveolar and pulmonary interstitial macrophages. In vivo pulmonary inflammation was assessed in male BALB/c mice. Results Activation of DP1, DP2, or both receptors on human macrophages induced strong intracellular Ca2+ flux, cytokine release, and migration of macrophages. In a murine model of LPS-induced pulmonary inflammation, activation of each PGD2 receptor resulted in aggravated airway neutrophilia, tissue myeloperoxidase activity, cytokine contents, and decreased lung compliance. Selective depletion of alveolar macrophages abolished the PGD2-enhanced inflammatory response. Activation of PGD2 receptors on human macrophages enhanced the migratory capacity and prolonged the survival of neutrophils in vitro. In human lung tissue specimens both DP1 and DP2 receptors were located on alveolar macrophages along with hematopoietic PGD synthase, the rate-limiting enzyme of PGD2 synthesis. Conclusion For the first time, our results show that PGD2 markedly augments disease activity through its ability to enhance the proinflammatory actions of macrophages and subsequent neutrophil activation. PMID:26792210

  9. Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils

    SciTech Connect

    Binet, Francois; Chiasson, Sonia; Girard, Denis

    2010-01-01

    Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenic trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2{alpha} are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.

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

    PubMed

    Damtew, B; Spagnuolo, P J

    1992-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2008-06-01

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

  13. Inactivated pepsin inhibits neutrophil activation by Fcgamma-receptor-dependent and independent stimuli.

    PubMed

    Kustiawan, Iwan; Derksen, Ninotska; Rispens, Theo

    2016-08-01

    Pepsin is widely used to produce F(ab')2 fragments of immunoglobulin G (IgG). In many cases, at least part of the pepsin will remain present in the F(ab')2 preparation, albeit in (irreversibly) inactivated form. Here we report on a potent immunomodulatory effect of irreversibly inactivated pepsin on activated human neutrophils. Degranulation, induced by coated IgG or via cytochalasin B/N-formyl-Met-Leu-Phe, was measured by quantifying elastase release, and was found to be inhibited in a dose-dependent manner by inactivated pepsin. Since a number of intravenous immunoglobulin (IVIg) products are also treated by limited digestion with pepsin, we investigated if pepsin would be present in quantities large enough to inhibit neutrophil activation. The amounts of pepsin detected in three different pepsin-treated IVIg products were found to be too low to induce an effect, at least in an in vitro setting. PMID:27368805

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

    PubMed

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

    2016-01-01

    Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activate proteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD. PMID:26608308

  15. Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model.

    PubMed

    Ding, Wanjing; Shimada, Hiroyuki; Li, Lin; Mittal, Rahul; Zhang, Xiaokun; Shudo, Koichi; He, Qiaojun; Prasadarao, Nemani V; Wu, Lingtao

    2013-02-01

    Despite advances in the therapeutic use of recombinant granulocyte colony-stimulating factor (G-CSF) to promote granulopoiesis of human hematopoietic stem cells (HSCs), neutropenia remains one of the most serious complications of cancer chemotherapy. We discovered that retinoid agonist Am80 (tamibarotene) is more potent than G-CSF in coordinating neutrophil differentiation and immunity development. Am80-induced neutrophils (AINs) either in vitro or in neutropenic mouse model displayed strong bactericidal activities, similar to those of human peripheral blood neutrophils (PBNs) or mouse peripheral blood neutrophils (MPBNs) but markedly greater than did G-CSF–induced neutrophils (GINs). In contrast to GINs but similar to PBNs, the enhanced bacterial killing by AINs accompanied both better granule maturation and greater coexpression of CD66 antigen with the integrin β2 subunit CD18. Consistently, anti-CD18 antibody neutralized Am80-induced bactericidal activities of AINs. These studies demonstrate that Am80 is more effective than G-CSF in promoting neutrophil differentiation and bactericidal activities, probably through coordinating the functional interaction of CD66 with CD18 to enhance the development of neutrophil immunity during granulopoiesis. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80 as a cost-effective treatment option. PMID:23243275

  16. Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model

    PubMed Central

    Ding, Wanjing; Shimada, Hiroyuki; Li, Lin; Mittal, Rahul; Zhang, Xiaokun; Shudo, Koichi; He, Qiaojun; Prasadarao, Nemani V.

    2013-01-01

    Despite advances in the therapeutic use of recombinant granulocyte colony-stimulating factor (G-CSF) to promote granulopoiesis of human hematopoietic stem cells (HSCs), neutropenia remains one of the most serious complications of cancer chemotherapy. We discovered that retinoid agonist Am80 (tamibarotene) is more potent than G-CSF in coordinating neutrophil differentiation and immunity development. Am80-induced neutrophils (AINs) either in vitro or in neutropenic mouse model displayed strong bactericidal activities, similar to those of human peripheral blood neutrophils (PBNs) or mouse peripheral blood neutrophils (MPBNs) but markedly greater than did G-CSF–induced neutrophils (GINs). In contrast to GINs but similar to PBNs, the enhanced bacterial killing by AINs accompanied both better granule maturation and greater coexpression of CD66 antigen with the integrin β2 subunit CD18. Consistently, anti-CD18 antibody neutralized Am80-induced bactericidal activities of AINs. These studies demonstrate that Am80 is more effective than G-CSF in promoting neutrophil differentiation and bactericidal activities, probably through coordinating the functional interaction of CD66 with CD18 to enhance the development of neutrophil immunity during granulopoiesis. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80 as a cost-effective treatment option. PMID:23243275

  17. The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation

    PubMed Central

    Kumar, Sachin; Xu, Juying; Kumar, Rupali Sani; Lakshmikanthan, Sribalaji; Kapur, Reuben; Kofron, Matthew; Chrzanowska-Wodnicka, Magdalena

    2014-01-01

    Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation. PMID:25092872

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

    PubMed Central

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

    2014-01-01

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

  19. Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.

    PubMed

    Finley, Matthew J; Rauova, Lubica; Alferiev, Ivan S; Weisel, John W; Levy, Robert J; Stachelek, Stanley J

    2012-08-01

    CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils. PMID:22613135

  20. Bactericidal/permeability-increasing protein promotes complement activation for neutrophil-mediated phagocytosis on bacterial surface

    PubMed Central

    Nishimura, H; Gogami, A; Miyagawa, Y; Nanbo, A; Murakami, Y; Baba, T; Nagasawa, S

    2001-01-01

    The neutrophil bactericidal/permeability-increasing protein (BPI) has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Flow cytometric analysis indicated that fluorescein-labelled E. coli treated with BPI were phagocytosed in the presence of serum at two- to five-fold higher levels than phagocytosis of the bacteria without the treatment. In contrast, phagocytosis of the fluoresceined bacteria with or without treatment by BPI did not occur at all in the absence of serum. The phagocytosis stimulated by BPI and serum was dose-dependent. The effect of BPI on phagocytosis in the presence of serum was not observed on Gram-positive bacteria (Staphylococcus aureus). Interestingly, the complement C3b/iC3b fragments were deposited onto the bacterial surface also as a function of the BPI concentration under conditions similar to those for phagocytosis. Furthermore, the BPI-promoted phagocytosis was blocked completely by anti-C3 F(ab′)2 and partially by anti-complement receptor (CR) type 1 and/or anti-CR type 3. These findings suggest that BPI accelerates complement activation to opsonize bacteria with complement-derived fragments, leading to stimulation of phagocytosis by neutrophils via CR(s). PMID:11529944

  1. Oxidation of propylthiouracil to reactive metabolites by activated neutrophils. Implications for agranulocytosis.

    PubMed

    Waldhauser, L; Uetrecht, J

    1991-01-01

    Propylthiouracil (PTU) is associated with idiosyncratic agranulocytosis that may be due to reactive metabolites generated from oxidative metabolism by neutrophils. Therefore, the metabolism of PTU was investigated in activated neutrophils. Three oxidized metabolites were observed on HPLC: PTU-disulfide, propyluracil-2-sulfinate, and propyluracil-2-sulfonate (PTU-SO3-). No metabolism was detected in cells that had not been activated. Metabolism was inhibited by sodium azide and by catalase. The same products were produced by myeloperoxidase (MPO) in an MPO/H2O2/Cl- system. PTU inhibited its own metabolism; however, complete conversion to PTU-SO3- could be achieved with optimal PTU concentrations. MPO/H2O2 without Cl- produced only slight metabolism. The PTU-sulfenyl chloride is a postulated intermediate. In the absence of chloride, oxidation might proceed through propyluracil-2-sulfenic acid. The sulfenyl chloride and PTU-SO3- are both chemically reactive with sulfhydryl compounds such as N-acetylcysteine. Such reactive metabolites, generated by activated neutrophils, may be involved in hypersensitivity reactions associated with PTU, such as agranulocytosis. PMID:1676636

  2. Helicobacter pylori neutrophil-activating protein: From molecular pathogenesis to clinical applications

    PubMed Central

    Fu, Hua-Wen

    2014-01-01

    Helicobacter pylori (H. pylori) neutrophil-activating protein (HP-NAP) was originally identified as a virulence factor of H. pylori for its ability to activate neutrophils to generate respiratory burst by releasing reactive oxygen species. Later on, HP-NAP was also found to be involved in the protection of H. pylori from DNA damage, supporting the survival of H. pylori under oxidative stress. This protein is highly conserved and expressed by virtually all clinical isolates of H. pylori. The majority of patients infected with H. pylori produced antibodies specific for HP-NAP, suggesting its important role in immunity. In addition to acting as a pathogenic factor by activating the innate immunity through a wide range of human leukocytes, including neutrophils, monocytes, and mast cells, HP-NAP also mediates adaptive immunity through the induction of T helper cell type I responses. The pro-inflammatory and immunomodulatory properties of HP-NAP not only make it play an important role in disease pathogenesis but also make it a potential candidate for clinical use. Even though there is no convincing evidence to link HP-NAP to a disease outcome, recent findings supporting the pathogenic role of HP-NAP will be reviewed. In addition, the potential clinical applications of HP-NAP in vaccine development, clinical diagnosis, and drug development will be discussed. PMID:24833859

  3. Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection.

    PubMed

    Simpson, A J; Wallace, W A; Marsden, M E; Govan, J R; Porteous, D J; Haslett, C; Sallenave, J M

    2001-08-01

    During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils. PMID:11466403

  4. Clinical Efficacy of Andrographolide Sulfonate in the Treatment of Severe Hand, Foot, and Mouth Disease (HFMD) is Dependent upon Inhibition of Neutrophil Activation.

    PubMed

    Wen, Tao; Xu, Wenjun; Liang, Lianchun; Li, Junhong; Ding, Xiaorong; Chen, Xiao; Hu, Jianhua; Lv, Aiping; Li, Xiuhui

    2015-08-01

    Andrographolide sulfonate treatment has been shown to improve clinical severe hand, foot, and mouth disease (HFMD) efficacies when combined with conventional therapy. However, the mechanisms for its therapeutic effects remain elusive. In this study, we aimed to investigate whether andrographolide sulfonate exerts its efficacy by acting on neutrophil activation. We obtained serial plasma samples at two time points (before and after 5 days of therapy) from 28 HFMD patients who received conventional therapy and 18 patients who received combination therapy (andrographolide sulfonate plus conventional therapy). Then, we measured plasma myeloperoxidase (MPO), S100A8/A9, histone, and inflammatory cytokine levels. Furthermore, we examined if andrographolide sulfonate had direct effects on neutrophil activation in vitro. We observed that MPO and S100A8/A9 levels were markedly elevated in the HFMD patients before clinical treatment. At 5 days post-medication, the MPO, S100A8/A9, histone, and interleukin-6 levels were markedly lower in the combination therapy group compared with the conventional therapy group. In vitro studies showed that andrographolide sulfonate inhibited lipopolysaccharide-stimulated neutrophil activation, demonstrated by the decreased production of reactive oxygen species and cytokines. These data indicate that neutrophil activation modulation by andrographolide sulfonate may be a critical determinant for its clinical HFMD treatment efficacy. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25960284

  5. [The mechanism of bactericidal activity in phagosomes of neutrophils].

    PubMed

    Murav'ev, R A; But, P G; Fomina, V A; Rogovin, V V

    2002-01-01

    Myeloperoxidase plays the key role in antimicrobial of phagocytes. This enzyme uses hydrogen peroxide and chloride to catalyze hypochlorous acid formation. HOCl is the most probable agent in the oxygen-dependent bactericidal activity in the phagocyte phagosome. Chlorination markers indicate HOCl generation in the quantities lethal for bacteria. Enzymatic assay for myeloperoxidase indicates proceeding of other reactions involved in bactericidal activity. Superoxide integrates many activities of this kind and is important for physiological function of myeloperoxidase. Elucidation of phagosomes biochemistry can help us to understand why certain pathogens survive in such unfavorable environment. PMID:12180008

  6. Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

    SciTech Connect

    Nakashima, S.; Suganuma, A.; Sato, M.; Tohmatsu, T.; Nozawa, Y. )

    1989-08-15

    Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When (3H) AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of (3H)AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of (3H)AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate).

  7. Neutrophil's weapons in atherosclerosis.

    PubMed

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

    2015-12-01

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

  8. Ras regulates alveolar macrophage formation of CXC chemokines and neutrophil activation in streptococcal M1 protein-induced lung injury.

    PubMed

    Zhang, Songen; Hwaiz, Rundk; Rahman, Milladur; Herwald, Heiko; Thorlacius, Henrik

    2014-06-15

    Streptococcal toxic shock syndrome (STSS) is associated with a high mortality rate. The M1 serotype of Streptococcus pyogenes is most frequently associated with STSS. Herein, we examined the role of Ras signaling in M1 protein-induced lung injury. Male C57BL/6 mice received the Ras inhibitor (farnesylthiosalicylic acid, FTS) prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Administration of FTS reduced M1 protein-induced neutrophil recruitment, edema formation and tissue damage in the lung. M1 protein challenge increased Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Ras activity decreased M1 protein-induced expression of Mac-1 on neutrophils and secretion of CXC chemokines in the lung. Moreover, FTS abolished M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. Ras inhibition decreased chemokine-mediated neutrophil migration in vitro. Taken together, our novel findings indicate that Ras signaling is a potent regulator of CXC chemokine formation and neutrophil infiltration in the lung. Thus, inhibition of Ras activity might be a useful way to antagonize streptococcal M1 protein-triggered acute lung injury. PMID:24704370

  9. Heme oxygenase inhibition enhances neutrophil migration into the bronchoalveolar spaces and improves the outcome of murine pneumonia-induced sepsis.

    PubMed

    Czaikoski, Paula Giselle; Nascimento, Daniele Carvalho; Sônego, Fabiane; de Freitas, Andressa; Turato, Walter Miguel; de Carvalho, Michel A; Santos, Raquel Souza; de Oliveira, Gisele Pena; dos Santos Samary, Cynthia; Tefe-Silva, Cristiane; Alves-Filho, José C; Ferreira, Sérgio Henrique; Rossi, Marcos Antonio; Rocco, Patricia Rieken Macedo; Spiller, Fernando; Cunha, Fernando Queiroz

    2013-04-01

    A reduction of the neutrophil migration into the site of infection during cecal ligation and puncture-induced sepsis increases host mortality. Inhibition of heme oxygenase (HO) prevents this neutrophil paralysis and improves host survival in the cecal ligation and puncture model. Taking into account that almost 50% of all sepsis cases are a consequence of pneumonia, we designed the present study to determine the role of HO in an experimental model of pneumonia-induced sepsis. The objective of this study was to evaluate whether the inhibition of HO improves the outcome and pathophysiologic changes of sepsis induced by an intratracheal instillation of Klebsiella pneumoniae. The pretreatment of mice subjected to pneumonia-induced sepsis with ZnDPBG (zinc deuteroporphyrin 2,4-bis glycol), a nonspecific HO inhibitor, increased the number of neutrophils in the bronchoalveolar spaces, reduced the bacterial load at the site of infection, and prevented the upregulation of CD11b and the downregulation of CXCR2 on blood neutrophils. Moreover, the pretreatment with ZnDPBG decreased alveolar collapse, attenuating the deleterious changes in pulmonary mechanics and gas exchanges and, as a consequence, improved the survival rate of mice from 0% to ∼20%. These results show that heme oxygenase is involved in the pathophysiology of pneumonia-induced sepsis and suggest that HO inhibitors could be helpful for the management of this disease. PMID:23481491

  10. Ambroxol inhibits neutrophil respiratory burst activated by alpha chain integrin adhesion.

    PubMed

    Peroni, D G; Moser, S; Gallo, G; Pigozzi, R; Tenero, L; Zanoni, L; Boner, A L; Piacentini, G L

    2013-01-01

    The purpose of the present study was to investigate the possible anti-oxidant effect(s) of Ambroxol on neutrophils activated by ligand-binding of the drug with membrane-associated adhesion integrin CD11a and to estimate dose-response changes in oxygen free radical production. The amount of free radical production by anti-CD11a- and anti-CD4-coated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and challenged with increasing concentration of Ambroxol, was evaluated within a time frame of 90 minutes. A significant dose-dependent effect response of Ambroxol on O2‾ production by cells coated with anti-CD11a antibody was observed. This preliminary study opens a new perspective on the therapeutic role of Ambroxol as an antioxidant drug and for its potential use in controlling oxidative stress, particularly in leukocyte-dependent inflammation. PMID:24355223

  11. Generation and secretion of eosinophilotactic activity from human polymorphonuclear neutrophils by various mechanisms of cell activation.

    PubMed Central

    König, W; Frickhofen, N; Tesch, H

    1979-01-01

    An eosinophil chemotactic factor(s) (ECF) can be generated from human polymorphonuclear neutrophils by the calcium ionophore, phagocytosis, arachidonic acid and hypotonic lysis. In kinetic studies it is observed that peak ECF activity is released prior to the maximum of lysosomal enzyme release with the calcium ionophore, phagocytosis and arachidonic acid, while under conditions of hypotonic exposure ECF activity appears after the maximum of enzyme release. The ECF obtained by hypotonic exposure shows a fluctuating pattern with sharp peaks and steep fall-offs in activity. The ECF-release for each stimulus is temperature dependent; extracellular calcium is required when the ionophore or phagocytosis are used as stimuli, while with arachidonic acid and hypotonic exposure no extracellular calcium is necessary for ECF-release. On Sephadex G-25 each preparation of ECF eluted in the low molecular weight range at approximately 500 daltons. Eosinophils can be deactivated and cross-deactivated with the various ECF-preparations indicating either a molecular identity or a common mode of action on eosinophils. PMID:437847

  12. Ursolic Acid Inhibits Superoxide Production in Activated Neutrophils and Attenuates Trauma-Hemorrhage Shock-Induced Organ Injury in Rats

    PubMed Central

    Hwang, Tsong-Long; Shen, Hsin-I; Liu, Fu-Chao; Tsai, Hsin-I; Wu, Yang-Chang; Chang, Fang-Rong; Yu, Huang-Ping

    2014-01-01

    Neutrophil activation is associated with the development of organ injury after trauma–hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma–hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma–hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma–hemorrhagic shock-induced organ injury in rats. PMID:25360589

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

    SciTech Connect

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

    2012-10-01

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

  14. Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis

    PubMed Central

    Lin, Tian; Liu, Jialin; Huang, Feng; van Engelen, Tjitske SR; Thundivalappil, Sujatha R; Riley, Frank E; Super, Michael; Watters, Alexander L; Smith, Ann; Brinkman, Nathan; Ingber, Donald E; Warren, H Shaw

    2016-01-01

    Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme. PMID:26772775

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

  17. A2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

    PubMed Central

    Bazzichi, Laura; Trincavelli, Letizia; Rossi, Alessandra; De Feo, Francesca; Lucacchini, Antonio; Bombardieri, Stefano; Martini, Claudia

    2005-01-01

    We conducted the present study to investigate protein expression and functioning of A2A and A2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A2A and A2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A2A and A2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A2A and A2B ARs in human neutrophils. No significant changes in A2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A2B AR density and signalling in SSc. PMID:15743465

  18. Activated protein C inhibits neutrophil migration in allergic asthma: a randomised trial.

    PubMed

    de Boer, J Daan; Berger, Marieke; Majoor, Christof J; Kager, Liesbeth M; Meijers, Joost C M; Terpstra, Sanne; Nieuwland, Rienk; Boing, Anita N; Lutter, René; Wouters, Diana; van Mierlo, Gerard J; Zeerleder, Sacha S; Bel, Elisabeth H; van't Veer, Cornelis; de Vos, Alex F; van der Zee, Jaring S; van der Poll, Tom

    2015-12-01

    Asthma patients show evidence of a procoagulant state in their airways, accompanied by an impaired function of the anticoagulant protein C system. We aimed to study the effect of recombinant human activated protein C (rhAPC) in allergic asthma patients.We conducted a randomised, double-blind, placebo-controlled, proof-of-concept study in house dust mite (HDM) allergic asthma patients. Patients were randomised to receive intravenous rhAPC (24 µg·kg(-1)·h(-1); n=12) or placebo (n=12) for 11 h. 4 h after the start of infusion, a first bronchoscopy was performed to challenge one lung segment with saline (control) and a contralateral segment with a combination of HDM extract and lipopolysaccharide (HDM+LPS), thereby mimicking environmental house dust exposure. A second bronchoscopy was conducted 8 h after intrabronchial challenge to obtain bronchoalveolar lavage fluid (BALF).rhAPC did not influence HDM+LPS induced procoagulant changes in the lung. In contrast, rhAPC reduced BALF leukocyte counts by 43% relative to placebo, caused by an inhibitory effect on neutrophil influx (64% reduction), while leaving eosinophil influx unaltered. rhAPC also reduced neutrophil degranulation products in the airways.Intravenous rhAPC attenuates HDM+LPS-induced neutrophil migration and protein release in allergic asthma patients by an effect that does not rely on coagulation inhibition. PMID:26381519

  19. In Vitro Oxidation of Collagen Promotes the Formation of Advanced Oxidation Protein Products and the Activation of Human Neutrophils.

    PubMed

    Bochi, Guilherme Vargas; Torbitz, Vanessa Dorneles; de Campos, Luízi Prestes; Sangoi, Manuela Borges; Fernandes, Natieli Flores; Gomes, Patrícia; Moretto, Maria Beatriz; Barbisan, Fernanda; da Cruz, Ivana Beatrice Mânica; Moresco, Rafael Noal

    2016-04-01

    The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions. Here, we investigated collagen as a potential source for AOPP formation and determined the effects of hypochlorous acid (HOCl)-treated collagen (collagen-AOPPs) on human neutrophil activity. We also assessed whether alpha-tocopherol could counteract these effects. Exposure to HOCl increased the levels of collagen-AOPPs. Collagen-AOPPs also stimulated the production of AOPPs, nitric oxide (NO), superoxide radicals (O2 (-)), and HOCl by neutrophils. Collagen-AOPPs induced apoptosis and decreased the number of viable cells. Alpha-tocopherol prevented the formation of collagen-AOPPs, strongly inhibited the collagen-AOPP-induced production of O2 (-) and HOCl, and increased the viability of neutrophils. Our results suggest that collagen is an important protein that interacts with HOCl to form AOPPs, and consequently, collagen-AOPP formation is related to human neutrophil activation and cell death. PMID:26920846

  20. Neutrophil-Derived Proteases in the Microenvironment of Pancreatic Cancer -Active Players in Tumor Progression

    PubMed Central

    Felix, Klaus; Gaida, Matthias M.

    2016-01-01

    A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the fibro-inflammatory microenvironment, consisting of activated pancreatic stellate cells, extracellular matrix proteins, and a variety of inflammatory cells, such as T cells, macrophages, or neutrophils. Tumor-infiltrating immune cells, which are found in nearly all cancers, including PDAC, often fail to eliminate the tumor, but conversely can promote its progression by altering the tumor microenvironment. Pancreatic cancer cells are able to attract polymorphonuclear neutrophils (PMN) via tumor secreted chemokines and in human PDAC, PMN infiltrates can be observed in the vicinity of tumor cells and in the desmoplastic tumor stroma, which correlate with undifferentiated tumor growth and poor prognosis. The behavior of tumor-infiltrating neutrophils in the tumor micromilieu is not yet understood at a mechanistic level. It has been shown that PMN have the potential to kill tumor cells, either directly or by antibody-dependent cell-mediated cytotoxicity, but on the other side various adverse effects of PMN, such as promotion of aggressive tumor growth with epithelial-to-mesenchymal transition and increased metastatic potential, have been described. Recent therapeutic approaches for PDAC focus not only the tumor cell itself, but also elements of the tumor microenvironment. Therefore, the role of PMN and their derived products (e.g. cytokines, proteases) as a new vein for a therapeutic target should be critically evaluated in this context. This review summarizes the current understanding of the interplay between proteases of tumor-infiltrating neutrophils and pancreatic tumor cells and elements of the desmoplastic stroma. PMID:26929737

  1. The Effects of Plantago major on the Activation of the Neutrophil Respiratory Burst.

    PubMed

    Reina, Elaine; Al-Shibani, Nouf; Allam, Eman; Gregson, Karen S; Kowolik, Michael; Windsor, L Jack

    2013-10-01

    Plantago major is a common plant that grows worldwide in temperate zones and is found in fields, lawns, and on the roadsides. Its leaves and seeds have been used in almost all parts of the world for centuries as a wound healer, analgesic, antioxidant, and antibiotic, as well as an immune system modulator, antiviral, antifungal, and anti-inflammatory agent. Baicalein and aucubin are the two most biologically active components of P. major, and both have been shown to have antioxidant, anti-inflammatory, and anticancer properties. Neutrophils have a pivotal role in wound healing and inflammation. Their principal mechanism of host defense is the killing of pathogens via the production of reactive oxygen species (ROS). The aim of the present study was to determine the in vitro effects of P. major extract, baicalein, and aucubin on human neutrophil respiratory burst activity. The cytotoxicity of the agents was assessed by lactate dehydrogenase (LDH) assays. A standard luminol-dependent chemiluminescence (CL) assay was utilized to monitor the respiratory burst of the neutrophils after exposure to P. major extract and its two active ingredients, baicalein and aucubin. Three replicates per group were included in each of the three runs of the experiments and analysis of variance (ANOVA) was used for statistical analysis. P. major and baicalein were not toxic to the cells at any of the concentrations examined. Aucubin was toxic to the cells only at the highest concentration tested (P = 0.0081). However, genistein was toxic to the cells at all of the concentrations examined except for the lowest concentration of 16.9 μg/ml (P = 0.985). P. major (-0.10 ± 0.11), aucubin (0.06 ± 0.16), baicalein (-0.10 ± 0.11), and genistein (-0.18 ± 0.07) all significantly (P < 0.0001) inhibited ROS production from the neutrophils. P. major extract inhibited neutrophil ROS production, as did aucubin and baicalein. Therefore, these components should be investigated further with relation to

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

    SciTech Connect

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

    1989-09-15

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

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

    PubMed

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

    1989-09-15

    We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation. PMID:2549071

  4. Degradation of Complement 3 by Streptococcal Pyrogenic Exotoxin B Inhibits Complement Activation and Neutrophil Opsonophagocytosis▿

    PubMed Central

    Kuo, Chih-Feng; Lin, Yee-Shin; Chuang, Woei-Jer; Wu, Jiunn-Jong; Tsao, Nina

    2008-01-01

    Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcus (GAS) infection. The inhibition of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we examined the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using an enzyme-linked immunosorbent assay, we found that SPE B-treated serum impaired the activation of the classical, the lectin, and the alternative complement pathways. In contrast, C192S, a SPE B mutant lacking protease activity, had no effect on complement activation. Further study showed that cleavage of serum C3 by SPE B, but not C192S, blocked zymosan-induced production of reactive oxygen species in neutrophils as a result of decreased deposition of C3 fragments on the zymosan surface. Reconstitution of C3 into SPE B-treated serum unblocked zymosan-mediated neutrophil activation dose dependently. SPE B-treated, but not C192S-treated, serum also impaired opsonization of C3 fragments on the surface of GAS strain A20. Moreover, the amount of C3 fragments on the A20 cell surface, a SPE B-producing strain, was less than that on its isogenic mutant strain, SW507, after opsonization with normal serum. A20 opsonized with SPE B-treated serum was more resistant to neutrophil killing than A20 opsonized with normal serum, and SPE B-mediated resistance was C3 dependent. These results suggest a novel SPE B mechanism, one which degrades serum C3 and enables GAS to resist complement damage and opsonophagocytosis. PMID:18174338

  5. Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

    PubMed Central

    Li, Jinquan; Garcia, Cristiana C.; Feng, Wenchao; Kirpotina, Liliya N.; Hilmer, Jonathan; Tavares, Luciana P.; Layton, Arthur W.; Quinn, Mark T.; Bothner, Brian; Teixeira, Mauro M.; Lei, Benfang

    2012-01-01

    The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (ΔsseMGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and kcat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of ΔsseMGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses. PMID:22496650

  6. Gene Expression during the Generation and Activation of Mouse Neutrophils: Implication of Novel Functional and Regulatory Pathways

    PubMed Central

    Ericson, Jeffrey A.; Duffau, Pierre; Yasuda, Kei; Ortiz-Lopez, Adriana; Rothamel, Katherine; Rifkin, Ian R.; Monach, Paul A.

    2014-01-01

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

  7. In vivo hydroquinone exposure alters circulating neutrophil activities and impairs LPS-induced lung inflammation in mice.

    PubMed

    Ribeiro, André Luiz Teroso; Shimada, Ana Lúcia Borges; Hebeda, Cristina Bichels; de Oliveira, Tiago Franco; de Melo Loureiro, Ana Paula; Filho, Walter Dos Reis Pereira; Santos, Alcinéa Meigikos Dos Anjos; de Lima, Wothan Tavares; Farsky, Sandra Helena Poliselli

    2011-10-01

    Hydroquinone (HQ) is an environmental contaminant which causes immune toxicity. In this study, the effects of exposure to low doses of HQ on neutrophil mobilization into the LPS-inflamed lung were investigated. Male Swiss mice were exposed to aerosolized vehicle (control) or 12.5, 25 or 50ppm HQ (1h/day for 5 days). One hour later, oxidative burst, cell cycle, DNA fragmentation and adhesion molecules expressions in circulating neutrophils were determined by flow cytometry, and plasma malondialdehyde (MDA) levels were measured by HPLC. Also, 1h later the last exposures, inflammation was induced by LPS inhalation (0.1mg/ml/10min) and 3h later, the numbers of leukocytes in peripheral blood and in the bronchoalveolar lavage fluid (BALF) were determined using a Neubauer chamber and stained smears; adhesion molecules expressed on lung microvessel endothelial cells were quantified by immunohistochemistry; myeloperoxidase (MPO) activity was measured in the lung tissue by colorimetric assay; and cytokines in the BALF were determined by ELISA. In vivo HQ exposure augmented plasma MDA levels and oxidative activity of neutrophils, but did not cause alterations in cell cycle and DNA fragmentation. Under these conditions, the number of circulating leukocytes was not altered, but HQ exposure reduced LPS-induced neutrophil migration into the alveolar space, as these cells remained in the lung tissue. The impaired neutrophil migration into BALF may not be dependent on reduced cytokines secretions in the BALF and lung endothelial adhesion molecules expressions. However, HQ exposure increased the expression of β(2) and β(3) integrins and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in neutrophils, which were not further enhanced by fMLP in vitro stimulation, indicating that HQ exposure activates circulating neutrophils, impairing further stimulatory responses. Therefore, it has been shown, for the first time, that neutrophils are target of lower levels of in vivo HQ

  8. Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

    PubMed

    Hamon, Yveline; Legowska, Monika; Hervé, Virginie; Dallet-Choisy, Sandrine; Marchand-Adam, Sylvain; Vanderlynden, Lise; Demonte, Michèle; Williams, Rich; Scott, Christopher J; Si-Tahar, Mustapha; Heuzé-Vourc'h, Nathalie; Lalmanach, Gilles; Jenne, Dieter E; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2016-04-15

    The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases. PMID:26884336

  9. One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

    PubMed Central

    Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection. PMID:23577158

  10. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium.

    PubMed

    Chen, Chun-Yu; Leu, Yann-Lii; Fang, Yu; Lin, Chwan-Fwu; Kuo, Liang-Mou; Sung, Wei-Che; Tsai, Yung-Fong; Chung, Pei-Jen; Lee, Ming-Chung; Kuo, Yu-Ting; Yang, Hsuan-Wu; Hwang, Tsong-Long

    2015-01-01

    The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton's tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca(2+) levels ([Ca(2+)]i), whereas PP2 prolonged the time required for [Ca(2+)]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca(2+). PMID:26659126

  11. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium

    PubMed Central

    Chen, Chun-Yu; Leu, Yann-Lii; Fang, Yu; Lin, Chwan-Fwu; Kuo, Liang-Mou; Sung, Wei-Che; Tsai, Yung-Fong; Chung, Pei-Jen; Lee, Ming-Chung; Kuo, Yu-Ting; Yang, Hsuan-Wu; Hwang, Tsong-Long

    2015-01-01

    The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton’s tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca2+ levels ([Ca2+]i), whereas PP2 prolonged the time required for [Ca2+]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca2+. PMID:26659126

  12. Activation of TAK1 by Chemotactic and Growth Factors, and Its Impact on Human Neutrophil Signaling and Functional Responses.

    PubMed

    Sylvain-Prévost, Stéphanie; Ear, Thornin; Simard, François A; Fortin, Carl F; Dubois, Claire M; Flamand, Nicolas; McDonald, Patrick P

    2015-12-01

    The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions. PMID:26491199

  13. 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.

  14. Activity of lung neutrophils and matrix metalloproteinases in cyclophosphamide-treated mice with experimental sepsis

    PubMed Central

    Hirsh, Mark; Carmel, Julie; Kaplan, Viktoria; Livne, Erella; Krausz, Michael M

    2004-01-01

    Sepsis in patients receiving chemotherapy may result in acute respiratory distress syndrome, despite decreased number of blood neutrophils [polymorphonuclear neutrophils (PMNs)]. In the present study, we investigated the correlation of cyclophosphamide (CY)-induced neutropenia with the destructive potential of lung PMN in respect to formation of septic acute lung injury (ALI). Mice were treated with 250 mg/kg of CY or saline (control) and subjected to cecal ligation and puncture (CLP) or sham operation. ALI was verified by histological examination. Lung PMNs and matrix metalloproteinases (MMPs) were assessed by flow cytometry and gelatin zymography. CLP in CY-treated mice induced a typical lung injury. Despite profound neutropenia, CY treatment did not attenuate CLP-induced ALI. This might relate to only a partial suppression of PMN: CY has significantly reduced PMN influx into the lungs (P = 0.008) and suppressed their oxidative metabolism, but had no suppressive effect on degranulation (P = 0.227) and even induced MMP-9 activity (P = 0.0003). In CY-untreated animals, peak of CLP-induced ALI coincided with massive PMN influx (P = 0.013), their maximal degranulation (P = 0.014) and activation of lung MMP-9 (P = 0.002). These findings may indicate an important role of the residual lung PMN and activation of MMP-9 in septic lung injury during CY chemotherapy. PMID:15255968

  15. Calcium mobilization and phosphoinositide turnover in fluoride-activated human neutrophils

    SciTech Connect

    Strnad, C.F.; Wong, K.

    1986-05-01

    Fluoride ion, at concentrations above 10 mM, has been found to activate a superoxide production response in human neutrophils which is strongly dependent on the presence of extracellular calcium. In an attempt to further explore the calcium requirement of fluoride-induced neutrophil activation, intracellular calcium concentrations were monitored through use of the fluorescent calcium probe, Quin 2. Fluoride ion, at concentrations between 10 and 20 mM, was found to elicit a rise in intracellular calcium levels which was characterized by a lag period of 4 to 10 min and a prolonged duration of action (greater than 20 min). In contrast, the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (FMLP), induced a rise in intracellular calcium concentration which peaked within 1 min. Preincubation of cells with 1 ..mu..g/ml pertussis toxin resulted in inhibition of the FMLP-induced response, but not that elicited by fluoride. Furthermore, anion exchange chromatography indicated that inositol phosphate accumulation occurred in fluoride-treated cells in association with calcium mobilization. Recent evidence suggests that the FMLP receptor is coupled to phospholipase C and phosphoinositide turnover through a guanine nucleotide binding protein susceptible to inhibition by pertussis toxin. Present results suggest that fluoride ion may serve to activate this protein in a manner resistant to inhibition by pertussis toxin.

  16. Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation.

    PubMed

    Masters, Seth L; Lagou, Vasiliki; Jéru, Isabelle; Baker, Paul J; Van Eyck, Lien; Parry, David A; Lawless, Dylan; De Nardo, Dominic; Garcia-Perez, Josselyn E; Dagley, Laura F; Holley, Caroline L; Dooley, James; Moghaddas, Fiona; Pasciuto, Emanuela; Jeandel, Pierre-Yves; Sciot, Raf; Lyras, Dena; Webb, Andrew I; Nicholson, Sandra E; De Somer, Lien; van Nieuwenhove, Erika; Ruuth-Praz, Julia; Copin, Bruno; Cochet, Emmanuelle; Medlej-Hashim, Myrna; Megarbane, Andre; Schroder, Kate; Savic, Sinisa; Goris, An; Amselem, Serge; Wouters, Carine; Liston, Adrian

    2016-03-30

    Pyrin responds to pathogen signals and loss of cellular homeostasis by forming an inflammasome complex that drives the cleavage and secretion of interleukin-1β (IL-1β). Mutations in the B30.2/SPRY domain cause pathogen-independent activation of pyrin and are responsible for the autoinflammatory disease familial Mediterranean fever (FMF). We studied a family with a dominantly inherited autoinflammatory disease, distinct from FMF, characterized by childhood-onset recurrent episodes of neutrophilic dermatosis, fever, elevated acute-phase reactants, arthralgia, and myalgia/myositis. The disease was caused by a mutation in MEFV, the gene encoding pyrin (S242R). The mutation results in the loss of a 14-3-3 binding motif at phosphorylated S242, which was not perturbed by FMF mutations in the B30.2/SPRY domain. However, loss of both S242 phosphorylation and 14-3-3 binding was observed for bacterial effectors that activate the pyrin inflammasome, such as Clostridium difficile toxin B (TcdB). The S242R mutation thus recapitulated the effect of pathogen sensing, triggering inflammasome activation and IL-1β production. Successful therapy targeting IL-1β has been initiated in one patient, resolving pyrin-associated autoinflammation with neutrophilic dermatosis. This disease provides evidence that a guard-like mechanism of pyrin regulation, originally identified for Nod-like receptors in plant innate immunity, also exists in humans. PMID:27030597

  17. Ascorbic acid supplementation diminishes microparticle elevations and neutrophil activation following SCUBA diving.

    PubMed

    Yang, Ming; Barak, Otto F; Dujic, Zeljko; Madden, Dennis; Bhopale, Veena M; Bhullar, Jasjeet; Thom, Stephen R

    2015-08-15

    Predicated on evidence that diving-related microparticle generation is an oxidative stress response, this study investigated the role that oxygen plays in augmenting production of annexin V-positive microparticles associated with open-water SCUBA diving and whether elevations can be abrogated by ascorbic acid. Following a cross-over study design, 14 male subjects ingested placebo and 2-3 wk later ascorbic acid (2 g) daily for 6 days prior to performing either a 47-min dive to 18 m of sea water while breathing air (∼222 kPa N2/59 kPa O2) or breathing a mixture of 60% O2/balance N2 from a tight-fitting face mask at atmospheric pressure for 47 min (∼40 kPa N2/59 kPa O2). Within 30 min after the 18-m dive in the placebo group, neutrophil activation, and platelet-neutrophil interactions occurred, and the total number of microparticles, as well as subgroups bearing CD66b, CD41, CD31, CD142 proteins or nitrotyrosine, increased approximately twofold. No significant elevations occurred among divers after ingesting ascorbic acid, nor were elevations identified in either group after breathing 60% O2. Ascorbic acid had no significant effect on post-dive intravascular bubble production quantified by transthoracic echocardiography. We conclude that high-pressure nitrogen plays a key role in neutrophil and microparticle-associated changes with diving and that responses can be abrogated by dietary ascorbic acid supplementation. PMID:26084697

  18. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. Azurocidin, a natural antibiotic from human neutrophils: expression, antimicrobial activity, and secretion.

    PubMed

    Almeida, R P; Vanet, A; Witko-Sarsat, V; Melchior, M; McCabe, D; Gabay, J E

    1996-06-01

    The azurophil granules of human PMN contain four antibiotic proteins, the serprocidins, which have extensive homology to one another and to serine proteases. Azurocidin, a member of this family, is a 29-kDa glycoprotein with broad spectrum antimicrobial activity and chemotactic activity toward monocytes. Insect cells transfected with a baculovirus vector carrying azurocidin cDNA produced a recombinant azurocidin protein. We purified the recombinant azurocidin protein from the culture medium of the infected cells and showed that it retained the antimicrobial activity of the native neutrophil-derived molecule. In addition, we present evidence that a 49-amino-acid region of the recombinant azurocidin protein is required for its secretion from insect cells. PMID:8776752

  3. GDF-15 inhibits integrin activation and mouse neutrophil recruitment through the ALK-5/TGF-βRII heterodimer.

    PubMed

    Artz, Annette; Butz, Stefan; Vestweber, Dietmar

    2016-07-28

    Growth differentiation factor 15 (GDF-15) is the first cytokine known to counteract chemokine-induced activation of leukocyte integrins. We showed recently that this activity dampens neutrophil recruitment into inflamed tissue and is required for survival of myocardial infarction in mice. The receptor responsible for this GDF-15-triggered anti-inflammatory mechanism on myeloid cells is not known. Here, we identify this receptor as transforming growth factor β receptor I (TGF-βRI) (activin receptor-like kinase 5 [ALK-5]) and TGF-β receptor II (TGF-βRII). We show that interference with these receptors by small-molecule inhibitors, antibodies, or small interfering RNA, blocked the GDF-15 effect on leukocyte integrin activation. Likewise, gene inactivation of each of the 2 receptors in neutrophils isolated from conditional gene-deficient mice abolished the inhibitory effect of GDF-15 on CXCL1-induced β2-integrin activation and neutrophil diapedesis. Rapid neutrophil arrest induced by CXCL1 in vivo was inhibited by GDF-15 in an ALK-5 and TGF-βRII dependent way. As for GDF-15 gene-deficient mice, we found that extravasation of neutrophils deficient for ALK-5 or TGF-βRII was strongly increased in the interleukin-1β inflamed cremaster. The inhibitory effects of GDF-15 on neutrophil integrin activation and in vivo neutrophil arrest were also found for TGF-β1. Mechanistically, GDF-15 and TGF-β1 interfered with integrin activation by inhibiting the activation of Ras-related protein 1 (Rap-1), an effect that depended on CalDAG- guanine nucleotide exchange factor 1 (GEF1) and cell division control protein 42 homolog. We conclude that both GDF-15 and TGF-β1 counteract chemokine-induced integrin activation on neutrophils via the ALK-5/TGF-βRII heterodimer. This represents a novel, rapid anti-inflammatory activity of the 2 TGF-β receptors and of TGF-β1. PMID:27235139

  4. Attenuated viral hepatitis in Trem1-/- mice is associated with reduced inflammatory activity of neutrophils.

    PubMed

    Kozik, Jan-Hendrik; Trautmann, Tanja; Carambia, Antonella; Preti, Max; Lütgehetmann, Marc; Krech, Till; Wiegard, Christiane; Heeren, Joerg; Herkel, Johannes

    2016-01-01

    TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils. PMID:27328755

  5. Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils.

    PubMed

    Gennaro, R; Skerlavaj, B; Romeo, D

    1989-10-01

    Extracts of granules of bovine neutrophils are known to exhibit a marked antibacterial activity in vitro. By a simple, two-step chromatographic procedure, we have resolved two peptide components of the antibacterial system. They were named Bac-5 and Bac-7 from the general term bactenecin and had molecular masses of about 5 and 7 kilodaltons, respectively. Over 45 and 20% of the amino acid residues in the two bactenecins are proline and arginine, respectively. The remaining amino acids are mainly hydrophobic (isoleucine, leucine, and phenylalanine). Both Bac-5 and Bac-7 efficiently kill Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae. They also arrest the growth of Enterobacter cloacae (MICs, 25 to 200 micrograms/ml) but not of Proteus vulgaris, Staphylococcus aureus, and Streptococcus agalactiae (MIC, greater than 200 micrograms/ml). Finally, Bac-7 but not Bac-5 has MICs of less than or equal to 200 micrograms/ml for Pseudomonas aeruginosa and Staphylococcus epidermidis. From the comparison between the efficient bactericidal concentrations in vitro and the estimated content of bactenecins in neutrophils (125 ng of Bac-5 and Bac-7 each per 10(6) cells), it is reasonable to conclude that the two cationic peptides may exert a major role in host defense against at least some microorganisms. PMID:2777377

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

    PubMed

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

    2016-06-01

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

  7. Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase–β

    PubMed Central

    Cheung, Yuk Yin; Kim, So Youn; Yiu, Wai Han; Pan, Chi-Jiunn; Jun, Hyun-Sik; Ruef, Robert A.; Lee, Eric J.; Westphal, Heiner; Mansfield, Brian C.; Chou, Janice Y.

    2007-01-01

    Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase–α (G6Pase-α), whose activity — limited to the liver, kidney, and intestine — was solely responsible for the final stages of gluconeogenesis and glycogenolysis, in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, that of G6Pase-β (also known as G6PC), which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-β led to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with this, G6Pase-β–deficient (G6pc3–/–) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow, and the G6pc3–/– neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these conditions. PMID:17318259

  8. Myeloperoxidase Stimulates Neutrophil Degranulation.

    PubMed

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

    2016-08-01

    Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation. PMID:27597056

  9. Mechanism of neutrophil recruitment to the lung after pulmonary contusion.

    PubMed

    Hoth, J Jason; Wells, Jonathan D; Hiltbold, Elizabeth M; McCall, Charles E; Yoza, Barbara K

    2011-06-01

    Blunt chest trauma resulting in pulmonary contusion is a common but poorly understood injury. We previously demonstrated that lung contusion activates localized and systemic innate immune mechanisms and recruits neutrophils to the injured lung. We hypothesized that the innate immune and inflammatory activation of neutrophils may figure prominently in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function and pulmonary neutrophil recruitment. Comparisons were made between injured mice with and without neutrophil depletion. We further examined the role of chemokines and adhesion receptors in neutrophil recruitment to the injured lung. We found that lung injury and resultant physiological dysfunction after contusion were dependent on the presence of neutrophils in the alveolar space. We show that CXCL1, CXCL2/3, and CXCR2 are involved in neutrophil recruitment to the lung after injury and that intercellular adhesion molecule 1 is locally expressed and actively participates in this process. Injured gp91-deficient mice showed improved lung function, indicating that oxidant production by neutrophil NADPH oxidase mediates lung dysfunction after contusion. These data suggest that both neutrophil presence and function are required for lung injury after lung contusion. PMID:21330942

  10. Effects of cytochalasin B on the intrcellular bactericidal activity of human neutrophils.

    PubMed

    Okuda, K

    1975-06-01

    Cytochalasin B (CB) is known to have some inhibitory effects on cytokinesis, single-cell movement, bacterial uptake by phagocytes, and many other processes. The effects of CB on intraleukocytic bactericidal activities in human leukocytes were studied, and the results were summarized as follows. (i) CB inhibited the early stage of the intracellular bactericidal activity of human leukocytes against Streptococcus pyogenes D58 (group A). The effect was rapidly eliminated by rinsing the CB solution. (ii) In the late stage of the intracellular bactericidal process, however, CB possessed no effect against S. pyogenes D58 (group A) and Staphylococcus aureus 209P. (iii) CB also inhibited the translocation of myeloperoxidase granules to the phagosomes of human neutrophils. PMID:1155917

  11. Suppression of lymphokine-activated killer (LAK) cell function by neutrophil polymorphonuclear leukocytes.

    PubMed

    Ottonello, L; Dallegri, F; Dapino, P; Patrone, F; Sacchetti, C

    1991-01-01

    Peripheral blood neutrophil polymorphonuclear leukocytes (PMN) from healthy donors were found to inhibit the cytolytic efficiency of interleukin 2 (IL-2)-activated lymphocytes (LAK cells) in a dose-dependent manner. The inhibitory activity of PMN was not merely due to PMN acting as cold alternative targets, PMN ingestion of the label released by target cells or cell overcrowding in test wells. Heat-treated (50 degrees C, 30 min) lysates from PMN maintained their ability to inhibit LAK cell cytotoxicity, whereas PMN supernatants were completely ineffective. Oxidant scavengers (catalase, superoxide, dismutase) did not affect the PMN-mediated inhibition of LAK cell function. The results suggest that PMN contain heat-stable factor(s) able to suppress LAK cytotoxicity and potentially capable of limiting the therapeutic efficacy of IL-2 and/or LAK cells. PMID:1667940

  12. NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A*S⃞

    PubMed Central

    Frasch, S. Courtney; Berry, Karin Zemski; Fernandez-Boyanapalli, Ruby; Jin, Hyun-Sun; Leslie, Christina; Henson, Peter M.; Murphy, Robert C.; Bratton, Donna L.

    2008-01-01

    Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation. PMID:18824544

  13. Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers

    PubMed Central

    Concha, Claudia; Carretta, María Daniella; Alarcón, Pablo; Conejeros, Ivan; Gallardo, Diego; Hidalgo, Alejandra Isabel; Tadich, Nestor; Cáceres, Dante Daniel; Hidalgo, María Angélica

    2014-01-01

    Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response. PMID:25013355

  14. Neutrophil priming occurs in a sequential manner and can be visualized in living animals by monitoring IL-1β promoter activation

    PubMed Central

    Yao, Yi; Matsushima, Hironori; Ohtola, Jennifer A.; Geng, Shuo; Lu, Ran; Takashima, Akira

    2014-01-01

    Rapid enhancement of phagocyte functionality is a hallmark of neutrophil priming. GeneChip analyses unveiled elevated CD54, dectin-2, and IL-1β mRNA expression by neutrophils isolated from inflammatory sites. In fact, CD54 and dectin-2 protein expression was detected on neutrophils recovered from skin, peritoneal and lung inflammation lesions, but not on those in bone marrow or peripheral blood. Neutrophils elevated CD54 and dectin-2 mRNA during migration in Boyden chambers and acquired CD54 and dectin-2 surface expression after subsequent exposure to GM-CSF. Neutrophils purified from IL-1β promoter-driven DsRed transgenic mice acquired DsRed signals during cell migration or exposure to GM-CSF. CD54 and dectin-2 were expressed by DsRed+ (but not DsRed–) neutrophils in GM-CSF-supplemented culture, and neutrophils recovered from inflammatory sites exhibited strong DsRed signals. The dynamic process of neutrophil priming was then studied in chemically induced inflammatory skin lesions by monitoring DsRed expression under confocal microscopy. A majority (>80%) of Ly6G+ neutrophils expressed DsRed, and those DsRed+/Ly6G+ cells exhibited crawling motion with a higher velocity compared to the DsRed–/Ly6G+ counterpart. This is the first report showing motile behaviors of primed neutrophils in living animals. We propose that neutrophil priming occurs in a sequential manner with rapid enhancement of phagocyte functionality followed by CD54 and dectin-2 mRNA and protein expression, IL-1β promoter activation, and accelerated motility. Not only do these findings provide a new conceptual framework for our understanding of the process of neutrophil priming, they also unveil new insights into the pathophysiology of many inflammatory disorders characterized by neutrophil infiltration. PMID:25527787

  15. Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation.

    PubMed

    Li, Jing; Kim, Kyungho; Hahm, Eunsil; Molokie, Robert; Hay, Nissim; Gordeuk, Victor R; Du, Xiaoping; Cho, Jaehyung

    2014-04-01

    Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD. PMID:24642468

  16. Immunoreceptors on neutrophils.

    PubMed

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

    2016-04-01

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

  17. Nonsteroidal anti-inflammatory drug reduces neutrophil and macrophage accumulation but does not improve tendon regeneration.

    PubMed

    Marsolais, David; Côté, Claude H; Frenette, Jérôme

    2003-07-01

    Whether nonsteroidal anti-inflammatory drugs have a beneficial effect on tendon regeneration is still a matter of debate. Given that inflammatory cells are thought to induce nonspecific damage following an injury, we tested the hypothesis that a 3-day treatment with diclofenac would protect tendons from inflammatory cell injury and would promote healing. Neutrophil and ED1(+) macrophage concentrations were determined in the paratenon and the core of the rat Achilles tendon following collagenase-induced injury. Hydroxyproline content, edema, and mechanical properties were also evaluated at 1, 3, 7, 14, and 28 days post-trauma. Collagenase injections induced a 70% decrease in the ultimate rupture point at Day 3. Diclofenac treatments (1 mg/kg bid) selectively decreased the accumulation of neutrophils and ED1(+) macrophages by 59% and 35%, respectively, in the paratenon, where blood vessels are numerous, but did not reduce the accumulation of neutrophils and ED1(+) macrophages in the core of the tendon. Edema was significantly reduced on Day 3 but persisted during the remodeling phase in the diclofenac-treated group only. The inhibition of leukocyte accumulation by diclofenac did not translate into a reduction of tissue damage or a promotion of tissue healing, because the mechanical properties of injured Achilles tendons were identical in placebo and diclofenac-treated groups. These results indicate that diclofenac reduced both edema and the accumulation of inflammatory cells within the paratenon but provided no biochemical or functional benefits for the Achilles tendon. PMID:12861039

  18. Evaluation of the Microbicidal Activity and Cytokines/Chemokines Profile Released by Neutrophils from HTLV-1-Infected Individuals

    PubMed Central

    Bezerra, Caroline A.; Cardoso, Thiago M.; Giudice, Angela; Porto, Aurélia F.; Santos, Silvane B.; Carvalho, Edgar M.; Bacellar, Olívia

    2011-01-01

    Human T cell lymphotropic virus type-1 (HTLV-1) induces activation and spontaneous proliferation of T cells with production of type-1 pro-inflammatory cytokines. It modifies the immune response to other antigens and increases susceptibility to infectious diseases. However, little is known about innate immunity in HTLV-1 infection. HTLV-1-infected individuals have higher spontaneous neutrophil activation than HTLV-1-seronegative individuals, as shown by the nitroblue tetrazolium (NBT) assay. This study was conducted to evaluate neutrophil function in HTLV-1-infected individuals. Participants in the study included 18 HTLV-1-infected individuals and 14 HTLV-1-seronegative controls. We evaluated the ability of neutrophils (PMNs) to control a parasite infection, to produce peroxynitrite, cytokines and chemokines and to express activation markers in cultures when stimulated with LPS or infected with Leishmania. When compared with the control group, there was no difference in the percentage of PMNs infected with Leishmania or in the number of amastigotes/100 PMNs in HTLV-1-infected individuals. The microbicidal activity of the PMNs and the levels of CXCL8 and CCL4 released by these cells did not show a difference between HTLV-1-infected individuals and the control group. In both the HTLV-1 group and the control group, infection with Leishmania or stimulation of PMNs led to cellular activation. These observations suggest that neutrophils from HTLV-1-infected individuals have preserved their ability to become activated and to produce chemokines and peroxynitrite after stimulation and that the susceptibility to infection by intracellular Leishmania amazonensis in HTLV-1-infected individuals does not depend on impairment of neutrophil function. PMID:21595736

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

    PubMed

    McCarty, Mark F

    2004-01-01

    Neutrophils are activated in the coronary circulation during acute coronary events (unstable angina and myocardial infarction), often prior to the onset of ischemic damage. Moreover, neutrophils infiltrate coronary plaque in these circumstances, and may contribute to the rupture or erosion of this plaque, triggering thrombosis. Activated neutrophils secrete proteolytic enzymes in latent forms which are activated by the hypochlorous acid (HOCl) generated by myeloperoxidase. These phenomena may help to explain why an elevated white cell count has been found to be an independent coronary risk factor. Low-fat vegan diets can decrease circulating leukocytes--neutrophils and monocytes--possibly owing to down-regulation of systemic IGF-I activity. Thus, a relative neutropenia may contribute to the coronary protection afforded by such diets. However, vegetarian diets are devoid of taurine - the physiological antagonist of HOCl--and tissue levels of this nutrient are relatively low in vegetarians. Taurine has anti-atherosclerotic activity in animal models, possibly reflecting a role for macrophage-derived myeloperoxidase in the atherogenic process. Taurine also has platelet-stabilizing and anti-hypertensive effects that presumably could reduce coronary risk. Thus, it is proposed that a taurine-supplemented low-fat vegan diet represents a rational strategy for diminishing the contribution of activated neutrophils to acute coronary events; moreover, such a regimen would work in a number of other complementary ways to promote cardiovascular health. Moderate alcohol consumption, the well-tolerated drug pentoxifylline, and 5-lipoxygenase inhibitors--zileuton, boswellic acids, fish oil--may also have potential in this regard. PMID:15288360

  20. Plasma ATP is required for neutrophil activation in a mouse sepsis model

    PubMed Central

    Sumi, Yuka; Woehrle, Tobias; Chen, Yu; Bao, Yi; Li, Xiaoou; Yao, Yongli; Inoue, Yoshiaki; Tanaka, Hiroshi; Junger, Wolfgang G.

    2014-01-01

    Our previous work has shown that polymorphonuclear neutrophils (PMN) require cellular ATP release and autocrine purinergic signaling for their activation. Here we studied in a mouse model of cecal ligation and puncture (CLP) whether sepsis affects this purinergic signaling process and thereby alters PMN responses after sepsis. Using high performance liquid chromatography, we found that plasma ATP, ADP, and AMP concentrations increased up to 6 fold during the first 8 h after CLP, reaching top levels that were significantly higher than those in sham control animals without CLP. While leukocyte and PMN counts in sham animals increased significantly after 4 h, these blood cell counts decreased in sepsis animals. CD11b expression on the cell surface of PMN of septic animals was significantly higher compared to sham and untreated control animals. These findings suggest increased PMN activation and sequestration of PMN from the circulation after sepsis. Plasma ATP levels correlated with CD11b expression, suggesting that increased ATP concentrations in plasma contribute to PMN activation. We found that treatment of septic mice with the ATP receptor antagonist suramin diminished CD11b expression, indicating that plasma ATP contributs to PMN activation by stimulating P2 receptors of PMN. Increased PMN activation can protect the host from invading microorganisms. However, increased PMN activation can also be detrimental by promoting secondary organ damage. We conclude that pharmacological targeting of P2 receptors may allow modulation of PMN responses in sepsis. PMID:24675414

  1. [Neutrophilic functional heterogeneity].

    PubMed

    2006-02-01

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

  2. Assessment of biological activity of immunoglobulin preparations by using opsonized micro-organisms to stimulate neutrophil chemiluminescence.

    PubMed Central

    Munro, C S; Stanley, P J; Cole, P J

    1985-01-01

    We have used the ability of opsonised bacteria to stimulate luminol enhanced chemiluminescence of human neutrophils to examine the opsonic capabilities of normal and hypogammaglobulinaemic sera for four common bacterial pathogens. Preparations of human immunoglobulin modified for i.v. use have then been compared with unmodified Cohn Fraction II for their effectiveness in improving opsonization when added to antibody deficient sera in vitro. Hypogammaglobulinaemic sera exhibited impaired opsonisation of Haemophilus influenzae, and severely antibody deficient sera also opsonized Streptococcus pneumoniae and Pseudomonas aeruginosa poorly. The opsonization of these organisms was improved by Cohn Fraction II, and by pH 4 and beta-propionolactone treated immunoglobulins, in descending order of effectiveness. Pepsin digested immunoglobulin was inactive, and in some cases impaired opsonic capacity. The opsonisation of Staphylococcus aureus by hypogammaglobulinaemic sera was near normal, and was not improved by any immunoglobulin. This technique, which assesses biological activity of immunoglobulin, is useful in comparing preparations, and may help to establish appropriate dosage and frequency for intravenous immunoglobulin replacement therapy. PMID:3930107

  3. Single-dose intravenous gammaglobulin can stabilize neutrophil Mac-1 activation in sickle cell pain crisis

    PubMed Central

    Manwani, Deepa; Chen, Grace; Carullo, Veronica; Serban, Stelian; Olowokure, Olugbenga; Jang, Jungeun; Huggins, Matthew; Cohen, Hillel W.; Billett, Henny; Atweh, George F.; Frenette, Paul S.; Shi, Patricia A.

    2015-01-01

    Intravenous immunoglobulin (IVIG) decreases neutrophil adhesion to endothelium and red blood cell-neutrophil interactions in sickle cell mice undergoing vaso-occlusion. In this Phase I clinical trial of sickle cell anemia (SCA) patients admitted with pain crisis, we evaluated the status of adhesion molecules on neutrophils in control and IVIG-treated subjects pre- and post-infusion up to 800 mg/kg, the same dose used in murine studies. Mac-1 function significantly decreased from baseline in the low-dose IVIG (200–400 mg/kg) cohorts. IVIG-related adverse events may have occurred in the high-dose (600–800 mg/kg) cohorts. There were no significant increases in neutrophil and leukocyte counts, suggesting that IVIG may more selectively inhibit Mac-1 function as opposed to neutrophil adhesion. This study provides the first in-human validation of pre-clinical murine studies that IVIG can decrease Mac-1 function. PMID:25616042

  4. Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils.

    PubMed

    Hulme-Moir, K Lisa; Clark, Phillip

    2011-07-15

    Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA. PMID:21596444

  5. Neutrophil elastase activity in differentiating HL-60 promyelocytes is decreased by culture with ethanol and elastase deficient neutrophils are produced in alcoholics

    SciTech Connect

    Sachs, C.; Christianson, R.; Pratt, P.; Lynn, W.

    1987-05-01

    Serum-free culture of HL-60 in the presence of recombinant Granulocyte-Macrophage Colony Stimulating Factor in four days elicits a five-fold increase in esterolytic neutrophil elastase (NE) like activity measured with methoxy-succinyl-ala-ala-pro-val p-nitroanilide and purified NE standard but does not cause terminal differentiation. Simultaneous exposure to 0.2, 0.4, or 0.6% (vol./vol.) ethanol blocks this increase in NE activity. Exposure to 0.85% ethanol promotes terminal differentiation to elastase-deficient granulocytes which as been described using DMSO. To ascertain if ethanol may have similar effects on granulocytic differentiation in vivo, they compared oxidase and elastase activities of PMN's in male alcoholics on a binge (ethanol > 200 mg/dl.). In 29 patients an average of 872 (+/- 237) (SD) ng./10/sup 6/ PMN's of active NE was found compared to 1571 (+/- 177) in 13 controls. Patients admitted for treatment of alcoholism had similar NE activity in 3-4 days, showed a slight increase in activity within one week and had NE activity comparable to controls within 2-3 weeks. These findings support the previous observation that smoking related emphysema is less prevalent and severe in patients who regularly consume alcohol. They conclude that ethanol may visibly alter responsiveness of promyelocytic precursors to regulatory differentiating factors.

  6. Kinetics of LFA-1 mediated adhesion of human neutrophils to ICAM-1-role of E-selectin signaling post-activation.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LFA-1 and Mac-1 are the two integrins involved in the arrest and firm adhesion of neutrophils. LFA-1 plays a role in the early stage of cell arrest while Mac-1 stabilizes firm adhesion. Here, we further elucidated the kinetics of LFA-1 activation and its role in mediating neutrophil adhesion to ICAM...

  7. Regulatory effect of cytokine-induced neutrophil chemoattractant, epithelial neutrophil-activating peptide 78 and pyrrolidine dithiocarbamate on pulmonary neutrophil aggregation mediated by nuclear factor-κB in lipopolysaccharide-induced acute respiratory distress syndrome mice

    PubMed Central

    Wang, Hongman; Zhao, Jiping; Xue, Guansheng; Wang, Junfei; Wu, Jinxiang; Wang, Donghui; Dong, Liang

    2016-01-01

    In the present study, the regulatory effect of cytokine-induced neutrophil chemoattractant (CINC) and epithelial neutrophil-activating peptide 78 (ENA-78) on pulmonary neutrophil (PMN) accumulation in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) mice, and the therapeutic effect of pyrrolidine dithiocarbamate (PDTC), was investigated. BALB/c mice were divided into control, LPS and PDTC + LPS groups using a random number table. The phosphorylation of nuclear factor-κB (NF-κB) was detected using a western blot, and the mRNA expression levels of CINC were evaluated using reverse transcription-quantitative polymerase chain reaction. The expression of NF-κB, CINC and ENA-78 was detected using immunohistochemistry. The production of interleukin (IL)-8 and IL-10 in serum and broncho-alveolar lavage fluid (BALF) was analyzed using an enzyme-linked immunosorbent assay. The total number of leukocytes and proportion of PMNs in BALF was also determined. Following injection with LPS (20 mg/kg), the expression levels of p-NF-κB, CINC and ENA-78 were increased in lung tissue, and the expression levels of IL-8, IL-10 and the number of PMNs increased in serum and BALF. However, in comparison with the LPS group, the degree of lung injury was reduced in ARDS mice that were treated with PDTC. In addition, the expression level of p-NF-κB and the production of chemokines in lung tissue decreased in ARDS mice that were treated with PDTC, and the number of PMNs in BALF also decreased. In conclusion, the results of the present study suggest that the LPS-induced phosphorylation of NF-κB may result in the synthesis and release of CINC and ENA-78, which induce the accumulation of PMNs in the lung. Therefore, PDTC may be used to reduce the production of chemokines and cytokines, thereby decreasing the activation of PMNs in lung tissue and reducing the damage of lung tissue in ARDS. PMID:27602092

  8. Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

    PubMed Central

    Abrahamson, M; Wikström, M; Potempa, J; Renvert, S; Hall, A

    1997-01-01

    AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in

  9. Neutrophil IL-1β processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation, and is dependent on K+ efflux

    PubMed Central

    Karmakar, Mausita; Katsnelson, Michael; Malak, Hesham A.; Greene, Neil G.; Howell, Scott J.; Hise, Amy G.; Camilli, Andrew; Kadioglu, Aras; Dubyak, George R.; Pearlman, Eric

    2014-01-01

    Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1β processing. In the current study, we investigated the mechanism by which neutrophils process IL-1β in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1β in bacterial clearance, and showed that NLRP3, ASC and caspase-1 are essential for IL-1β production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (FLICA-660+), and bone marrow neutrophils stimulated with heat killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks after staining with FLICA-660, NLRP3 or ASC. High molecular weight ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K+ efflux in neutrophils, and blocking K+ efflux inhibited caspase-1 activation and IL-1β processing; however, neutrophils did not undergo pyroptosis, indicating that K+ efflux and IL-1β processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin mediated IL-1β processing in neutrophils. Together, these findings demonstrate an essential role for neutrophil derived IL-1β in S. pneumoniae infection, and elucidate the role of the NLRP3 inflammasome in neutrophil cleavage and secretion of mature IL-1β. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases. PMID:25609842

  10. Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases.

    PubMed

    Baker, Paul R S; Owen, John S; Nixon, Andrew B; Thomas, Leslie N; Wooten, Rhonda; Daniel, Larry W; O'Flaherty, Joseph T; Wykle, Robert L

    2002-10-21

    Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2). PMID:12379481

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

    PubMed Central

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

    2012-01-01

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

  12. Cationic additives in nanosystems activate cytotoxicity and inflammatory response of human neutrophils: lipid nanoparticles versus polymeric nanoparticles.

    PubMed

    Hwang, Tsong-Long; Aljuffali, Ibrahim A; Lin, Chwan-Fwu; Chang, Yuan-Ting; Fang, Jia-You

    2015-01-01

    This report compares the effect of lipid and polymeric nanoparticles upon human neutrophils in the presence of cationic surfactants. Nanostructured lipid carriers and poly(lactic-co-glycolic) acid nanoparticles were manufactured as lipid and polymeric systems, respectively. Some cytotoxic and proinflammatory mediators such as lactate dehydrogenase (LDH), elastase, O2(•-), and intracellular Ca(2+) were examined. The nanoparticles showed a size of 170-225 nm. Incorporation of cetyltrimethylammonium bromide or soyaethyl morpholinium ethosulfate, the cationic surfactant, converted zeta potential from a negative to a positive charge. Nanoparticles without cationic surfactants revealed a negligible change on immune and inflammatory responses. Cationic surfactants in both nanoparticulate and free forms induced cell death and the release of mediators. Lipid nanoparticles generally demonstrated a greater response compared to polymeric nanoparticles. The neutrophil morphology observed by electron microscopy confirmed this trend. Cetyltrimethylammonium bromide as the coating material showed more significant activation of neutrophils than soyaethyl morpholinium ethosulfate. Confocal microscope imaging displayed a limited internalization of nanoparticles into neutrophils. It is proposed that cationic nanoparticles interact with the cell membrane, triggering membrane disruption and the following Ca(2+) influx. The elevation of intracellular Ca(2+) induces degranulation and oxidative stress. The consequence of these effects is cytotoxicity and cell death. Caution should be taken when selecting feasible nanoparticulate formulations and cationic additives for consideration of applicability and toxicity. PMID:25609950

  13. A phospholipase A₂ from Bothrops asper snake venom activates neutrophils in culture: expression of cyclooxygenase-2 and PGE₂ biosynthesis.

    PubMed

    Moreira, Vanessa; Gutiérrez, José María; Amaral, Rafaela Bacci; Lomonte, Bruno; Purgatto, Eduardo; Teixeira, Catarina

    2011-02-01

    In this study, the production of prostaglandin E₂ (PGE₂) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A₂ (PLA₂), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA₂s (cytosolic PLA₂ and Ca(2+)-independent PLA₂) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE₂ levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE₂ production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF₃), an intracellular PLA₂ inhibitor, but not bromoenol lactone (BEL), an iPLA₂ inhibitor, suppressed the MT-III-induced AA and PGE₂ release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE₂. COX-2 isoform is preeminent over COX-1 for production of PGE₂ stimulated by MT-III. PGE₂ and AA release by MT-III probably is related to cPLA₂ activation. PMID:21147147

  14. Cationic additives in nanosystems activate cytotoxicity and inflammatory response of human neutrophils: lipid nanoparticles versus polymeric nanoparticles

    PubMed Central

    Hwang, Tsong-Long; Aljuffali, Ibrahim A; Lin, Chwan-Fwu; Chang, Yuan-Ting; Fang, Jia-You

    2015-01-01

    This report compares the effect of lipid and polymeric nanoparticles upon human neutrophils in the presence of cationic surfactants. Nanostructured lipid carriers and poly(lactic-co-glycolic) acid nanoparticles were manufactured as lipid and polymeric systems, respectively. Some cytotoxic and proinflammatory mediators such as lactate dehydrogenase (LDH), elastase, O2•−, and intracellular Ca2+ were examined. The nanoparticles showed a size of 170–225 nm. Incorporation of cetyltrimethylammonium bromide or soyaethyl morpholinium ethosulfate, the cationic surfactant, converted zeta potential from a negative to a positive charge. Nanoparticles without cationic surfactants revealed a negligible change on immune and inflammatory responses. Cationic surfactants in both nanoparticulate and free forms induced cell death and the release of mediators. Lipid nanoparticles generally demonstrated a greater response compared to polymeric nanoparticles. The neutrophil morphology observed by electron microscopy confirmed this trend. Cetyltrimethylammonium bromide as the coating material showed more significant activation of neutrophils than soyaethyl morpholinium ethosulfate. Confocal microscope imaging displayed a limited internalization of nanoparticles into neutrophils. It is proposed that cationic nanoparticles interact with the cell membrane, triggering membrane disruption and the following Ca2+ influx. The elevation of intracellular Ca2+ induces degranulation and oxidative stress. The consequence of these effects is cytotoxicity and cell death. Caution should be taken when selecting feasible nanoparticulate formulations and cationic additives for consideration of applicability and toxicity. PMID:25609950

  15. Heterogeneity of functional responses in differentiated myeloid cell lines reveals EPRO cells as a valid model of murine neutrophil functional activation.

    PubMed

    Gaines, Peter; Chi, Jeffrey; Berliner, Nancy

    2005-05-01

    Mature neutrophils display multiple functional responses upon activation that include chemotaxis, adhesion to and transmigration across endothelial cells, phagocytosis, and pathogen destruction via potent microbicidal enzymes and reactive oxygen species. We are using myeloid cell line models to investigate the signaling pathways that govern neutrophil functional activation. To facilitate these studies, we have performed a direct comparison of functional responses of human and murine myeloid cell line models upon neutrophil differentiation. Our results show that EPRO cells, promyelocytes that undergo complete neutrophil maturation, demonstrate a full spectrum of functional responses, including respiratory burst, chemotaxis toward two murine chemokines, and phagocytosis. We also extend previous studies of granulocyte-colony stimulating factor-induced 32Dcl3 cells, showing they demonstrate chemotaxis and phogocytosis but completely lack a respiratory burst as a result of the absent expression of a critical oxidase subunit, gp91(phox). Induced human leukemic NB4 and HL-60 cells display a respiratory burst and phagocytosis but have defective chemotaxis to multiple chemoattractants. We also tested each cell line for the ability to up-regulate cell-surface membrane-activated complex-1 (Mac-1) expression upon activation, a response mediating neutrophil adhesion and a surrogate marker for degranulation. We show that EPRO cells, but not 32Dcl3 or NB4, significantly increase Mac-1 surface expression upon functional activation. Together, these data show that EPRO and MPRO cells demonstrate complete, functional activation upon neutrophil differentiation, suggesting these promyelocytic models accurately reflect the functional capacity of mature murine neutrophils. PMID:15673544

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

    PubMed Central

    Sotnikova, Ruzena

    2015-01-01

    The aim of the study was to analyze the involvement of the endothelium in the effects of neutrophils (PMNL) on phenylephrine-precontracted isolated rings of the rat thoracic aorta and to compare their effects with those of peroxynitrite (ONOO−) and hypochlorous acid (HOCl). Activated PMNL-induced contraction of the precontracted aorta was prevented by the blockade of NO-synthase and by endothelium removal. In the endothelium-free preparations, the effect of PMNL reappeared in the presence of sodium nitroprusside. The effect of ONOO– and HOCl significantly differed from that of activated PMNL both in the presence and absence of the endothelium. It is therefore likely that neither ONOO– nor HOCl generated by transformation of superoxide anion radical (O2•–) produced by PMNL is involved in their action. Reduction of the relaxant effect of nitric oxide derived from the endothelium by O2•– seems to be the keystone mechanism in generation of PMNL-induced contraction. PMID:27486359

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

    PubMed

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

    2016-10-01

    Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis. PMID:27333499

  18. Autocrine enhancement of leukotriene synthesis by endogenous leukotriene B4 and platelet-activating factor in human neutrophils.

    PubMed Central

    McDonald, P. P.; McColl, S. R.; Braquet, P.; Borgeat, P.

    1994-01-01

    1. Platelet-activating factor (PAF) and leukotriene B4 (LTB4), two potent lipid mediators synthesized by activated neutrophils, are known to stimulate several neutrophil functional responses. In this study, we have determined that endogenous LTB4 and PAF exert autocrine effects on LT synthesis, as well as the underlying mechanism involved. 2. Pretreatment of neutrophils with either pertussis toxin (PT), or with receptor antagonists for LTB4 and PAF, resulted in an inhibition of LT synthesis induced by calcium ionophore, A23187. This inhibition was most marked at submaximal (100-300 nM) A23187 concentrations, whilst it was least at ionophore concentrations which induce maximal LT synthesis (1-3 microM). Thus newly-synthesized PAF and LTB4 can enhance LT synthesis induced by A23187 under conditions where the LT-generating system is not fully activated. 3. In recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, LT synthesis in response to chemoattractants (fMet-Leu-Phe or rhC5a) was also significantly inhibited by the LTB4 receptor antagonist, and to a lesser extent by PAF receptor antagonists. 4. Further investigation revealed that LTB4 and/or PAF exert their effects on LT synthesis via an effect on arachidonic acid (AA) availability, as opposed to 5-lipoxygenase (5-LO) activation. Indeed, the receptor antagonists, as well as PT, inhibited LT synthesis and AA release to a similar extent, whereas 5-LO activation (assessed with an exogenous 5-LO substrate) was virtually unaffected under the same conditions. Accordingly, we showed that addition of exogenous LTB4 could enhance AA availability in response to chemoattractant challenge in rhGM-CSF-primed cells, without significantly affecting the 5-LO activation status.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8019762

  19. Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.

    PubMed Central

    Campbell, A K; Patel, A K; Razavi, Z S; McCapra, F

    1988-01-01

    1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells. PMID:3421897

  20. Regulation of membrane associated protein kinase C activity by guanine nucleotide in rabbit peritoneal neutrophils

    SciTech Connect

    Huang, C.K.; Devanney, J.F.

    1986-03-05

    Addition of phorbol myristate acetate (PMA) (0.1 ..mu..g/ml) or guanosine-5'-0-(3-thiotriphosphate) (GTP..gamma..S) (10..mu..M) to the membrane fraction from rabbit peritoneal neutrophils results in an increase of phosphorylation of several membrane proteins. To test whether membrane associated protein kinase C is involved in the activation, histone is added to the membrane as a substrate for protein kinase C. Phosphorylation of histone is determined by counting the gel pieces containing histone IIIS after separation from other membrane components by SDS-gel electrophoresis. In the presence of CaC12 (20 ..mu..M), GTP..gamma..S (10 ..mu..M) or PMA (0.1 ..mu..g/ml) stimulates the phosphorylation of histone IIIS (40% to 70% increase). To achieve this effect calcium is required for GTP..gamma..S but not for PMA. The effect of GTP..gamma..S but not PMA is inhibited in membranes obtained from cells pretreated with pertussis toxin. Membrane protein kinase C is solubilized with Triton X-100 (1%) and then applied to a DEAE-52 cellulose column chromatography. Two peaks of protein kinase C activity are observed. Peak one is eluted at 40 mM NaCl, peak two is eluted at 140 mM NaCl. The activity of peak one is stimulated with phosphatidylserine (PS) and PMA but not with PS and calcium. The activity of peak two is stimulated with either PS and PMA or PS and calcium. The results suggest that GTP binding protein is involved in the activation of membrane associated protein kinase C and the kinase may exist in two forms, calcium sensitive and calcium insensitive.

  1. Modulation of signalling in neutrophils activated by a chemotactic peptide: calcium regulates diacyl glycerol metabolism

    SciTech Connect

    Korchak, H.M.; Vosshall, L.B.; Lundquist, K.F.

    1987-05-01

    Neutrophils activated by ligands such as the chemotactic peptide f-Met-Leu-Phe (FMLP) generate superoxide anion (O/sub 2//sup -/) and release specific and azurophil granule contents. The signalling for this response is thought to involve both elevated cytosolic Ca and protein kinase C activity. Receptor-occupation triggers a phospholipase C to cleave phosphatidyl inositol 4,5 bisphosphate (PIP/sub 2/) yielding inositol 1,4,5 trisphosphate, (IP/sub 3/), a trigger for intracellular Ca release, and diacyl glycerol (DG), which together with Ca activates protein kinase C. The DG can be metabolized to phosphatidic acid (PA). FMLP triggered a rapid increase in cytosolic Ca (fura-2). Loading cells with MAPTAM, and intracellular Ca buffer, suppressed this Ca transient in FMLP activated cells and inhibited O/sub 2//sup -/ generation to 12.5% of control, beta-glucuronidase release to 40.3% of control and lysozyme release to 55.1% of control. FMLP triggered a prompt decrease in PIP/sub 2/ in cells pre-labelled with /sup 32/P or /sup 3/H-inositol and an increase in PA and release of /sup 3/H-IP/sub 3/. A rapid increase in /sup 14/C-DG levels was also observed in /sup 14/C-glycerol pre-loaded cells activated by FMLP. Suppression of the Ca transient by buffering with MAPTAM inhibited elevation of /sup 14/C-DG. Breakdown of PIP/sub 2/ was not inhibited and elevation of /sup 32/P-PA was enhanced in MAPTAM loaded cells. Conversely, 200nM ionomycin which elevated cytosolic Ca to an equivalent level to 10/sup -7/M FMLP, triggered a rise in /sup 14/C-DG but not in PA.

  2. Chemotactic and enzyme-releasing activity of amphipathic proteins for neutrophils. A possible role for protease in chemotaxis on substratum-bound protein gradients.

    PubMed Central

    Wilkinson, P C; Bradley, G R

    1981-01-01

    The purified amphipathic proteins, alpha s 1-casein, beta-casein, and alkali-denatured serum albumin were studied for chemotactic and enzyme-releasing effects on human neutrophil leucocytes. Evidence for chemotaxis both in fluid-phase gradients and on solid-phase gradients was obtained using visual assays. In fluid-phase gradients, neutrophils showed good orientation to gradient sources of these proteins at concentrations of 10(-4) to 10(-5) M. Solid-phase gradients of casein and of denatured albumin were prepared on glass coverslips, and the locomotion of neutrophils attached to these coverslips was filmed by time-lapse cinematography. Displacement of neutrophils towards the highest concentration of substratum-bound protein was observed, suggesting that neutrophils can show true chemotaxis on a solid-phase gradient. All three proteins induced enzyme release from neutrophils in the absence of cytochalasin B. Lysozyme release was equivalent to that released by stimulation with formyl methionyl peptide in the presence of cytochalasin B, but the proteins stimulated a smaller release of beta-glucuronidase than the peptide. The proteins stimulated release of neutrophil proteases which were able to digest both casein and denatured albumin extracellularly. It is suggested that this proteolytic activity may assist locomotion of neutrophils, especially on solid-phase protein gradients, by cleaving membrane-attached protein, thus both freeing cell-surface receptors and allowing the cell to detach itself from the substratum and continue movement. Images Figure 1 PMID:7016748

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

    PubMed Central

    Bay, Boon Huat

    2016-01-01

    This study evaluates the ability of Lactobacillus rhamnosus GG (LGG) to activate DC and neutrophils and modulate T cell activation and the impact of bacterial dose on these responses. Murine bone marrow derived DC or neutrophils were stimulated with LGG at ratios of 5 : 1, 10 : 1, and 100 : 1 (LGG : cells) and DC maturation (CD40, CD80, CD86, CD83, and MHC class II) and cytokine production (IL-10, TNF-α, and IL-12p70) were examined after 2 h and 18 h coculture and compared to the ability of BCG (the present immunotherapeutic agent for bladder cancer) to stimulate these cells. A 2 h exposure to 100 : 1 (high dose) or an 18 h exposure to 5 : 1 or 10 : 1 (low dose), LGG : cells, induced the highest production of IL-12 and upregulation of CD40, CD80, CD86, and MHC II on DC. In DCs stimulated with LGG activated neutrophils IL-12 production decreased with increasing dose. LGG induced 10-fold greater IL-12 production than BCG. T cell IFNγ and IL-2 production was significantly greater when stimulated with DC activated with low dose LGG. In conclusion, DC or DC activated with neutrophils exposed to low dose LGG induced greater Th1 polarization in T cells and this could potentially exert stronger antitumor effects. Thus the dose of LGG used for immunotherapy could determine treatment efficacy. PMID:27525288

  4. Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor.

    PubMed

    Goy, Sebastian D; Olling, Alexandra; Neumann, Detlef; Pich, Andreas; Gerhard, Ralf

    2015-06-01

    Clostridium difficile may induce antibiotic-associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB-induced rise in intracellular-free Ca(2+) and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1. Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus, non-cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved 'non-cytotoxic' fragments and (iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal receptor binding domain of TcdB with target cells. PMID:25529763

  5. Incidence of mastitis and activity of milk neutrophils in Tharparkar cows reared under semi-arid conditions.

    PubMed

    Alhussien, Mohanned; Manjari, P; Mohammed, Seid; Sheikh, Aasif Ahmad; Reddi, Srinu; Dixit, Satpal; Dang, Ajay K

    2016-08-01

    Rearing of indigenous Tharparkar (TP) cows (native of arid Thar deserts) under high humid conditions (>75 % humidity) has increased the incidence of mammary infections in them. A study was undertaken to see the number, activity, and expression of milk neutrophils isolated from healthy and mastitic cows. There was a significant (P < 0.05) influx in milk somatic cell counts (SCC) and neutrophils in sub-clinical and clinical mastitis cows. No change was observed in the phagocytic activity (PA) of milk neutrophils between healthy and sub-clinical mastitis (SCM) cows, but these activities decreased significantly (P < 0.05) in clinical cases. Chemotactic activity showed a significant difference between all the groups. Lactose varied significantly (P < 0.05) between healthy, sub-clinical, and clinical mastitis (CM) cows. Expression of chemokine receptor (CXCR1) was more in mastitis cows and also higher as compared to CXCR2. No change was observed in cluster of differentiation molecule (CD62L) among all the three groups of TP cows. Expression of interleukin (IL-8) and CD11b was low in healthy cows, increased significantly (P < 0.05) in both sub-clinical and mastitis cows. This study indicates that low producing TP cows are also prone to mammary infections when reared under semi-arid conditions. PMID:27154217

  6. Tumor Exosomal RNAs Promote Lung Pre-metastatic Niche Formation by Activating Alveolar Epithelial TLR3 to Recruit Neutrophils.

    PubMed

    Liu, Yanfang; Gu, Yan; Han, Yanmei; Zhang, Qian; Jiang, Zhengping; Zhang, Xiang; Huang, Bo; Xu, Xiaoqing; Zheng, Jianming; Cao, Xuetao

    2016-08-01

    The pre-metastatic niche educated by primary tumor-derived elements contributes to cancer metastasis. However, the role of host stromal cells in metastatic niche formation and organ-specific metastatic tropism is not clearly defined. Here, we demonstrate that lung epithelial cells are critical for initiating neutrophil recruitment and lung metastatic niche formation by sensing tumor exosomal RNAs via Toll-like receptor 3 (TLR3). TLR3-deficient mice show reduced lung metastasis in the spontaneous metastatic models. Mechanistically, primary tumor-derived exosomal RNAs, which are enriched in small nuclear RNAs, activate TLR3 in lung epithelial cells, consequently inducing chemokine secretion in the lung and promoting neutrophil recruitment. Identification of metastatic axis of tumor exosomal RNAs and host lung epithelial cell TLR3 activation provides potential targets to control cancer metastasis to the lung. PMID:27505671

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

    PubMed

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

    2015-10-01

    Neutrophil infiltration and activation in the lung are important pathophysiological features in COPD, severe asthma and bronchiectasis mostly mediated by CXCL8 and CXCL1 via CXCR1 and CXCR2. No thorough study to date has been performed to compare the anti-inflammatory effect profile of dual CXCR1/2 vs. selective CXCR2 antagonists in relevant human neutrophil assays and pulmonary inflammation models. Dual CXCR1/2 (SCH527123, diaminocyclobutandione-1) and selective CXCR2 (SB265610, thiopyrimidine-1) antagonist activity and receptor residence time were determined by [(35)S]GTPγS binding in human (h)- and guinea pig (gp)-CXCR1 and CXCR2 overexpressing membranes. h-neutrophil chemotaxis, degranulation and ROS production were established using CXCL8 or CXCL1 to evaluate dual CXCR1/2- or selective CXCR2-dependent activities. LPS-induced lung inflammation in gp was selected to assess in vivo potency. Dual CXCR1/2 antagonists blocked both CXCL8 and CXCL1-induced h-neutrophil functions and [(35)S]GTPγS binding. In contrary, selective CXCR2 antagonists displayed significantly reduced potency in CXCL8 -mediated h-neutrophil responses despite being active in CXCR2 assays. Upon LPS challenge in gp, administration of SCH527123 inhibited the increase of neutrophils in BALF, modestly reduced blood neutrophils and induced minor neutrophil accumulation in bone marrow. Differentiation of CXCR1/2 vs. CXCR2 antagonists could not be extended to in vivo due to differences in CXCR1 receptor homology between h and gp. Dual CXCR1/2 therapy may represent a promising anti-inflammatory treatment for respiratory diseases reducing more effectively neutrophil migration and activation in the lung than a CXCR2 selective treatment. However, the in vivo confirmation of this claim is still missing due to species differences in CXCR1. PMID:26271598

  8. Effect of activation on adhesion of flowing neutrophils to cultured endothelium: time course and inhibition by a calcium channel blocker (nitrendipine).

    PubMed Central

    Perry, I.; Buttrum, S. M.; Nash, G. B.

    1993-01-01

    1. Adhesion of neutrophils to vascular endothelium plays an important role in inflammation and thrombosis. Modulation of adhesion may be therapeutic in these conditions. 2. A flow model was used to quantify adhesion of neutrophils to human cultured umbilical vein endothelial cells. The time course of the neutrophil response to activation by N-formyl-methionyl-leucylphenylalanine (fMLP, 10(-7) M) was studied and the inhibitory effects of the calcium-channel blockers, nitrendipine and nifedipine, were investigated. 3. Neutrophils adhered firmly to the endothelial cells without rolling, but initial attachment was highly dependent on shear stress; doubling the stress from 0.05 to 0.1Pa decreased the number of neutrophils adhering by over 80%. 4. Adhesion rapidly increased after activation of neutrophils by fMLP, peaking at 1-3 min post-treatment, and then decreased over the next 10-12 min. A monoclonal antibody to the beta 2-integrin component CD18 inhibited adhesion by over 80% for activated or unactivated cells. 5. The Ca-channel blocker, nitrendipine, but not nifedipine, significantly inhibited the fMLP-induced increase of adhesion in a dose-dependent manner (10(-8) to 10(-6) M). Dihydropyridines may be useful agents for modifying neutrophil function. PMID:7905773

  9. NMR structure and dynamics of monomeric neutrophil-activating peptide 2.

    PubMed Central

    Young, H; Roongta, V; Daly, T J; Mayo, K H

    1999-01-01

    Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the alpha-chemokine family. Although NAP-2, like other alpha-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel beta-sheet on which is folded a C-terminal alpha-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other alpha-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the beta-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from beta-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other alpha-chemokines. PMID:10051427

  10. Increased oxidative activity in human blood neutrophils and monocytes after spinal cord injury.

    PubMed

    Bao, Feng; Bailey, Christopher S; Gurr, Kevin R; Bailey, Stewart I; Rosas-Arellano, M Patricia; Dekaban, Gregory A; Weaver, Lynne C

    2009-02-01

    Traumatic injury can cause a systemic inflammatory response, increasing oxidative activity of circulating leukocytes and potentially exacerbating the original injury, as well as causing damage to initially unaffected organs. Although the importance of intraspinal inflammation after human spinal cord injury is appreciated, the role of the systemic inflammatory response to this injury is not widely recognised. We investigated oxidative activity of blood leukocytes from nine cord-injured subjects and six trauma controls (bone fractures without CNS injury) at 6 h-2 weeks after injury, comparing values to those of ten uninjured subjects. Neutrophil and monocyte free radical production, evaluated by flow cytometry, increased significantly more in cord injury subjects than in trauma controls (6-fold vs 50% increases). In leukocyte homogenates, the concentration of free radicals increased significantly more in cord injury subjects (2-fold) than in the trauma controls (1.6-fold) as did activity of myeloperoxidase (2.3-fold vs. 1.7-fold). Moreover, in homogenates and blood smears, expression of the NADPH oxidase subunit gp91(phox) and of the oxidative enzyme, inducible nitric oxide synthetase was 20-25% greater in cord injury subjects than in trauma controls. Expression of the pro-inflammatory transcription factor NF-kappaB and of cyclooxygenase-2 increased similarly after both injuries. Finally, aldehyde products of tissue-damaging lipid peroxidation also increased significantly more in the plasma of spinal cord injury subjects than in trauma controls (2.6 fold vs. 1.9-fold). Spinal cord injury causes a particularly intense systemic inflammatory response. Limiting this response briefly after cord injury should protect the spinal cord and tissues/organs outside the CNS from secondary damage. PMID:19056384

  11. Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.

    PubMed Central

    Curnutte, J T

    1985-01-01

    Sonicates from unstimulated human neutrophils produce no measurable superoxide since the superoxide-generating enzyme, NADPH oxidase, is inactive in these preparations. Previous attempts to activate the oxidase in disrupted cells with conventional neutrophil stimuli have been unsuccessful. This report describes a cell-free system in which arachidonic acid (82 microM) was able to activate superoxide generation that was dependent upon the presence of NADPH and the sonicate. For activation to occur, both the particulate and supernatant fractions of the sonicate must be present. Calcium ions, which are required for activation of intact neutrophils by arachidonate, were not necessary in the cell-free system. In quantitative terms, the superoxide-generating activity in the cell-free system could account for at least 20-50% of the superoxide rate observed in intact neutrophils stimulated with arachidonate. Sonicates from patients with chronic granulomatous disease (CGD) could not be activated by arachidonic acid in the cell-free system. In three patients representing both genetic forms of CGD, the defect appeared to reside in the particulate fraction. The soluble cofactor was normal in all three patients and could be used to activate normal neutrophil pellets in the presence of arachidonic acid. Thus, at least a portion of the activation mechanism in the neutrophil, that residing in the soluble phase, appeared to be normal in patients with CGD. PMID:2987311

  12. Short-Term Heat Exposure Inhibits Inflammation by Abrogating Recruitment of and Nuclear Factor-κB Activation in Neutrophils Exposed to Chemotactic Cytokines

    PubMed Central

    Choi, Mira; Salanova, Birgit; Rolle, Susanne; Wellner, Maren; Schneider, Wolfgang; Luft, Friedrich C.; Kettritz, Ralph

    2008-01-01

    Cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-8 attract neutrophils into inflammatory sites. During emigration from the blood neutrophils interact with extracellular matrix proteins such as fibronectin. Fibronectin provides β2-integrin co-stimulation, allowing GM-CSF and IL-8 to activate nuclear factor (NF)-κB, an effect that does not occur in suspension. We tested the hypothesis that exposure of mice to fever-like temperatures abrogates neutrophil recruitment and NF-κB activation in a mouse model of skin inflammation. Mice that were exposed to 40°C for 1 hour showed strongly reduced GM-CSF- and IL-8-induced neutrophilic skin inflammation. In vitro heat exposure did not interfere with neutrophil adhesion or spreading on fibronectin but strongly inhibited migration toward both cytokines. Using specific inhibitors, we found that PI3-K/Akt was pivotal for neutrophil migration and that heat down-regulated this pathway. Furthermore, neutrophils on fibronectin showed abrogated NF-κB activation in response to GM-CSF and IL-8 after heat. In vivo heat exposure of mice followed by ex vivo stimulation of isolated bone marrow neutrophils confirmed these results. Finally, less NF-κB activation was seen in the inflammatory lesions of mice exposed to fever-like temperatures as demonstrated by in situ hybridization for IκBα mRNA. These new findings suggest that heat may have anti-inflammatory effects in neutrophil-dependent inflammation. PMID:18187571

  13. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

    PubMed

    Manna, Sunil K; Sarkar, Abira; Sreenivasan, Yashin

    2006-03-01

    Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress. PMID:16479540

  14. Purification of equine neutrophil lysozyme and its antibacterial activity against gram-positive and gram-negative bacteria.

    PubMed

    Pellegrini, A; Waiblinger, S; Von Fellenberg, R

    1991-01-01

    Lysozyme from equine neutrophil granulocytes was isolated in a pure form by fast performance liquid chromatography, i.e. ion-exchange chromatography and reversed-phase chromatography. The lysozyme lysed Micrococcus luteus, Bacillus subtilis and Staphylococcus lentus and was also bactericidal against the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Bordetella bronchiseptica, and Serratia marcescens. Staphylococcus aureus and Staphylococcus epidermidis were not lysed. The lysozyme was only very slightly bactericidal for S. epidermidis and S. aureus. Equine neutrophil lysozyme was found to be bactericidal for Gram-positive as well as for Gram-negative bacteria without further treatment. Equine and chicken egg white lysozymes were found to be immunologically related when examined using specific antisera against each of them. Both lysozymes also had very similar specific enzymatic activities against M. luteus membranes. PMID:1803722

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

    SciTech Connect

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

    1987-01-01

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

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

    PubMed

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

    1993-06-01

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

  17. Critical COPD respiratory illness is linked to increased transcriptomic activity of neutrophil proteases genes

    PubMed Central

    2012-01-01

    Background Gene expression profiling (GEP) in cells obtained from peripheral blood has shown that this is a very useful approach for biomarker discovery and for studying molecular pathogenesis of prevalent diseases. While there is limited literature available on gene expression markers associated with Chronic Obstructive Pulmonary Disease (COPD), the transcriptomic picture associated with critical respiratory illness in this disease is not known at the present moment. Findings By using Agilent microarray chips, we have profiled gene expression signatures in the whole blood of 28 COPD patients hospitalized with different degrees of respiratory compromise.12 of them needed of admission to the ICU, whilst 16 were admitted to the Respiratory Medicine Service. GeneSpring GX 11.0 software was used for performing statistical comparisons of transcript levels between ICU and non-ICU patients. Ingenuity pathway analysis 8.5 (IPA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to select, annotate and visualize genes by function and pathway (gene ontology). T-test showed evidence of 1501 genes differentially expressed between ICU and non-ICU patients. IPA and KEGG analysis of the most representative biological functions revealed that ICU patients had increased levels of neutrophil gene transcripts, being [cathepsin G (CTSG)], [elastase, neutrophil expressed (ELANE)], [proteinase 3 (PRTN3)], [myeloperoxidase (MPO)], [cathepsin D (CTSD)], [defensin, alpha 3, neutrophil-specific (DEFA3)], azurocidin 1 (AZU1)], and [bactericidal/permeability-increasing protein (BPI)] the most representative ones. Proteins codified by these genes form part of the azurophilic granules of neutrophils and are involved in both antimicrobial defence and tissue damage. This “neutrophil signature” was paralleled by the necessity of advanced respiratory and vital support, and the presence of bacterial infection. Conclusion Study of transcriptomic signatures in blood suggests an

  18. Participation of Mammalian Target of Rapamycin Complex 1 in Toll-Like Receptor 2– and 4–Induced Neutrophil Activation and Acute Lung Injury

    PubMed Central

    Lorne, Emmanuel; Zhao, Xia; Zmijewski, Jaroslaw W.; Liu, Gang; Park, Young-Jun; Tsuruta, Yuko; Abraham, Edward

    2009-01-01

    mTOR complex 1 (mTORC1) plays a central role in cell growth and cellular responses to metabolic stress. Although mTORC1 has been shown to be activated after Toll-like receptor (TLR)-4 engagement, there is little information concerning the role that mTORC1 may play in modulating neutrophil function and neutrophil-dependent inflammatory events, such as acute lung injury. To examine these issues, we determined the effects of rapamycin-induced inhibition of mTORC1 on TLR2- and TLR4-induced neutrophil activation. mTORC1 was dose- and time-dependently activated in murine bone marrow neutrophils cultured with the TLR4 ligand, LPS, or the TLR2 ligand, Pam3 Cys-Ser-(Lys)4 (PAM). Incubation of PAM- or LPS-stimulated neutrophils with rapamycin inhibited expression of TNF-α and IL-6, but not IκB-α degradation or nuclear translocation of NF-κB. Exposure of PAM or LPS-stimulated neutrophils to rapamycin inhibited phosphorylation of serine 276 in the NF-κB p65 subunit, a phosphorylation event required for optimal transcriptional activity of NF-κB. Rapamycin pretreatment inhibited PAM- or LPS-induced mTORC1 activation in the lungs. Administration of rapamycin also decreased the severity of lung injury after intratracheal LPS or PAM administration, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-α and IL-6 in bronchoalveolar lavage fluid. These results indicate that mTORC1 activation is essential in TLR2- and TLR4-induced neutrophil activation, as well as in the development and severity of acute lung injury. PMID:19131641

  19. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

    PubMed Central

    Duchez, Anne-Claire; Boudreau, Luc H.; Naika, Gajendra S.; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E.; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J.; Poubelle, Patrice E.; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. PMID:26106157

  20. Comparison of activation process of platelets and neutrophils after coronary stent implantation versus balloon angioplasty for stable angina pectoris.

    PubMed

    Inoue, T; Sohma, R; Miyazaki, T; Iwasaki, Y; Yaguchi, I; Morooka, S

    2000-11-15

    The pathophysiologic features of stent-induced cellular responses of platelets and leukocytes have not been established. This study was designed to clinically investigate the activation of platelets and neutrophils after coronary stenting and to identify its effects on the long-term results of coronary stents. Forty-eight consecutive patients with left anterior descending coronary artery disease indicating coronary intervention were randomly assigned to either a balloon angioplasty group or a coronary stent group. Flow cytometric analysis demonstrated that the transcardiac gradient (the value of coronary sinus blood minus the value of peripheral blood) of platelet surface expression of CD62P (p < 0.001) and CD63 (p < 0.01) increased immediately after coronary stenting, but increased less significantly immediately after balloon angioplasty (CD62P, p < 0.01; CD63, p < 0.05). These increases were persistently observed after coronary stenting but transiently after balloon angioplasty alone during a 48-hour observation period after the procedures. The gradient for neutrophil surface expression of CD11b increased, and that of CD62 L decreased 48 hours after coronary stenting (CD11b, p < 0.001; CD62 L, p < 0.05), but these changes showed less significance 48 hours after balloon angioplasty alone (CD11b, p < 0.05; CD62 L, p = NS). The gradients 48 hours after the procedures for both CD62P (r = 0.39, p < 0.05) and CD11b (r = 0.44, p < 0.01) were independently correlated with the late loss in the stent group, whereas the correlation was seen only for CD11b (r = 0.38, p < 0.05) in the balloon angioplasty group. Both platelet and neutrophil activation was greater after coronary stenting than after balloon angioplasty. Cellular interactions between platelets and neutrophils may be related to the progression of neointimal proliferation leading to restenosis after coronary stent implantation. PMID:11074199

  1. Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases.

    PubMed Central

    Sorsa, T; Ingman, T; Suomalainen, K; Haapasalo, M; Konttinen, Y T; Lindy, O; Saari, H; Uitto, V J

    1992-01-01

    Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in

  2. Apoptotic human neutrophil peptide-1 anti-tumor activity revealed by cellular biomechanics.

    PubMed

    Gaspar, Diana; Freire, João M; Pacheco, Teresa R; Barata, João T; Castanho, Miguel A R B

    2015-02-01

    Cancer remains a major cause of morbidity and mortality worldwide. Although progress has been made regarding chemotherapeutic agents, new therapies that combine increased selectivity and efficacy with low resistance are still needed. In the search for new anticancer agents, therapies based on biologically active peptides, in particular, antimicrobial peptides (AMPs), have attracted attention for their decreased resistance development and low cytotoxicity. Many AMPs have proved to be tumoricidal agents against human cancer cells, but their mode of action is still controversial. The existence of common properties shared by the membranes of bacteria and tumor cells points to similar lipid-targeting mechanisms in both cases. On the other hand, anticancer peptides (ACPs) also induce apoptosis and inhibit angiogenesis. Human neutrophil peptide-1 (HNP-1) is an endogenous AMP that has been implicated in different cellular phenomena such as tumor proliferation. The presence of HNP-1 in the serum/plasma of oncologic patients turns this peptide into a potential tumor biomarker. The present work reveals the different effects of HNP-1 on the biophysical and nanomechanical properties of solid and hematological tumor cells. Studies on cellular morphology, cellular stiffness, and membrane ultrastructure and charge using atomic force microscopy (AFM) and zeta potential measurements show a preferential binding of HNP-1 to solid tumor cells from human prostate adenocarcinoma when compared to human leukemia cells. AFM also reveals induction of apoptosis with cellular membrane defects at very low peptide concentrations. Understanding ACPs mode(s) of action will certainly open innovative pathways for drug development in cancer treatment. PMID:25447543

  3. Epithelial neutrophil-activating peptide (ENA-78), acute coronary syndrome prognosis, and modulatory effect of statins.

    PubMed

    Zineh, Issam; Beitelshees, Amber L; Welder, Gregory J; Hou, Wei; Chegini, Nasser; Wu, Jun; Cresci, Sharon; Province, Michael A; Spertus, John A

    2008-01-01

    Endothelial inflammation with chemokine involvement contributes to acute coronary syndromes (ACS). We tested the hypothesis that variation in the chemokine gene CXCL5, which encodes epithelial neutrophil-activating peptide (ENA-78), is associated with ACS prognosis. We also investigated whether statin use, a potent modulator of inflammation, modifies CXCL5's association with outcomes and characterized the in vitro effect of atorvastatin on endothelial ENA-78 production. Using a prospective cohort of ACS patients (n = 704) the association of the CXCL5 -156 G>C polymorphism (rs352046) with 3-year all-cause mortality was estimated with hazard ratios (HR). Models were stratified by genotype and race. To characterize the influence of statins on this association, a statin*genotype interaction was tested. To validate ENA-78 as a statin target in inflammation typical of ACS, endothelial cells (HUVECs) were treated with IL-1beta and atorvastatin with subsequent quantification of CXCL5 expression and ENA-78 protein concentrations. C/C genotype was associated with a 2.7-fold increase in 3-year all-cause mortality compared to G/G+G/C (95%CI 1.19-5.87; p = 0.017). Statins significantly reduced mortality in G/G individuals only (58% relative risk reduction; p = 0.0009). In HUVECs, atorvastatin dose-dependently decreased IL-1beta-stimulated ENA-78 concentrations (p<0.0001). Drug effects persisted over 48 hours (p<0.01). CXCL5 genotype is associated with outcomes after ACS with potential statin modification of this effect. Atorvastatin lowered endothelial ENA-78 production during inflammation typical of ACS. These findings implicate CXCL5/ENA-78 in ACS and the statin response. PMID:18769620

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

    PubMed Central

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

    1993-01-01

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

  5. Characterization of a polysaccharide from Rosa davurica and inhibitory activity against neutrophil migration.

    PubMed

    Tong, Haibin; Jiang, Guiquan; Guan, Xingang; Wu, Hong; Song, Kangxing; Cheng, Kuan; Sun, Xin

    2016-08-01

    The rapid recruitment of neutrophils from peripheral blood into infected sites is critical step for inflammatory responses; however, the excessive and improper recruitment can lead to serious tissue damages. Thus, it is a promising strategy to inhibit their excessive recruitment for treating inflammation-related disease. Here, we isolated a polysaccharide (RDPA1) from Rosa davurica, to evaluate its physicochemical property and inhibitory effects on neutrophil migration. RDPA1 was obtained by hot-water extraction, ethanol precipitation, and fractionated by DEAE-cellulose and Sepharose CL-6B columns. RDPA1 significantly inhibited in vitro migration of human neutrophils evaluated by transwell chamber and impacted the migratory behavior observed by time-lapsed microscopy, we found the migrated distance and average velocity of RDPA1-treated cells were greatly reduced. In addition, RDPA1 treatment impaired in vivo neutrophil infiltration in the peritonitis mice. RDPA1 exhibited significant blocking capacity of the interaction between β2 integrins and ICAM-1 evaluated by flow cytometry and in vitro protein binding assay. Together, these results suggest RDPA1 could be considered as a potential candidate for developing a novel anti-inflammatory agent. PMID:27112979

  6. [The phagocytosis of polymorphonuclear neutrophilic granulocytes in progressive periodontitis].

    PubMed

    Konopka, T; Zietek, M

    1995-01-01

    The aim of this paper was the evaluation of the phagocytic activity of neutrophils in blood and in gingival pocket fluid in patients suffering from rapidly progressive periodontitis (RPP) and postjuvenile periodontitis (PJP). Prior to periodontal treatment the authors evaluated the capacity to phagocytose latex particles of peripheral blood neutrophils from 21 patients with RPP, 51 with PJP and 59 healthy subjects (control group) as well as the phagocytic activity of neutrophils in pocket fluid from 21 patients with RPP, 14 with PJP and from 20 healthy subjects. This phagocytic activity was significantly lower in all examined groups in comparison with the control group. A similar evaluation executed 3 months after treatment revealed normal phagocytosis of blood neutrophils from patients with RPP. In patients receiving complementary pharmacotherapy (spiramycine combined with metronidazol), a better improvement of phagocytosis was noted, than that observed in patients treated only surgically. PMID:7481699

  7. Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

    PubMed Central

    Davidson-Moncada, Jan K; Lopez-Lluch, Guillermo; Segal, Anthony W; Dekker, Lodewijk V

    2002-01-01

    Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase. PMID:11903052

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

    PubMed

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

    2015-09-01

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

  9. Microbicidal activity of neutrophils is inhibited by isolates from recurrent vaginal candidiasis (RVVC) caused by Candida albicans through fungal thioredoxin reductase.

    PubMed

    Ratti, Bianca Altrão; Godoy, Janine Silva Ribeiro; de Souza Bonfim Mendonça, Patrícia; Bidóia, Danielle Lazarin; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Lopes Consolaro, Marcia Edilaine; Estivalet Svidzinski, Terezinha Inez; de Oliveira Silva, Sueli

    2015-01-01

    Vulvovaginal candidiasis (VVC) is characterized by an infection of the vulva and vagina, mainly caused by Candida albicans, a commensal microorganism that inhabits the vaginal, digestive, and respiratory mucosae. Vulvovaginal candidiasis affects approximately 75% of women, and 5% develop the recurrent form (RVVC). The aim of the present study was to evaluate whether neutrophils microbicidal response is triggered when activated with RVVC isolates caused by C. albicans. Our results showed that RVVC isolates induced neutrophil migration but significantly decrease the microbicidal activity of neutrophils, compared with VVC and ASS isolates. The microbicidal activity of neutrophils is highly dependent on the production of reactive oxygen species/reactive nitrogen species (ROS/RNS). However, this isolate induced detoxification of ROS/RNS produced by neutrophils, reflected by the high level of thiol groups and by the oxygen consumption. Therefore, RVVC isolates induced biochemical changes in the inflammatory response triggered by neutrophils, and these effects were mainly related to the detoxification of ROS/RNS through the thioredoxin reductase (TR), a key antioxidant enzyme in fungi. This might be one of the resistance mechanisms triggered by RVVC caused by C. albicans. PMID:25497972

  10. JAK/STAT regulation of Aspergillus fumigatus corneal infections and IL-6/23-stimulated neutrophil, IL-17, elastase, and MMP9 activity.

    PubMed

    Taylor, Patricia R; Roy, Sanhita; Meszaros, Evan C; Sun, Yan; Howell, Scott J; Malemud, Charles J; Pearlman, Eric

    2016-07-01

    IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage. PMID:27034404