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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.

  2. Improved recovery of functionally active eosinophils and neutrophils using novel immunomagnetic technology.

    PubMed

    Son, Kiho; Mukherjee, Manali; McIntyre, Brendan A S; Eguez, Jose C; Radford, Katherine; LaVigne, Nicola; Ethier, Caroline; Davoine, Francis; Janssen, Luke; Lacy, Paige; Nair, Parameswaran

    2017-10-01

    Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P<0.001, n=8, Wilcoxon paired test), with high isolation purities for both eosinophils (95.0±1.7%) and neutrophils (94.2±10.1%) assessed by two methods: Wright's staining and flow cytometry. In addition, enumeration of CD63(+) (marker for eosinophil activation) and CD66b(+) (marker for neutrophil activation) cells within freshly isolated granulocytes, respectively, confirmed that conventional protocols using density-gradient centrifugation caused cellular activation of the granulocytes at baseline compared to the MACSxpress method. In conclusion, MACSxpress isolation kits were found to be superior to conventional techniques for consistent purifications of eosinophils and neutrophils that were suitable for activation

  3. Neutrophil adhesion and activation under flow

    PubMed Central

    Zarbock, Alexander; Ley, Klaus

    2009-01-01

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

  4. On-chip evaluation of neutrophil activation and neutrophil-endothelial cell interaction during neutrophil chemotaxis.

    PubMed

    Kim, Donghyuk; Haynes, Christy L

    2013-11-19

    Neutrophils are always surrounded by/interacting with other components of the immune system; however, the current mechanistic understanding of neutrophil function is largely based on how neutrophils respond to a single chemical signal in a simplified environment. Such approaches are unable to recapitulate the in vivo microenvironment; thus, cell behavior may not fully represent the physiological behavior. Herein, we exploit a microfluidic model of the complex in vivo milieu to investigate how cell-cell interactions influence human neutrophil migration and surface marker expression. Neutrophil migration against a bacterially derived chemoattractant (formyl-met-leu-phe, fMLP), with and without preactivation by interleukins (interleukin-2 or interleukin-6), was evaluated in the presence and absence of endothelial support cells. Preactivation by interleukins or interaction with endothelial cells resulted in altered migration rates compared to naïve neutrophils, and migration trajectories deviated from the expected movement toward the fMLP signal. Interestingly, interaction with both interleukins and endothelial cells simultaneously resulted in a slight compensation in the deviation-on endothelial cells, 34.4% of untreated neutrophils moved away from the fMLP signal, while only 15.2 or 22.2% (interleukin-2-or interleukin-6-activated) of preactivated cells moved away from fMLP. Neutrophils interacting with interleukins and/or endothelial cells were still capable of prioritizing the fMLP signal over a competing chemoattractant, leukotriene B4 (LTB4). Fluorescence imaging of individual human neutrophils revealed that neutrophils treated with endothelial-cell-conditioned media showed up-regulation of the surface adhesion molecules cluster determinant 11b and 66b (CD11b and CD66b) upon stimulation. On the other hand, CD11b and CD66b down-regulation was observed in untreated neutrophils. These results leverage single cell analysis to reveal that the interaction between

  5. CFTR targeting during activation of human neutrophils.

    PubMed

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

    2016-12-01

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

  6. 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.

  7. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

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

  10. 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.

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

    PubMed

    Falcão, Sarah A C; Weinkopff, Tiffany; Hurrell, Benjamin P; Celes, Fabiana S; Curvelo, Rebecca P; Prates, Deboraci B; Barral, Aldina; Borges, Valeria M; Tacchini-Cottier, Fabienne; de Oliveira, Camila I

    2015-03-01

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

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. Biologically active neutrophil chemokine pattern in tonsillitis

    PubMed Central

    RUDACK, C; JÖRG, S; SACHSE, F

    2004-01-01

    To gain an insight into the mechanisms of chronic and acute inflammation, the production of neutrophil chemokines in different types of tonsillitis – hyperplastic tonsillitis (HT), recurrent tonsillitis (RT) and peritonsillar abscesses (PA) – was investigated. The chemokines interleukin-8 (IL-8), growth-related oncogene-α (GRO-α), epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2) were detected and shown to have different biological activities. With respect to the biological properties of CXC chemokines, the biological activity of the chemokines was identified using a three-step high-performance liquid chromatography (HPLC) technique, a bioassay involving measurement of neutrophil chemotaxis in a single Boyden chamber in tissue of HT, RT and PA. Using reverse transcription-polymerase chain reaction (RT-PCR), the chemokine concentrations were determined in the different tonsillitis entities. The chemokine pattern was dominated in PA by IL-8 and GRO-α and in RT by GRO-α. Hyperplastic tonsils of patients without a history of infection generated about five times lower IL-8 than PA. A protein concentration of GCP-2 was induced in PA and RT, whereas ENA-78 remained the same in all entities. In conclusion, it would appear that IL-8 was up-regulated in acute inflammation, whereas GRO-α dominated in chronic inflammation. ENA-78 seems not to play a pivotal role in inflammatory processes in tonsils. GCP-2 may serve as a substitute chemokine in certain inflammatory conditions as its quantity of mRNA and protein was higher in RT and PA than in HT. PMID:15008987

  15. In vitro interferon γ improves the oxidative burst activity of neutrophils in patients with chronic granulomatous disease with a subtype of gp91phox deficiency

    PubMed Central

    Uygun, Dilara F. Kocacik; Köksoy, Sadi; Şahin, Emel; Yeğin, Olcay

    2015-01-01

    Aim of this study Chronic granulomatous disease (CGD) is a genetically heterogeneous primary immunodeficiency caused by a defect in phagocyte production of oxygen metabolites, and resulting in infections produced by catalase-positive microorganisms and fungi. Interferon γ (IFN-γ) has a multitude of effects on the immune system. Although preliminary studies with CGD patients on treatment with IFN-γ showed that it enhanced phagocytosis and superoxide production, ongoing studies did not reveal a significant increase of this function. Here we investigated the oxidative capacity of phagocytes in different subtypes of CGD patients on treatment with IFN-γ in vitro. Material and methods Fifty-seven patients with CGD from 14 immunology centres were enrolled to our multi-centre study. Twenty-one patients were studied as controls. Oxidative burst assay with dihydrorhodamine 123 (DHR) was used and the stimulation index (SI) was calculated with respect to CGD subtypes in both neutrophils and monocytes before, and then one and 24 hours after adding IFN-γ. Results Upon comparison of the SIs of the patients’ neutrophils before in vitro IFN-γ at hour 0, and after adding IFN-γ at hour 1 and 24 were compared, and the differences were determined between hours 0-24 and hours 1-24. This difference was especially apparent between hours 1-24. In CGD subtypes, particularly in gp91phox subtype, it was seen that, following in vitro IFN-γ, SIs of neutrophils began to increase after hour 1, and that increase became more apparent at hour 24. Conclusions Our study showed that IFN-γ treatment may increase the oxidative bursting activity by increasing the superoxide production in neutrophils, particularly in gp91phox subtype. PMID:26155184

  16. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed Central

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

    2014-01-01

    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

  19. 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. © 2014 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.

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

    PubMed

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

    1999-03-05

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

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

    PubMed

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

    2013-11-01

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

  2. P-SELECTIN MEDIATED PLATELET-NEUTROPHIL AGGREGATE FORMATION ACTIVATES NEUTROPHILS IN MOUSE AND HUMAN SICKLE CELL DISEASE

    PubMed Central

    Polanowska-Grabowska, Renata; Wallace, Kori; Field, Joshua J.; Chen, Lanlin; Marshall, Melissa A.; Figler, Robert; Gear, Adrian R. L.; Linden, Joel

    2010-01-01

    Objective Both platelet and neutrophil activation occur in sickle cell disease (SCD) but the interdependence of these events is unknown. The goal of this study was to determine the role of platelets in stimulating mouse and human neutrophil activation and pulmonary injury in SCD. Methods and Results Platelet activation and binding to leukocytes was measured in control and SCD mice and patients. Relative to controls, blood obtained from SCD mice or patients contained significantly elevated platelet-neutrophil aggregates (PNAs). Both platelets and neutrophils found in sickle PNAs were activated. Multi-spectral imaging (ImageStream) and conventional flow cytometry revealed a subpopulation of activated neutrophils with multiple adhered platelets that expressed significantly more CD11b and exhibited greater oxidative activity than single neutrophils. On average, wild type and sickle PNAs contained 1.1 and 2.6 platelets per neutrophil, respectively. Hypoxia/reoxygenation induced a further increase in platelet-neutrophil aggregates in SCD mice and additional activation of both platelets and neutrophils. Pretreatment of SCD mice with clopidogrel or P-selectin antibody reduced the formation of PNAs and neutrophil activation and decreased lung vascular permeability. Conclusions In sum, our findings suggest that platelet binding activates neutrophils and contributes to a chronic inflammatory state and pulmonary dysfunction in SCD. Inhibition of platelet activation may be useful to decrease tissue injury in SCD, particularly during the early stages of vaso-occlusive crises. PMID:21071696

  3. Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation.

    PubMed

    Duarte, Tonya Azevedo; Noronha-Dutra, Alberto Augusto; Nery, Joilda Silva; Ribeiro, Samantha Brum; Pitanga, Thassila Nogueira; Lapa E Silva, José R; Arruda, Sérgio; Boéchat, Neio

    2012-05-01

    The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2010-09-01

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

  5. Luminol chemiluminescence and active oxygen generation by activated neutrophils.

    PubMed

    Takahashi, R; Edashige, K; Sato, E F; Inoue, M; Matsuno, T; Utsumi, K

    1991-03-01

    Upon stimulation by various ligands and membrane perturbers, neutrophils produce various active oxygen species. Since luminol chemiluminescence (LCL) in neutrophils can be blocked by azide, an inhibitor of myeloperoxidase, LCL has been believed to reflect mainly the myeloperoxidase-catalyzed reaction. When cells were stimulated by formyl-methionyl-leucyl-phenylalanine, LCL was strongly inhibited by superoxide dismutase (SOD) and uric acid, a scavenger for hydroxy radical (.OH) and singlet oxygen, whereas it was stimulated by azide. LCL was also inhibited by .OH scavengers, such as mannitol, ethanol, and dimethylsulfoxide. However, when stimulated by phorbol myristate acetate or opsonized zymosan, LCL was strongly inhibited by azide but not by uric acid, and the inhibitory action of SOD was low. Thus, the qualitative and quantitative aspects of reactive oxygen generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the metabolic fate of active oxygens in neutrophils and, hence, their effect on microorganisms and the surrounding tissues might differ depending on the stimulus.

  6. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

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

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

    PubMed

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

    2007-07-27

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

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

    PubMed

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

    2015-07-31

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

  9. Fish oil supplementation improves neutrophil function during cancer chemotherapy.

    PubMed

    Bonatto, Sandro J R; Oliveira, Heloisa H P; Nunes, Everson A; Pequito, Daniele; Iagher, Fabiola; Coelho, Isabela; Naliwaiko, Katya; Kryczyk, Marcelo; Brito, Gleisson A P; Repka, João; Sabóia, Luciano V; Fukujima, George; Calder, Philip C; Fernandes, Luiz C

    2012-04-01

    Cancer chemotherapy is associated with neutropenia and impaired neutrophil function. This study aimed to investigate whether supplementation with low dose fish oil (FO), providing n-3 polyunsaturated fatty acids, in cancer patients receiving chemotherapy after surgical tumor (mainly gastrointestinal) removal is able to improve the function of blood neutrophils. Patients (n = 38) receiving chemotherapy (5-fluorouracil and leucovorin) were randomized into two groups; one group (control) did not receive a supplement, while the other group (FO) received 2 g FO/day for 8 weeks; the FO provided 0.3 g eicosapentaenoic acid plus 0.4 g docosahexaenoic acid per day. Patients in the control group lost an average of 2.5 kg of weight over the 8 weeks of the study. The number of blood polymorphonuclear cells (PMNC), mainly neutrophils, and their functions (phagocytosis and hydrogen peroxide production) decreased in the control group (average decreases of approximately 30, 45 and 17%, respectively). FO prevented these decreases and actually increased body weight (average of 1.7 kg weight gain; p < 0.002 vs. control group), PMNC number (average 29% increase), phagocytosis (average 14% increase) and superoxide production (average 28% increase). FO may be useful in preventing chemotherapy-induced decline in neutrophil number and function.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2001-08-01

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

  12. Neutrophils scan for activated platelets to initiate inflammation

    PubMed Central

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

    2014-01-01

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

  13. FOXO1 Regulates Bacteria-Induced Neutrophil Activity

    PubMed Central

    Dong, Guangyu; Song, Liang; Tian, Chen; Wang, Yu; Miao, Fang; Zheng, Jiabao; Lu, Chanyi; Alsadun, Sarah; Graves, Dana T.

    2017-01-01

    Neutrophils play an essential role in the innate immune response to microbial infection and are particularly important in clearing bacterial infection. We investigated the role of the transcription factor FOXO1 in the response of neutrophils to bacterial challenge with Porphyromonas gingivalis in vivo and in vitro. In these experiments, the effect of lineage-specific FOXO1 deletion in LyzM.Cre+FOXO1L/L mice was compared with matched littermate controls. FOXO1 deletion negatively affected several critical aspects of neutrophil function in vivo including mobilization of neutrophils from the bone marrow (BM) to the vasculature, recruitment of neutrophils to sites of bacterial inoculation, and clearance of bacteria. In vitro FOXO1 regulated neutrophil chemotaxis and bacterial killing. Moreover, bacteria-induced expression of CXCR2 and CD11b, which are essential for several aspects of neutrophil function, was dependent on FOXO1 in vivo and in vitro. Furthermore, FOXO1 directly interacted with the promoter regions of CXCR2 and CD11b. Bacteria-induced nuclear localization of FOXO1 was dependent upon toll-like receptor (TLR) 2 and/or TLR4 and was significantly reduced by inhibitors of reactive oxygen species (ROS and nitric oxide synthase) and deacetylases (Sirt1 and histone deacetylases). These studies show for the first time that FOXO1 activation by bacterial challenge is needed to mobilize neutrophils to transit from the BM to peripheral tissues in response to infection as well as for bacterial clearance in vivo. Moreover, FOXO1 regulates neutrophil function that facilitates chemotaxis, phagocytosis, and bacterial killing. PMID:28928749

  14. Adhesive dynamics simulation of neutrophil arrest with deterministic activation.

    PubMed

    Krasik, Ellen F; Yee, Ka Lai; Hammer, Daniel A

    2006-08-15

    The transition from rolling to firm adhesion is a key element of neutrophil activation and essential to the inflammatory response. Although the molecular mediators of rolling and firm adhesion are known to be selectins and beta2 -integrins, respectively, the precise dynamic mechanism by which these ligands facilitate neutrophil arrest remains unknown. Recently, it has been shown that ligation of E-selectin can stimulate the firm adhesion of neutrophils via a MAP-kinase cascade. To study the possible mechanism by which neutrophil arrest could occur, we created an integrated model by combining two methodologies from computational biology: a mechanics-based modeling of leukocyte adhesion (adhesive dynamics) and signal transduction pathway modeling. Within adhesive dynamics, a computational method our group has shown to accurately recreate rolling dynamics, we include a generic, tunable integrin activation module that links selectin engagement to integrin and activity. This model allows us to relate properties of the activation function to the dynamics of rolling and the time and distance rolled before arrest. This integrated model allows us to understand how intracellular signaling activity can set the timescale of neutrophil activation, adhesion, and diapedesis.

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

    PubMed Central

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

    2016-01-01

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

  16. Mitraphylline inhibits lipopolysaccharide-mediated activation of primary human neutrophils.

    PubMed

    Montserrat-de la Paz, Sergio; Fernandez-Arche, Angeles; de la Puerta, Rocío; Quilez, Ana M; Muriana, Francisco J G; Garcia-Gimenez, Maria Dolores; Bermudez, Beatriz

    2016-02-15

    Mitraphylline (MTP) is the major pentacyclic oxindolic alkaloid presented in Uncaria tomentosa. It has traditionally been used to treat disorders including arthritis, heart disease, cancer, and other inflammatory diseases. However, the specific role of MTP is still not clear, with more comprehensivestudies, our understanding of this ancient herbal medicine will continue growing. Some studies provided its ability to inhibit proinflamatory cytokines, such as TNF-α, through NF-κB-dependent mechanism. TNF-α primes neutrophils and modulates phagocytic and oxidative burst activities in inflammatory processes. Since, neutrophils represent the most abundant pool of leukocytes in human blood and play a crucial role in inflammation, we aimed to determine the ability of MTP to modulate neutrophil activation and differentially regulate inflammatory-related cytokines. To determine the mechanism of action of MTP, we investigated the effects on LPS-activated human primary neutrophils responses including activation surface markers by FACS and the expression of inflammatory cytokines, measured by real time PCR and ELISA. Treatment with MTP reduced the LPS-dependent activation effects. Activated neutrophils (CD16(+)CD62L(-)) diminished after MTP administration. Moreover, proinflamatory cytokines (TNF-α, IL-6 or IL-8) expression and secretion were concomitantly reduced, similar to basal control conditions. Taken together, our results demonstrate that MTP is able to elicit an anti-inflammatory response that modulates neutrophil activation contributing to the attenuation of inflammatory episodes. Further studies are need to characterize the mechanism by which MTP can affect this pathway that could provide a means to develop MTP as new candidate for inflammatory disease therapies. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Effect of temperature on phagocytic activity of neutrophils.

    PubMed

    Pramanik, Tapas; Thapa, Manoj; Saikia, Tolan Chandra

    2004-06-01

    The effect of temperature on phagocytic activity of neutrophils has been evaluated. Freshly collected heparinised blood samples from young healthy volunteers were incubated with heat killed Staphylococcus aureus at 37 degrees C, 38 degrees C, 39 degrees C and 40 degrees C for 20 minutes. Some of the neutrophils engulfed some heat killed bacteria. Then the blood smears were prepared and stained by Leishman's stain. Engulfed bacteria within the neutrophils stained intensely were observed and counted to find out the phagocytic index and avidity index of the neutrophils. It was found that phagocytic index increased significantly at 38 degrees C and 39 degrees C in comparison to that of at 37 degrees C but did not show significant increase when incubated at 40 degrees C. It seems that contractile elements responsible for the movement of the neutrophils through the formation of pseudopod is more activated at higher temperature (38 degrees C and 39 degrees C) in comparison to that of at normal body temperature (37 degrees C). Temperature higher than 39 degrees C may cause impairment in enzyme function responsible for assembly and disassembly of actin and myosin filaments in the cell causing decreased movement and decreased rate of formation of psudopod resulting in decreased phagocytic activity.

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

    PubMed

    Martinova, E A; Baranov, V I

    2014-01-01

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

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

    PubMed

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

    2006-01-01

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

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

    PubMed

    Mukaida, N

    2000-12-01

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

  1. Cyanate-mediated inhibition of neutrophil myeloperoxidase activity.

    PubMed Central

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

    1997-01-01

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

  2. Activation of the neutrophil bactericidal activity for nontypable Haemophilus influenzae by tumor necrosis factor and lymphotoxin.

    PubMed

    Tan, A M; Ferrante, A; Goh, D H; Roberton, D M; Cripps, A W

    1995-02-01

    Previous studies have suggested that, in vivo, activated T lymphocytes and neutrophils are important in immunity to nontypable Haemophilus influenzae. We now extend this work by showing that neutrophils pretreated with products of activated T lymphocytes or activated macrophages show significantly enhanced killing of nontypable H. influenzae. Lymphotoxin, a product of activated T lymphocytes, significantly enhanced the neutrophil-mediated killing of nontypable H. influenzae, and tumor necrosis factor, produced by activated T lymphocytes as well as macrophages stimulated by activated T lymphocytes, also significantly increased the bactericidal activity of neutrophils. These cytokine-induced effects were seen with short pretreatment times of neutrophils and were maximal by 30 min. The killing of H. influenzae by neutrophils required the presence of heat-labile opsonins. In the absence of these opsonins, both tumor necrosis factor and lymphotoxin were unable to promote the killing of the bacteria by neutrophils. Furthermore, the results showed that tumor necrosis factor-primed neutrophils displayed significantly increased expression of CR3 and CR4 that was associated with increased phagocytosis of complement-opsonized nontypable H. influenzae. These cytokines may play an important role in immunity toward nontypable H. influenzae by stimulating neutrophil bactericidal activity.

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

    PubMed Central

    Kusaka, Y; Donaldson, K

    1990-01-01

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

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

    PubMed

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

    2016-02-16

    RBC sequestration. There were approximately 198 million cases of malaria worldwide in 2013, with an estimated 584,000 deaths occurring mostly in sub-Saharan African children. CM is a severe and rare form of Plasmodium falciparum infection and is associated with high rates of mortality and neurological morbidity, despite antimalarial treatment. A greater understanding of the pathophysiology of CM would allow the development of adjunctive therapies to improve clinical outcomes. A hallmark of CM is cerebral microvasculature sequestration of P. falciparum-infected red blood cells (iRBCs), which results in vasculopathy in some patients. Our data provide a global analysis of the host pathways associated with CM and newly identify an association of activated neutrophils with brain iRBC sequestration. Products of activated neutrophils could alter endothelial cell receptors and coagulation to facilitate iRBC adherence. Future studies can now examine the role of neutrophils in CM pathogenesis to improve health outcomes. Copyright © 2016 Feintuch et al.

  5. Innate immune activation in neutrophilic asthma and bronchiectasis

    PubMed Central

    Simpson, Jodie L; Grissell, Terry V; Douwes, Jeroen; Scott, Rodney J; Boyle, Michael J; Gibson, Peter G

    2007-01-01

    Background The role of the innate immune system in the pathogenesis of asthma is unclear. Activation of innate immune receptors in response to bacterial lipopolysaccharide, viral infection and particulate matter triggers a pre‐programmed inflammatory response, which involves interleukin (IL)8 and neutrophil influx. The inflammatory response in asthma is heterogeneous. Aim To test the hypothesis that innate immune activation may be a relevant inflammatory mechanism in neutrophilic asthma where IL8 levels are increased. Methods Induced sputum was obtained from non‐smoking adults with asthma (n = 49), healthy controls (n = 13) and a positive reference group with bronchiectasis (n = 9). Subjects with asthma were classified into inflammatory subtypes using induced sputum cell counts. Sputum was examined for mRNA expression of the innate immune receptors toll‐like receptor (TLR)2, TLR4 and CD14, and inflammatory cytokines. A separate sputum portion was dispersed and the supernatant assayed for surfactant protein A, IL8, soluble CD14 and endotoxin. Results Expression of innate immune receptors was increased in subjects with bronchiectasis and neutrophilic asthma compared with other asthma subtypes and controls. Increased expression of the receptors TLR2, TLR4 and CD14, as well as the pro‐inflammatory cytokines IL8 and IL1β, was observed. Subjects with neutrophilic asthma had higher airway levels of endotoxin than the other groups studied. Conclusion There is evidence of activation of the innate immune system in asthma which results in the production of pro‐inflammatory cytokines and may contribute to the pathogenesis of neutrophilic asthma. PMID:16844729

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

    PubMed

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

    2017-02-01

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

  7. Nicotinamide Effects Oxidative Burst Activity of Neutrophils in Patients with Poorly Controlled Type 2 Diabetes Mellitus

    PubMed Central

    Samanci, Tülay; Demirel, Gülderen Yanikkaya; Damci, Taner; Ilkova, Hasan

    2004-01-01

    , 1994) and compared in diabetic subjects and healthy controls. Diabetic patients were grouped to receive either 50 mg/kg oral nicotinamide (n = 15) or placebo (n = 15) for a period of 1 month. The 2 groups did not differ in terms of treatment, frequency of hypertension, BMI, diabetes duration, age, fasting plasma glucose (FPG), HbA1c, CRP, ESR, polymorphonuclear leukocyte (PNL) and neutrophil counts. Neutrophil functions were reassessed after the treatment period. Phagocytic activity represented as indexes were lower in diabetic patients when compared to healthy subjects, but the differences were not statistically significant (P > .05). Patients with diabetes mellitus had significantly lower oxidative burst indexes when compared to healthy controls (P values < .05). In diabetic patients, a negative correlation between neutrophil functions and HbA1c was found which was not statistically significant (P values > .05). Phagocytic indexes were similar in nicotinamide and placebo groups after treatment period (P > .05). But oxidative burst activity in patients receiving nicotinamide was greater when compared with placebo and the difference was statistically significant at 30 and 45 minutes (P values .04 and .03). This effect of nicotinamide may be due to increased NADH content and NADPH oxidase activity of the cell, which needs to be further studied. Impaired neutrophil functions may aggravate various infections in patients with diabetes mellitus and blood glucose regulation is an important target of treatment to improve neutrophil functions. But nicotinamide treatment may help to improve prognosis in diabetic patients with severe infections. PMID:15203886

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

    PubMed Central

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

    2016-01-01

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

  9. Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

    SciTech Connect

    Ahluwalia, Jatinder

    2008-10-31

    Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I{sub e}. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 {mu}M), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

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

    PubMed

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

    2017-01-01

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

  11. [Metronidazole effect on active oxygen production by human blood neutrophils].

    PubMed

    Shchepetkin, I A

    1997-01-01

    The in vitro effect of metronidazole on production of active oxygen by neutrophila and in the enzymatic system of glucose-glucose oxidase-peroxidase was studied by luminol-dependent chemiluminescence. An increase in the spontaneous and zymozan-stimulated chemiluminescence and a decrease in the phorbolmyristate acetate (PMA)-stimulated chemiluminescence after 2-hour preincubation of the neutrophils with 8.5 mM of metronidazole were observed. In concentrations of 0.9 to 8.7 mM metronidazole (without washing) dose-dependently lowered the neutrophil chemiluminescence in response to the effect of PMA and ionophore A23187 and to a lesser degree to that of zymozan. In doses of 20 to 100 mM the drug had an insignificant effect on production of active oxygen by the neutrophils in response to the cell stimulation by PMA, ionophore A23187 and zymozan. The data are in conformity with the scavenger effect of metronidazole on active oxygen radicals generating in the cell-free enzymatic system both in the presence and in the absence of superoxide dismutase.

  12. 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. Copyright © 2015 the American Physiological Society.

  13. Comparison of respiratory burst activity of inflammatory neutrophils in ayu (Plecoglossus altivelis) and carp (Cyprinus carpio).

    PubMed

    Serada, Ken; Moritomo, Tadaaki; Teshirogi, Kyosuke; Itou, Takuya; Shibashi, Takashi; Inoue, Yuuki; Nakanishi, Teruyuki

    2005-10-01

    Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

  14. Repurposing Treatments to Enhance Innate Immunity. Can Statins Improve Neutrophil Functions and Clinical Outcomes in COPD?

    PubMed Central

    Walton, Georgia M.; Stockley, James A.; Griffiths, Diane; Sadhra, Charandeep S.; Purvis, Thomas; Sapey, Elizabeth

    2016-01-01

    Drug classes used in the treatment of Chronic Obstructive Pulmonary Disease (COPD) have not changed for many years, and none to date have shown disease-modifying activity. Statins are used to help reduce cardiovascular risk, which is high in many patients with COPD. Their use has been associated with improvements in some respiratory manifestations of disease and reduction in all-cause mortality, with greatest reductions seen in patients with the highest inflammatory burden. The mechanism for these effects is poorly understood. Neutrophils are key effector cells in COPD, and correlate with disease severity and inflammation. Recent in vitro studies have shown neutrophil functions are dysregulated in COPD and this is thought to contribute both to the destruction of lung parenchyma and to the poor responses seen in infective exacerbations. In this article, we will discuss the potential utility of statins in COPD, with a particular emphasis on their immune-modulatory effects as well as presenting new data regarding the effects of statins on neutrophil function in vitro. PMID:27727158

  15. Depletion of tissue plasminogen activator attenuates lung ischemia-reperfusion injury via inhibition of neutrophil extravasation

    PubMed Central

    Zhao, Yunge; Sharma, Ashish K.; LaPar, Damien J.; Kron, Irving L.; Ailawadi, Gorav; Liu, Yuan; Jones, David R.; Laubach, Victor E.

    2011-01-01

    Ischemia-reperfusion (IR) injury following lung transplantation remains a major source of early morbidity and mortality. Histologically, this inflammatory process is characterized by neutrophil infiltration and activation. We previously reported that lung IR injury was significantly attenuated in plasminogen activator inhibitor-1-deficient mice. In this study, we explored the potential role of tissue plasminogen activator (tPA) in a mouse lung IR injury model. As a result, tPA knockout (KO) mice were significantly protected from lung IR injury through several mechanisms. At the cellular level, tPA KO specifically blocked neutrophil extravasation into the interstitium, and abundant homotypic neutrophil aggregation (HNA) was detected in the lung microvasculature of tPA KO mice after IR. At the molecular level, inhibition of neutrophil extravasation was associated with reduced expression of platelet endothelial cell adhesion molecule-1 mediated through the tPA/ LDL receptor-related protein/NF-κB signaling pathway, whereas increased P-selectin triggered HNA. At the functional level, tPA KO mice incurred significantly decreased vascular permeability and improved lung function following IR. Protection from lung IR injury in tPA KO mice occurs through a fibrinolysis-independent mechanism. These results suggest that tPA could serve as an important therapeutic target for the prevention and treatment of acute IR injury after lung transplantation. PMID:21378024

  16. Granulocyte and monocyte adsorption apheresis for refractory skin diseases due to activated neutrophils, psoriasis, and associated arthropathy.

    PubMed

    Sakanoue, Masanao; Takeda, Koichiro; Kawai, Kazuhiro; Kanekura, Takuro

    2013-10-01

    Granulocyte and monocyte adsorption apheresis (GMA), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is designed to remove activated granulocytes and monocytes. We previously demonstrated that GMA was useful for treating neutrophilic dermatoses and associated arthropathy as it adsorbs Mac-1 (CD11b/CD18)-expressing neutrophils to the CA beads by the binding of complement component (iC3b) and CD11b expressed on activated neutrophils. The objective of this study is to further assess the clinical effectiveness of GMA in the treatment of neutrophilic dermatoses and associated arthropathy. The effect of GMA for skin lesions and joint lesions was assessed in 44 and 23 patients, respectively. Mac-1 expression on peripheral neutrophils was measured by flow cytometry. Skin lesions and arthropathy improved in 39 of 44 patients (88.6%) and 22 of 23 (95.6%), respectively. Mac-1 (CD11b/CD18) expression on the peripheral neutrophils, 27.1 ± 6.66 MFI (mean fluorescence intensity) before treatment, was reduced to 17.9 ± 3.02 MFI by GMA (P < 0.05). Clinical effectiveness of GMA for the treatment of intractable neutrophilic dermatoses and associated arthropathy was further confirmed. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

  17. 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

  18. 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

  19. Peripheral Neutrophil to Lymphocyte Ratio Improves Prognostication in Colon Cancer.

    PubMed

    Rashtak, Shahrooz; Ruan, Xiaoyang; Druliner, Brooke R; Liu, Hongfang; Therneau, Terry; Mouchli, Mohamad; Boardman, Lisa A

    2017-06-01

    We studied the role of peripheral neutrophil to lymphocyte ratio (NLR) on survival outcomes in colon and rectal cancer to determine if its inclusion improved prognostication within existing staging systems. Disease-free (DFS) and overall survival (OS) hazard ratios (HRs) of pretreatment NLR were calculated for 2536 patients with stage I to III colon or rectal cancer and adjusted for age, positive/total number of nodes, T stage, and grade. The association of NLR with clinicopathologic features and survival was evaluated and compared with the American Joint Committee on cancer (AJCC) TNM staging and Memorial Sloan Kettering Cancer Center (MSKCC) models. High NLR was significantly associated with worse DFS (HR, 1.36; 95% confidence interval [CI], 1.08-1.70; P = .009) and OS (HR, 1.65; 95% CI, 1.29-2.10; P < .0005) in all stages for patients with colon, but not rectal, cancer. High NLR was significantly associated with site-specific worse prognosis, which was stronger in the left versus right colon; an inverse relationship with grade was found. The impact of high NLR on DFS and OS occurred early, with the majority of deaths within 2 years following surgery. Adjusted HRs for 5-year and 2-year outcomes in colon cancer per each additional 2-unit increase in NLR were 1.15 (95% CI, 1.08-1.23) and 1.20 (95% CI, 1.10-1.30), respectively. The addition of NLR enhanced the prognostic utility of TNM (TNM alone vs. TNM + NLR: concordance index, 0.60 vs. 0.68), and MSKCC (MSKCC alone vs. MSKCC + NLR: concordance index, 0.71 vs. 0.73) models for colon cancer patients. NLR is an independent prognostic variable for nonmetastatic colon cancer that enhances existing clinical staging systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Calcium pyrophosphate dihydrate crystals activate MAP kinase in human neutrophils: inhibition of MAP kinase, oxidase activation and degranulation responses of neutrophils by taxol.

    PubMed Central

    Jackson, J K; Tudan, C; Sahl, B; Pelech, S L; Burt, H M

    1997-01-01

    The activation of MAP kinase in human neutrophils stimulated by both uncoated and plasma-opsonized crystals of triclinic calcium pyrophosphate dihydrate (CPPD) was investigated. The effect of taxol on MAP kinase activation and on the responses of neutrophils stimulated by plasma-opsonized crystals was determined. MAP kinase activation was identified and quantified in Mono Q chromatography separated fractions of neutrophils that had been incubated with CPPD crystals by measuring [gamma-32P]adenosine triphosphate (ATP) phosphorylation of myelin basic protein and using immunoblotting techniques. Human neutrophils were incubated with taxol (0-50 microM), added to plasma-opsonized CPPD (50 mg/ml) and MAP kinase activation, chemiluminescence, superoxide anion generation, lysozyme and myeloperoxidase release were monitored. Both uncoated and plasma coated CPPD crystals induced a large increase in MAP kinase activity in neutrophils over control levels within 1 min of incubation. Pretreatment of neutrophils with taxol was able to suppress this activation of MAP kinase. Taxol produced a concentration-dependent inhibition of opsonized CPPD-induced neutrophil chemiluminescence, superoxide anion production and myeloperoxide release. Taxol at 28 microM also significantly inhibited chemiluminescence, superoxide anion production and myeloperoxidase release from neutrophils stimulated by opsonized zymosan. This is the first report of crystal-induced activation of MAP kinase in neutrophils. Microtubule-associated processes, such as signal transduction, secretion and phagocytosis are involved in particulate-induced neutrophil responses. We have suggested that the inhibitory effect of taxol observed in this work is due to its stabilizing effect on microtubules and disruption of MAP kinase activation associated with microtubules. Images Figure 1 Figure 3 PMID:9176102

  1. Human resistin promotes neutrophil pro-inflammatory activation, neutrophil extracellular trap formation, and increases severity of acute lung injury

    PubMed Central

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

    2014-01-01

    Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies have also suggested that resistin has proinflammatory properties. In these studies, we examined if the human specific variant of resistin affects neutrophil activation as well as the severity of LPS-induced acute lung injury (ALI). Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using resistin humanized 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+/−/−, compared to control Rtn−/−/− neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase (AMPK), a major sensor and regulator of cellular bioenergetics that is also implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin also enhanced neutrophil extracellular trap formation. In LPS-induced ALI, humanized resistin mice demonstrated enhanced production of pro-inflammatory cytokines, more severe pulmonary edema, increased NET 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 may be a target for future therapies aimed at diminishing the severity of acute lung injury and other inflammatory situations where neutrophils play a major role. PMID:24719460

  2. Neutrophil extracellular traps can activate alternative complement pathways.

    PubMed

    Wang, H; Wang, C; Zhao, M-H; Chen, M

    2015-09-01

    The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV. © 2015 British Society for Immunology.

  3. Neutrophil extracellular traps can activate alternative complement pathways

    PubMed Central

    Wang, H; Wang, C; Zhao, M-H; Chen, M

    2015-01-01

    The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV PMID:25963026

  4. Chloride transport in functionally active phagosomes isolated from Human neutrophils

    PubMed Central

    Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

    2012-01-01

    Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport

  5. Interference of Wegener's granulomatosis autoantibodies with neutrophil Proteinase 3 activity.

    PubMed Central

    van de Wiel, B A; Dolman, K M; van der Meer-Gerritsen, C H; Hack, C E; von dem Borne, A E; Goldschmeding, R

    1992-01-01

    Classic anti-neutrophil cytoplasmic autoantibodies (C-ANCA) are disease-specific markers of Wegener's granulomatosis (WG). The possible pathogenetic role of these autoantibodies, which are directed against Proteinase 3 (PR3), is not yet clear. We studied the effect of C-ANCA on PR3 proteolytic activity and on the complexation of PR3 with alpha 1-antitrypsin (alpha 1AT). C-ANCA IgG from eight patients with active WG significantly inhibited PR3 proteolytic activity, particularly towards elastin (median 84.2% inhibition). C-ANCA IgG significantly inhibited the complexation of PR3 with alpha 1AT (median 58.8% inhibition). Moreover, addition of purified PR3 to C-ANCA-positive sera from WG patients yielded less complexes with alpha 1AT (median 44.8%) compared with sera containing perinuclear anti-neutrophil cytoplasmic autoantibodies (P-ANCA) or ANCA-negative sera. These findings indicate the existence of a hitherto unknown property of C-ANCA, which may be of importance in the pathogenesis of WG. PMID:1458677

  6. 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

  7. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

    PubMed

    Noubouossie, Denis F; Whelihan, Matthew F; Yu, Yuan-Bin; Sparkenbaugh, Erica; Pawlinski, Rafal; Monroe, Dougald M; Key, Nigel S

    2017-02-23

    NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins. © 2017 by The American Society of Hematology.

  8. Activation of the human neutrophil respiratory burst with zymosan-activated serum.

    PubMed

    Smith, R J; Iden, S S; Bowman, B J

    1984-06-15

    Zymosan-activated serum ( ZAS ) stimulated a time- and concentration-dependent generation of superoxide anion (O-2) by human neutrophils. O-2 production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS . O-2 generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS . The amount of O-2 produced by ZAS stimulated neutrophils was enhanced in the presence of extra-cellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS -elicited O-2 production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O-2 in response to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC),N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), demonstrates the stimulus specific nature of ZAS -induced desensitization. Thus, ZAS , which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represents a relevant probe for investigating neutrophil function.

  9. Synergy between RU 28965 (roxithromycin) and human neutrophils for bactericidal activity in vitro.

    PubMed Central

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

    1986-01-01

    The in vitro effects of RU 28965 (roxithromycin), a new semisynthetic macrolide, on human neutrophil activity were compared with those of erythromycin. RU 28965, at a concentration as low as 0.1 microgram/ml, significantly enhanced the phagocytosis and killing of Staphylococcus aureus by neutrophils. Erythromycin displayed a less stimulating effect in a dose-dependent manner. Phagocytosis of Klebsiella pneumoniae was also increased after incubation of neutrophils with RU 28965, but killing was not altered. Neutrophil chemotaxis, myeloperoxidase activity, and O2 consumption were unchanged in the presence of RU 28965. PMID:3019233

  10. Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus

    PubMed Central

    Finsterbusch, Michaela; Hall, Pam; Li, Anqi; Devi, Sapna; Westhorpe, Clare L. V.; Kitching, A. Richard

    2016-01-01

    Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using β2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil

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

    PubMed

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

    2014-10-30

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

  12. Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

    PubMed Central

    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 (104−106). 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

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

    PubMed Central

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

    2016-01-01

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

  14. Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

    PubMed

    Jin, Rong; Yu, Shiyong; Song, Zifang; Zhu, Xiaolei; Wang, Cuiping; Yan, Jinchuan; Wu, Fusheng; Nanda, Anil; Granger, D Neil; Li, Guohong

    2013-01-01

    Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the

  15. Early Enhanced Local Neutrophil Recruitment in Peritonitis-Induced Sepsis Improves Bacterial Clearance and Survival

    PubMed Central

    Craciun, Florin L.; Schuller, Elizabeth R.; Remick, Daniel G.

    2017-01-01

    Neutrophils are critical for the rapid eradication of bacterial pathogens, but they also contribute to the development of multiple organ failure in sepsis. We hypothesized that increasing early recruitment of neutrophils to the focus of infection will increase bacterial clearance and improve survival. Sepsis was induced in mice, using cecal ligation and puncture (CLP); blood samples were collected at 6 and 24 h; and survival was followed for 28 d. In separate experiments, peritoneal bacteria and inflammatory cells were measured. Septic mice predicted to die based on IL-6 levels (Die-P) had higher concentrations of CXCL1 and CXCL2 in the peritoneum and plasma compared with those predicted to live (Live-P). At 6 h, Live-P and Die-P had equivalent numbers of peritoneal neutrophils and bacteria. In Die-P mice the number of peritoneal bacteria increased between 6 and 24 h post-CLP, whereas in Live-P it decreased. The i.p. injection of CXCL1 and CXCL2 in naive mice resulted in local neutrophil recruitment. When given immediately after CLP, CXC chemokines increased peritoneal neutrophil recruitment at 6 h after CLP. This early increase in neutrophils induced by exogenous chemokines resulted in significantly fewer peritoneal bacteria by 24 h [CFU (log) = 6.04 versus 4.99 for vehicle versus chemokine treatment; p < 0.05]. Chemokine treatment significantly improved survival at both 5 d (40 versus 72%) and 28 d (27 versus 52%; p < 0.02 vehicle versus chemokines). These data demonstrate that early, local treatment with CXC chemokines enhances neutrophil recruitment and clearance of bacteria as well as improves survival in the CLP model of sepsis. PMID:21041722

  16. Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival.

    PubMed

    Craciun, Florin L; Schuller, Elizabeth R; Remick, Daniel G

    2010-12-01

    Neutrophils are critical for the rapid eradication of bacterial pathogens, but they also contribute to the development of multiple organ failure in sepsis. We hypothesized that increasing early recruitment of neutrophils to the focus of infection will increase bacterial clearance and improve survival. Sepsis was induced in mice, using cecal ligation and puncture (CLP); blood samples were collected at 6 and 24 h; and survival was followed for 28 d. In separate experiments, peritoneal bacteria and inflammatory cells were measured. Septic mice predicted to die based on IL-6 levels (Die-P) had higher concentrations of CXCL1 and CXCL2 in the peritoneum and plasma compared with those predicted to live (Live-P). At 6 h, Live-P and Die-P had equivalent numbers of peritoneal neutrophils and bacteria. In Die-P mice the number of peritoneal bacteria increased between 6 and 24 h post-CLP, whereas in Live-P it decreased. The i.p. injection of CXCL1 and CXCL2 in naive mice resulted in local neutrophil recruitment. When given immediately after CLP, CXC chemokines increased peritoneal neutrophil recruitment at 6 h after CLP. This early increase in neutrophils induced by exogenous chemokines resulted in significantly fewer peritoneal bacteria by 24 h [CFU (log) = 6.04 versus 4.99 for vehicle versus chemokine treatment; p < 0.05]. Chemokine treatment significantly improved survival at both 5 d (40 versus 72%) and 28 d (27 versus 52%; p < 0.02 vehicle versus chemokines). These data demonstrate that early, local treatment with CXC chemokines enhances neutrophil recruitment and clearance of bacteria as well as improves survival in the CLP model of sepsis.

  17. 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.

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

    PubMed

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

    2001-12-01

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

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

    PubMed

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

    2017-04-13

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

  20. 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.

  1. Assessment of phagocytic activity of neutrophils in chronic obstructive pulmonary disease.

    PubMed

    Shanmugam, Lalitha; Ravinder, S Sheela; Johnson, Priscilla; Padmavathi, R; Rajagopalan, B; Kindo, Anupma Jyoti

    2015-01-01

    To assess the phagocytic activity of neutrophils in subjects with chronic obstructive pulmonary disease (COPD). There is a paucity of data in relation to phagocytic function in COPD. By this multidisciplinary study, a better understanding about the etiology of lung destruction among COPD patients is being sought. The study was conducted among 28 subjects with COPD and 25 controls in a private tertiary hospital in Chennai after obtaining Institutional Ethical Clearance. Known cases of COPD as proven by clinical findings and spirometry were included in the study, and subjects with any other source of infection, recent surgery, or chronic granulomatous disease were excluded. The study subjects were divided into three groups based on the severity of COPD as determined by spirometry, and healthy volunteers were taken as Group 4. After obtaining informed consent, validated respiratory health questionnaire was administered. The phagocytic function was assessed by Candida phagocytic test and Nitroblue Tetrazolium (NBT) Reduction Test. Significantly impaired phagocytic function as indicated by lower phagocytic, lytic indices and decreased NBT reduction of neutrophils was seen in COPD subjects compared to normal healthy controls (P < .001). This study showed that there is phagocytic dysfunction in COPD subjects when compared with normal subjects. This could be due to underlying inflammation in human airway. Understanding the role of neutrophils may lead to improved understanding of the pathogenesis of COPD, which in turn may pave way for implementing modified therapeutic intervention strategies.

  2. 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.

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

    PubMed Central

    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

    2016-01-01

    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

  4. Human Neutrophil-Mediated Nonoxidative Antifungal Activity against Cryptococcus neoformans

    PubMed Central

    Mambula, Salamatu S.; Simons, Elizabeth R.; Hastey, Ryan; Selsted, Michael E.; Levitz, Stuart M.

    2000-01-01

    It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percents inhibition of growth of 67.4 ± 3.4, 84.6 ± 4.4, and 29.2 ± 10.5 (mean ± standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C4 column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules. PMID:11035733

  5. CCR2 dependent neutrophil activation and mobilization rely on TLR4-p38 axis during liver ischemia-reperfusion injury

    PubMed Central

    Xu, Peng; Zhang, Junbin; Wang, Hui; Wang, Guoliang; Wang, Cong-Yi; Zhang, Jinxiang

    2017-01-01

    Liver ischemia-reperfusion injury (IRI) is a common clinical problem in which neutrophil recruitment is an essential event. Our previous study revealed the important role of C-C motif chemokine receptor 2 (CCR2) in neutrophils during liver IRI. The aim of the present study was to further investigate the underlying mechanisms mediating the changes in CCR2 expression in neutrophils during this pathophysiological process. Herein, we found that TLR4 ablation reduced neutrophil mobilization from the bone marrow and the subsequent infiltration into the liver during liver IRI; neutrophil-derived CCR2 expression was also repressed. In addition, neutrophil mobilization was dependent on CCR2 expression in neutrophils, which in turn relied on activation of the TLR4-p38 axis during liver IRI. In conclusion, neutrophil-derived CCR2 expression regulates neutrophil mobilization from the bone marrow and infiltration into the liver, which requires activation of the TLR4-p38 axis during liver IRI. PMID:28670376

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

    PubMed

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

    2017-07-27

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

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

    PubMed

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

    1991-06-15

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

  8. Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation.

    PubMed

    Rodriguez-Rodrigues, Nahuel; Castillo, Luis A; Landoni, Verónica I; Martire-Greco, Daiana; Milillo, M Ayelén; Barrionuevo, Paula; Fernández, Gabriela C

    2017-01-01

    Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria

  9. Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation

    PubMed Central

    Rodriguez-Rodrigues, Nahuel; Castillo, Luis A.; Landoni, Verónica I.; Martire-Greco, Daiana; Milillo, M. Ayelén; Barrionuevo, Paula; Fernández, Gabriela C.

    2017-01-01

    Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria

  10. 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.

  11. PTPN22 Is a Critical Regulator of Fcγ Receptor–Mediated Neutrophil Activation

    PubMed Central

    Miles, Katherine; Chu, Julia Y.; Salter, Donald; Zamoyska, Rose

    2016-01-01

    Neutrophils act as a first line of defense against bacterial and fungal infections, but they are also important effectors of acute and chronic inflammation. Genome-wide association studies have established that the gene encoding the protein tyrosine phosphatase nonreceptor 22 (PTPN22) makes an important contribution to susceptibility to autoimmune disease, notably rheumatoid arthritis. Although PTPN22 is most highly expressed in neutrophils, its function in these cells remains poorly characterized. We show in this article that neutrophil effector functions, including adhesion, production of reactive oxygen species, and degranulation induced by immobilized immune complexes, were reduced in Ptpn22−/− neutrophils. Tyrosine phosphorylation of Lyn and Syk was altered in Ptpn22−/− neutrophils. On stimulation with immobilized immune complexes, Ptpn22−/− neutrophils manifested reduced activation of key signaling intermediates. Ptpn22−/− mice were protected from immune complex–mediated arthritis, induced by the transfer of arthritogenic serum. In contrast, in vivo neutrophil recruitment following thioglycollate-induced peritonitis and in vitro chemotaxis were not affected by lack of PTPN22. Our data suggest an important role for PTPN22-dependent dephosphorylation events, which are required to enable full FcγR-induced activation, pointing to an important role for this molecule in neutrophil function. PMID:27807193

  12. PTPN22 Is a Critical Regulator of Fcγ Receptor-Mediated Neutrophil Activation.

    PubMed

    Vermeren, Sonja; Miles, Katherine; Chu, Julia Y; Salter, Donald; Zamoyska, Rose; Gray, Mohini

    2016-12-15

    Neutrophils act as a first line of defense against bacterial and fungal infections, but they are also important effectors of acute and chronic inflammation. Genome-wide association studies have established that the gene encoding the protein tyrosine phosphatase nonreceptor 22 (PTPN22) makes an important contribution to susceptibility to autoimmune disease, notably rheumatoid arthritis. Although PTPN22 is most highly expressed in neutrophils, its function in these cells remains poorly characterized. We show in this article that neutrophil effector functions, including adhesion, production of reactive oxygen species, and degranulation induced by immobilized immune complexes, were reduced in Ptpn22(-/-) neutrophils. Tyrosine phosphorylation of Lyn and Syk was altered in Ptpn22(-/-) neutrophils. On stimulation with immobilized immune complexes, Ptpn22(-/-) neutrophils manifested reduced activation of key signaling intermediates. Ptpn22(-/-) mice were protected from immune complex-mediated arthritis, induced by the transfer of arthritogenic serum. In contrast, in vivo neutrophil recruitment following thioglycollate-induced peritonitis and in vitro chemotaxis were not affected by lack of PTPN22. Our data suggest an important role for PTPN22-dependent dephosphorylation events, which are required to enable full FcγR-induced activation, pointing to an important role for this molecule in neutrophil function.

  13. Leishmania donovani promastigotes evade the antimicrobial activity of neutrophil extracellular traps.

    PubMed

    Gabriel, Christelle; McMaster, W Robert; Girard, Denis; Descoteaux, Albert

    2010-10-01

    Upon their recruitment to a site of infection and their subsequent activation, neutrophils release DNA and a subset of their granule content to form filamentous structures, known as neutrophil extracellular traps, which capture and kill microorganisms. In this study, we show that Leishmania promastigotes induced the rapid release of neutrophil extracellular traps from human neutrophils and were trapped by these structures. The use of Leishmania mutants defective in the biosynthesis of either lipophosphoglycan or GP63 revealed that these two major surface promastigote virulence determinants were not responsible for inducing the release of the surface protease neutrophil extracellular traps. We also demonstrate that this induction was independent of superoxide production by neutrophils. Finally, in contrast to wild-type Leishmania donovani promastigotes, mutants defective in lipophosphoglycan biosynthesis were highly susceptible to the antimicrobial activity of neutrophil extracellular traps. Altogether, our data suggest that neutrophil extracellular traps may contribute to the containment of L. donovani promastigotes at the site of inoculation, thereby facilitating their uptake by mononuclear phagocytes.

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

    PubMed Central

    Vaidyanathan, Ashwin; Damodar, Komaladevi Sampath

    2015-01-01

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

  15. Hypertonic saline up-regulates A3 adenosine receptors expression of activated neutrophils and increases acute lung injury after sepsis

    PubMed Central

    Inoue, Yoshiaki; Chen, Yu; Pauzenberger, Reinhard; Mark, Hirsh I.; Junger, Wolfgang G.

    2008-01-01

    receptor knockout mice remained only 50% regardless of timing of hypertonic saline administration. Conclusions Polymorphonuclear neutrophils A3 receptors expression determines whether hypertonic saline resuscitation inhibits or aggravates polymorphonuclear neutrophils-induced acute lung injury. These findings suggest that A3 antagonists could improve the efficacy of hypertonic saline resuscitation by reducing side effects in patients whose polymorphonuclear neutrophils are activated before hypertonic saline treatment. PMID:18679117

  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. 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

  18. Modulation of γδ T-cell activation by neutrophil elastase.

    PubMed

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

    2017-09-09

    γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation. © 2017 John Wiley & Sons Ltd.

  19. The Role of Interleukin-1β in Direct and Toll-Like Receptor 4-Mediated Neutrophil Activation and Survival

    PubMed Central

    Prince, Lynne R.; Allen, Lucy; Jones, Elizabeth C.; Hellewell, Paul G.; Dower, Steven K.; Whyte, Moira K.B.; Sabroe, Ian

    2004-01-01

    The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1β has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1β fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1β induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1β, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1−/− mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1β on neutrophils are indirect and mediated by other cells. PMID:15509550

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

    PubMed

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

    2011-08-01

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

  1. Interleukin-6 stimulates neutrophil production of platelet-activating factor.

    PubMed

    Biffl, W L; Moore, E E; Moore, F A; Barnett, C C; Silliman, C C; Peterson, V M

    1996-04-01

    Interleukin-6 (IL-6) is an integral mediator of the acute phase response to injury and infection; an exaggerated IL-6 response has been associated with adverse clinical events. The precise role of IL-6 is unclear, but it appears capable of modulating the functional repertoire of mature neutrophils (PMNs). Our previous work demonstrated that IL-6 -stimulated PMNs are primed by lower concentrations of platelet-activating factor (PAF) than nonstimulated PMNs. Recently, we have found that IL-6 suppresses PMN apoptosis via a PAF-like mechanism. We hypothesized that IL-6 stimulates PMNs to produce PAF. PMNs isolated from healthy human donors were incubated with IL-6 (0.1-100 ng/ml) at 37 degrees C. Lipid production was measured by use of thin-layer chromatography, and PAF quantitated with a scintillation proximity assay. IL-6 (1 and 10 ng/ml) stimulated PMNs to produce increase quantities of PAF. PAF production was associated with an increase in PMN cytosolic calcium. These data may provide mechanistic insight into IL-6 regulation of PMN-mediated cytotoxicity and the role of PAF in mediating IL-6 effects on PMNs.

  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. 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.

  4. Antiinflammatory Effects of Hydrogen Peroxide in Neutrophil Activation and Acute Lung Injury

    PubMed Central

    Zmijewski, Jaroslaw W.; Lorne, Emmanuel; Zhao, Xia; Tsuruta, Yuko; Sha, Yonggang; Liu, Gang; Abraham, Edward

    2009-01-01

    Rationale: Although reactive oxygen species (ROS) are generally considered to be proinflammatory and to contribute to cellular and organ dysfunction when present in excessive amounts, there is evidence that specific ROS, particularly hydrogen peroxide (H2O2), may have antiinflammatory properties. Objectives: To address the role that increases in intracellular H2O2 may play in acute inflammatory processes, we examined the effects of catalase inhibition or the absence of catalase on LPS-induced inflammatory responses. Methods: Neutrophils from control or acatalasemic mice, or control neutrophils incubated with the catalase inhibitor aminotriazole, were treated with LPS, and levels of reactive oxygen species, proteasomal activity, NF-κB activation, and proinflammatory cytokine expression were measured. Acute lung injury (ALI) was produced by intratracheal injection of LPS into control, acatalasemic-, or aminotriazole-treated mice. Measurements and Main Results: Intracellular levels of H2O2 were increased in acatalasemic neutrophils and in neutrophils exposed to aminotriazole. Compared with LPS-stimulated neutrophils from control mice, neutrophils from acatalasemic mice or neutrophils treated with aminotriazole demonstrated reduced 20S and 26S proteasomal activity, IκB-α degradation, NF-κB nuclear accumulation, and production of the proinflammatory cytokines TNF-α and macrophage inhibitory protein (MIP)-2. The severity of LPS-induced ALI was less in acatalasemic mice and in mice treated with aminotriazole as compared with that found in control mice. Conclusions: These results indicate that H2O2 has antiinflammatory effects on neutrophil activation and inflammatory processes, such as ALI, in which activated neutrophils play a major role. PMID:19151196

  5. Antiinflammatory effects of hydrogen peroxide in neutrophil activation and acute lung injury.

    PubMed

    Zmijewski, Jaroslaw W; Lorne, Emmanuel; Zhao, Xia; Tsuruta, Yuko; Sha, Yonggang; Liu, Gang; Abraham, Edward

    2009-04-15

    Although reactive oxygen species (ROS) are generally considered to be proinflammatory and to contribute to cellular and organ dysfunction when present in excessive amounts, there is evidence that specific ROS, particularly hydrogen peroxide (H(2)O(2)), may have antiinflammatory properties. To address the role that increases in intracellular H(2)O(2) may play in acute inflammatory processes, we examined the effects of catalase inhibition or the absence of catalase on LPS-induced inflammatory responses. Neutrophils from control or acatalasemic mice, or control neutrophils incubated with the catalase inhibitor aminotriazole, were treated with LPS, and levels of reactive oxygen species, proteasomal activity, NF-kappaB activation, and proinflammatory cytokine expression were measured. Acute lung injury (ALI) was produced by intratracheal injection of LPS into control, acatalasemic-, or aminotriazole-treated mice. Intracellular levels of H(2)O(2) were increased in acatalasemic neutrophils and in neutrophils exposed to aminotriazole. Compared with LPS-stimulated neutrophils from control mice, neutrophils from acatalasemic mice or neutrophils treated with aminotriazole demonstrated reduced 20S and 26S proteasomal activity, IkappaB-alpha degradation, NF-kappaB nuclear accumulation, and production of the proinflammatory cytokines TNF-alpha and macrophage inhibitory protein (MIP)-2. The severity of LPS-induced ALI was less in acatalasemic mice and in mice treated with aminotriazole as compared with that found in control mice. These results indicate that H(2)O(2) has antiinflammatory effects on neutrophil activation and inflammatory processes, such as ALI, in which activated neutrophils play a major role.

  6. Neutrophil elastase induces inflammation and pain in mouse knee joints via activation of proteinase-activated receptor-2.

    PubMed

    Muley, Milind M; Reid, Allison R; Botz, Bálint; Bölcskei, Kata; Helyes, Zsuzsanna; McDougall, Jason J

    2016-02-01

    Neutrophil elastase plays a crucial role in arthritis. Here, its potential in triggering joint inflammation and pain was assessed, and whether these effects were mediated by proteinase-activated receptor-2 (PAR2). Neutrophil elastase (5 μg) was injected into the knee joints of mice and changes in blood perfusion, leukocyte kinetics and paw withdrawal threshold were assessed. Similar experiments were performed in animals pretreated with the neutrophil elastase inhibitor sivelestat, the PAR2 antagonist GB83, the p44/42 MAPK inhibitor U0126 and in PAR2 receptor knockout (KO) mice. Neutrophil elastase activity was also evaluated in arthritic joints by fluorescent imaging and sivelestat was assessed for anti-inflammatory and analgesic properties. Intra-articular injection of neutrophil elastase caused an increase in blood perfusion, leukocyte kinetics and a decrease in paw withdrawal threshold. Sivelestat treatment suppressed this effect. The PAR2 antagonist GB83 reversed neutrophil elastase-induced synovitis and pain and these responses were also attenuated in PAR2 KO mice. The MAPK inhibitor U0126 also blocked neutrophil elastase-induced inflammation and pain. Active neutrophil elastase was increased in acutely inflamed knees as shown by an activatable fluorescent probe. Sivelestat appeared to reduce neutrophil elastase activity, but had only a moderate anti-inflammatory effect in this model. Neutrophil elastase induced acute inflammation and pain in knee joints of mice. These changes are PAR2-dependent and appear to involve activation of a p44/42 MAPK pathway. Blocking neutrophil elastase, PAR2 and p44/42 MAPK activity can reduce inflammation and pain, suggesting their utility as therapeutic targets. © 2015 The British Pharmacological Society.

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

    PubMed

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

    2012-04-01

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

  8. The Effects of Pterostilbene on Neutrophil Activity in Experimental Model of Arthritis

    PubMed Central

    Drabikova, Katarina; Lojek, Antonin; Ciz, Milan; Ponist, Silvester; Bauerova, Katarina; Nosal, Radomir; Harmatha, Juraj; Jancinova, Viera

    2013-01-01

    It has been demonstrated that pterostilbene inhibits reactive oxygen species production in neutrophils in vitro. However, little is known about its effects on neutrophils during inflammation in vivo. In this study, the effect of pterostilbene on neutrophil activity was investigated in experimental arthritis model. Lewis rats were injected by a single intradermal injection of heat-killed Mycobacterium butyricum in Freund's adjuvant to develop arthritis. Another group of arthritic animals received pterostilbene 30 mg/kg, daily, p.o. The number and activity of neutrophils in blood were measured on a weekly basis during the whole experiment. Moreover, the total radical trapping potential in plasma was measured at the end of the experiment. In the pterostilbene treated arthritic group, the treatment significantly lowered the number of neutrophils in blood on days 14 and 21 without significant downregulation of neutrophil oxidative burst. Pterostilbene nonsignificantly increased total radical trapping potential in arthritic animals. These results indicate that the promising effects of pterostilbene on reactive oxygen species operate by different mechanisms in vitro and in the animal model of inflammation. In conclusion, the positive effects of pterostilbene in the model of arthritis may be attributed to regulation of neutrophil number. PMID:24195064

  9. Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods.

    PubMed

    Mosca, Tainá; Forte, Wilma C N

    2016-01-01

    Studies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

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

    PubMed

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

    1999-01-01

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

  11. Interleukin-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

    PubMed Central

    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.

    2016-01-01

    A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multi-step process that involves sequential cell-cell interactions of circulating leukocytes with interleukin (IL)-1- or tumor necrosis factor-α (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 pro-inflammatory 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, while 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-seq analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media (CM) from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and also stimulate neutrophil production of pro-inflammatory 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 mitochondria outer membrane permeabilization and caspase 9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by CM 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

  12. 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...

  13. 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...

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

    PubMed Central

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

    2017-01-01

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

  15. Stress-induced adaptation of neutrophilic granulocyte activity in K and R3 carp lines.

    PubMed

    Pijanowski, L; Verburg-van Kemenade, B M L; Irnazarow, I; Chadzinska, M

    2015-12-01

    Both in mammals and fish, stress induces remarkable changes in the immune response. We focused on stress-induced changes in the activity of neutrophilic granulocytes in the R3 and K lines of common carp, which showed differential stress responses. Our study clearly demonstrates that a prolonged restraint stress differentially affects the activity of K and R3 carp neutrophils. In the K line, stress decreased the respiratory burst, while in the R3 line it reduced the release of extracellular DNA. Surprisingly, the stress-induced changes in ROS production and NET formation did not correlate with changes in gene expression of the inflammatory mediators and GR receptors. In neutrophilic granulocytes from K carp, gene expression of the stress-sensitive cortisol GR1 receptor was significantly higher than in neutrophils from R3 fish, which will make these cells more sensitive to high levels of cortisol. Moreover, upon stress, neutrophilic granulocytes of K carp up-regulated gene expression of the anti-inflammatory cytokine IL-10 while this was not observed in neutrophilic granulocytes of R3 carp. Therefore, we can hypothesize that, in contrast to R3 neutrophils, the more cortisol sensitive neutrophils from K carp respond to stress with up-regulation of IL-10 and consequently reduction of ROS production. Most probably the ROS-independent NET formation in K carp is not regulated by this anti-inflammatory cytokine. These data may indicate a predominantly ROS-independent formation of NETs by carp neutrophilic granulocytes. Moreover, they underline the important role of IL-10 in stress-induced immunoregulation.

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

    PubMed

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

    1994-09-15

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

  17. Neutrophils Activate Plasmacytoid Dendritic Cells by Releasing Self-DNA–Peptide Complexes in Systemic Lupus Erythematosus

    PubMed Central

    Lande, Roberto; Ganguly, Dipyaman; Facchinetti, Valeria; Frasca, Loredana; Conrad, Curdin; Gregorio, Josh; Meller, Stephan; Chamilos, Georgios; Sebasigari, Rosalie; Riccieri, Valeria; Bassett, Roland; Amuro, Hideki; Fukuhara, Shirou; Ito, Tomoki; Liu, Yong-Jun; Gilliet, Michel

    2012-01-01

    Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease. PMID:21389263

  18. Nanocrystal quantum dot-conjugated anti-myeloperoxidase antibody as the detector of activated neutrophils.

    PubMed

    Hoshino, Akiyoshi; Nagao, Tomokazu; Nakasuga, Akira; Ishida-Okawara, Akiko; Suzuki, Kazuo; Yasuhara, Masato; Yamamoto, Kenji

    2007-12-01

    Fluorescent nanocrystal quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. Here we show that QDs conjugated with antibody against neutrophil peroxidase, myeloperoxidase (MPO). We designed a novel method to conjugate QDs to antibody without losing any antibody function including their antigen recognizing and Fc-receptor binding activities. When we applied anti-MPO antibody (Ab) with conventional organic probes in the case of immunostaining of living cells, the antibodies lost their fluorescence because of MPO enzymic activity to produce reactive oxygen species. Our QD-conjugated anti-MPO (alpha-MPO-QDs) can detect MPO on the surface of activated neutrophils. In addition, anti-MPO-QDs did not react to the inactivated neutrophils. In conclusion, we demonstrated that antibody visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. Taken together, these techniques have the possibility that QDs can reveal the activation of neutrophils by immunostaining and flow cytometric analysis as a powerful tool for diagnosis of the neutrophil activation in vitro.

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

    PubMed

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

    2001-05-01

    We hypothesized that estrogen administration would attenuate skeletal muscle neutrophil infiltration, indices of muscle membrane disruption, and muscle calpain activity shortly after the termination of exercise. Ovariectomized female rats were implanted with either an estogen pellet (25 mg beta-estradiol) or a placebo pellet. Two weeks postimplant, animals were killed either at rest or 1 h after running exercise (60 min at 21 m x min(-1), 12% grade). The 4 experimental groups (n = 12) used were: unexercised placebo (UP), unexercised estrogen (UE), exercised placebo (EP), and exercised estrogen (EE). Blood samples were analyzed for creatine kinase (CK) activity and estradiol content. Plantaris and gastrocnemius muscles were removed and histochemical determination of neutrophil content or biochemical determination of myeloperoxidase (MPO), glucose-6-phosphate dehydrogenase (G6PD), and calpain-like activity determined. Estrogen supplemented animals had 10-20-fold higher circulating estradiol levels than placebo animals. EP animals had significantly higher (P < 0.05) circulating CK activities than EE or unexercised animals. Muscle neutrophil concentrations were significantly (P < 0.01) elevated in EP and EE groups compared with unexercised controls, with EP muscle neutrophil levels also being over 60% greater (P < 0.05) than in EE animals. EP animals also had higher (P < 0.05) muscle MPO activities than unexercised or EE animals. Muscle G6PD activities were not significantly different between any groups. Muscle caplain-like activities were 80% higher (P < 0.01) in EP animals than EE animals with calpain-like activities in EE animals similar to unexercised groups. These results indicate that estrogen supplementation in ovariectomized rats attenuated 1-h post-exercise serum CK activities, muscle neutrophil infiltration, MPO activities, and calpain-like activities when compared with exercised, unsupplemented animals. This supports the possibility of a relationship between

  20. 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.

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

    PubMed

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

    2008-09-15

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

  2. Activation of Adenosine A2A Receptors Inhibits Neutrophil Transuroepithelial Migration ▿

    PubMed Central

    Säve, Susanne; Mohlin, Camilla; Vumma, Ravi; Persson, Katarina

    2011-01-01

    Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A2A receptors. In this study, we examined the role of adenosine and A2A receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A1, A2A, A2B, and A3 receptors), but A3 receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A2A receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A2A receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A2A receptor activation, possibly through inhibition of NF-κB signaling pathways. PMID:21646447

  3. Shedding of tumor necrosis factor receptors by activated human neutrophils

    PubMed Central

    1990-01-01

    The capacity of human neutrophils (PMN) to bind tumor necrosis factor (TNF) was rapidly lost when the cells were incubated in suspension with agents that can stimulate their migratory and secretory responses. Both physiological (poly)peptides (FMLP, C5a, CSF-GM) and pharmacologic agonists (PMN, calcium ionophore A23187) induced the loss of TNF receptors (TNF-R) from the cell surface. Half-maximal loss in TNF-R ensued after only approximately 2 min with 10(-7) M FMLP at 37 degrees C, and required only 10(-9) M FMLP during a 30-min exposure. However, there were no such changes even with prolonged exposure of PMN to FMLP at 4 degrees or 16 degrees C. Scatchard analysis revealed loss of TNF- binding sites without change in their affinity (Kd approximately 0.4 nM) as measured at incompletely modulating concentrations of FMLP, C5a, PMA, or A23187. The binding of anti-TNF-R mAbs to PMN decreased in parallel, providing independent evidence for the loss of TNF-R from the cell surface. At the same time, soluble TNF-R appeared in the medium of stimulated PMN. This inference was based on the PMN- and FMLP-dependent generation of a nonsedimentable activity that could inhibit the binding of TNF to fresh human PMN or to mouse macrophages, and the ability of mAbs specific for human TNF-R to abolish inhibition by PMN-conditioned medium of binding of TNF to mouse macrophages. Soluble TNF-R activity was associated with a protein of Mr approximately 28,000 by ligand blot analysis of cell-free supernatants of FMLP-treated PMN. Thus, some portion of the FMLP-induced loss of TNF-R from human PMN is due to shedding of TNF-R. Shedding was unaffected by inhibitors of serine and thiol proteases and could not be induced with phosphatidylinositol- specific phospholipase C. Loss of TNF-R from PMN first stimulated by other agents may decrease their responsiveness to TNF. TNF-R shed by PMN may be one source of the TNF-binding proteins found in body fluids, and may blunt the actions of the

  4. Involvement of nitric oxide donor compounds in the bactericidal activity of human neutrophils in vitro.

    PubMed

    Klink, Magdalena; Cedzyński, Maciej; St Swierzko, Anna; Tchórzewski, Henryk; Sulowska, Zofia

    2003-04-01

    The bactericidal activity of human neutrophils against extracellular and facultatively intracellular bacteria was studied in the presence of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), a molsidomine metabolite. SNP and molsidomine are drugs commonly used as nitrovasodilators in coronary heart disease. It is demonstrated here that the NO donor compounds themselves did not affect the viability and survival of the bacterial strains tested. Neither SNP nor SIN-1 had any effect on the process of bacteria ingestion. In contrast, NO donors enhanced the ability of neutrophils to kill Escherichia coli, Proteus vulgaris and Salmonella Anatum. However, strains differed in their susceptibility to SNP- and SIN-1-mediated killing by neutrophils. Removal of the superoxide anion reduced the bactericidal activity of SNP- and SIN-1-treated neutrophils against E. coli and S. Anatum. This suggests that the NO derivatives formed in the reaction of NO generated from donors with the reactive oxygen species released by phagocytosed neutrophils potentiate the bactericidal activity of human neutrophils in vitro. The above original observation discussed here suggests clinical significance for the treatment of patients with nitrovasodilators in the course of coronary heart disease therapy.

  5. 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

  6. 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

    Ebaid, Hossam

    2014-03-04

    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. 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. 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. 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. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2100966986117779.

  7. Double dose plateletpheresis by continuous and intermittent flow devices increases platelet-neutrophil complex formation in healthy donors without noticeable neutrophil activation.

    PubMed

    Bilgin, Aynur Ugur; Karadogan, Ihsan; Yilmaz, Ferahnaz Gencay; Undar, Levent

    2007-02-01

    Several reports have demonstrated that during a single plateletpheresis procedure, platelets may form heterotypic aggregates which may predispose certain donors to thrombotic complications. In this study, changes in the expression of neutrophil adhesion molecules (CD11b/CD18, CD50/54, CD62L) and platelet-neutrophil complex (PNC) formation were investigated by a flow cytometric method in healthy donors following a double dose plateletpheresis (DDP) procedure. Our results show that DDP which are carried out by the Fresenius AS.TEC 204 and Haemonetics MCS+ cause a significant increase in PNC formation in donors. Additionally, the Fresenius AS.TEC 204 device caused a decrease in CD62L expression which is a sign of mild neutrophil activation. Although the clinical significance of these laboratory changes is not clear, the occurrence of neutrophil activation and increased PNC formation might predispose certain donors to thrombotic complications following DDP.

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

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

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

    PubMed Central

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

    1984-01-01

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

  10. Activated Human Valvular Interstitial Cells Sustain Interleukin-17 Production To Recruit Neutrophils in Infective Endocarditis

    PubMed Central

    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

    2015-01-01

    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 the

  11. Guinea Pig Neutrophils Infected with Mycobacterium tuberculosis Produce Cytokines Which Activate Alveolar Macrophages in Noncontact Cultures▿

    PubMed Central

    Sawant, Kirti V.; McMurray, David N.

    2007-01-01

    The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, we investigated the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce interleukin-8 (IL-8; CXCL8) and tumor necrosis factor alpha (TNF-α) and to activate alveolar macrophages. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12, and 24 h. IL-8 protein production in cocultures, as measured by using an enzyme-linked immunosorbent assay, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected cocultures than in uninfected cocultures and in cultures of the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time reverse transcription-PCR, were elevated at 24 h in infected cocultures and infected cells cultured alone. In order to elucidate the contributions of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner by using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper wells, and alveolar macrophages were cultured in the lower wells. The release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to that from unexposed alveolar macrophages. Significant up-regulation of IL-1β and TNF-α mRNA levels in alveolar macrophages was observed at 24 and 30 h, respectively, compared to those in cells not exposed to soluble neutrophil products. Treatment with anti-guinea pig TNF-α polyclonal antibody completely abolished the response of alveolar macrophages to neutrophil products. This finding suggests that TNF-α produced by infected neutrophils may be involved in the activation of

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

    PubMed

    McCarty, M F

    1999-10-01

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

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

    PubMed

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

    1978-10-10

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

  14. Pathogen response-like recruitment and activation of neutrophils by sterile immunogenic dying cells drives neutrophil-mediated residual cell killing.

    PubMed

    Garg, Abhishek D; Vandenberk, Lien; Fang, Shentong; Fasche, Tekele; Van Eygen, Sofie; Maes, Jan; Van Woensel, Matthias; Koks, Carolien; Vanthillo, Niels; Graf, Norbert; de Witte, Peter; Van Gool, Stefaan; Salven, Petri; Agostinis, Patrizia

    2017-05-01

    Innate immune sensing of dying cells is modulated by several signals. Inflammatory chemokines-guided early recruitment, and pathogen-associated molecular patterns-triggered activation, of major anti-pathogenic innate immune cells like neutrophils distinguishes pathogen-infected stressed/dying cells from sterile dying cells. However, whether certain sterile dying cells stimulate innate immunity by partially mimicking pathogen response-like recruitment/activation of neutrophils remains poorly understood. We reveal that sterile immunogenic dying cancer cells trigger (a cell autonomous) pathogen response-like chemokine (PARC) signature, hallmarked by co-release of CXCL1, CCL2 and CXCL10 (similar to cells infected with bacteria or viruses). This PARC signature recruits preferentially neutrophils as first innate immune responders in vivo (in a cross-species, evolutionarily conserved manner; in mice and zebrafish). Furthermore, key danger signals emanating from these dying cells, that is, surface calreticulin, ATP and nucleic acids stimulate phagocytosis, purinergic receptors and toll-like receptors (TLR) i.e. TLR7/8/9-MyD88 signaling on neutrophil level, respectively. Engagement of purinergic receptors and TLR7/8/9-MyD88 signaling evokes neutrophil activation, which culminates into H2O2 and NO-driven respiratory burst-mediated killing of viable residual cancer cells. Thus sterile immunogenic dying cells perform 'altered-self mimicry' in certain contexts to exploit neutrophils for phagocytic targeting of dead/dying cancer cells and cytotoxic targeting of residual cancer cells.

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

    PubMed Central

    Sunil, Vasanthi R; Connor, Agnieszka J; Zhou, Peihong; Gordon, Marion K; Laskin, Jeffrey D; Laskin, Debra L

    2002-01-01

    Background Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. Methods Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. Results Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein. Conclusion These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells. PMID:12204102

  16. TNFα-activated mesenchymal stromal cells promote breast cancer metastasis by recruiting CXCR2(+) neutrophils.

    PubMed

    Yu, P F; Huang, Y; Han, Y Y; Lin, L Y; Sun, W H; Rabson, A B; Wang, Y; Shi, Y F

    2017-01-26

    Mesenchymal stromal cells (MSCs) tend to infiltrate into tumors and form a major component of the tumor microenvironment. Our previous work demonstrated that tumor necrosis factor α (TNFα)-activated MSCs significantly promoted tumor growth. However, the role of TNFα-treated MSCs in tumor metastasis remains elusive. Employing a lung metastasis model of murine breast cancer, we found that TNFα-activated MSCs strikingly enhanced tumor metastasis compared with normal MSCs. We analyzed the chemokine profiles and found that the expression of CCL5, CCR2 and CXCR2 ligands were enhanced in TNFα-activated MSCs. Using genetic or pharmacological strategies to inhibit CCL5 or CCR2, we demonstrated that CCL5 and CCR2 ligands were indispensable in supporting TNFα-activated MSCs to promote tumor metastasis. Analysis of immune cells revealed that CXCR2 ligands (CXCL1, CXCL 2 and CXCL5) expressed by TNFα-activated MSCs efficiently recruited CXCR2(+) neutrophils into tumor. These neutrophils were responsible for the pro-metastatic effect of MSCs since inhibition of this chemotaxis abolished increased neutrophil recruitment and tumor metastasis. The interaction between neutrophils and tumor cells resulted in markedly elevated metastasis-related genes by tumor cells, including CXCR4, CXCR7, MMP12, MMP13, IL-6 and TGFβ. Importantly, in IL8(high) human breast cancer samples, we also observed similar alterations of gene expression. Collectively, our findings demonstrate that TNFα-activated MSCs promote tumor metastasis via CXCR2(+) neutrophil recruitment.

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

    PubMed

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

    2005-03-15

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

  18. 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

  19. Intravascular clearance of disseminating Cryptococcus neoformans in the brain can be improved by enhancing neutrophil recruitment in mice.

    PubMed

    Sun, Donglei; Zhang, Mingshun; Liu, Gongguan; Wu, Hui; Li, Chang; Zhou, Hong; Zhang, Xiquan; Shi, Meiqing

    2016-07-01

    Extrapulmonary dissemination of Cryptococcus neoformans (C. neoformans) is one of the most critical steps in the development of meningoencephalitis. Here, we report that clearance of the disseminating C. neoformans occurs within the brain microvasculature. Interestingly, the efficiency of the intravascular clearance in the brain is reduced compared to that in the lung. Intravascular clearance is mainly mediated by neutrophils, and complement C5a receptor signaling is crucial for mediating neutrophil recruitment in the vasculature. C. neoformans stimulated actin polymerization of neutrophils is critically involved in their recruitment to the lung, which is associated with the unique vascular structure detected in the lung. The relatively lower efficiency of fungal clearance in the brain vasculature correlates with less efficient recruitment of neutrophils. Accordingly, intravascular clearance of C. neoformans in the brain could be remarkably improved by increasing the recruitment of neutrophils. We conclude that neutrophils have the ability to eliminate C. neoformans arrested in the vasculature. However, insufficient recruitment of neutrophils limited the optimal clearance of this microorganism in the brain. These results imply that a therapeutic strategy aimed at enhancing the accumulation of neutrophils could help prevent cryptococcal meningoencephalitis.

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

    PubMed

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

    2008-09-01

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

  1. Inhibition of Neutrophil Adhesion and Antimicrobial Activity by Diluted Hydrosol Prepared from Rosa damascena.

    PubMed

    Maruyama, Naho; Tansho-Nagakawa, Shigeru; Miyazaki, Chizuru; Shimomura, Kazuyuki; Ono, Yasuo; Abe, Shigeru

    2017-01-01

    Hydrosol prepared from the flowers of Rosa damascena (rose water) has been traditionally used for various health-related issues, including skin troubles such as erythema, itchiness, swelling. For the care of these skin troubles caused by microbial infection, both antimicrobial and antiinflammatory effects are required. Here, we investigated the effects of rose water on the growth of Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), which cause skin infections, and on the function of neutrophils, which play a major role in the regulation of inflammatory reactions. To assess its modulatory effects on neutrophils, the effects of rose water against neutrophil adhesion response were evaluated. Rose water inhibited mycelial growth of C. albicans at a concentration of ca. 2.2%, and reduced viability of MRSA within 1 h. Rose water suppressed neutrophil activation induced by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and N-formyl-Met-Leu-Phe (fMLP) at 5-15%. It also reduced the LPS- and TNF-α-induced cell surface expression of the adhesion-related molecule, cluster of differentiation (CD) 11b, but did not affect the migratory capacity of neutrophils with or without chemoattractant. These results suggest that rose water may reduce the pathogenicity of microbes, and attenuate neutrophil stimulation, which is involved in inflammatory responses. These findings suggest that rose water has a potential effect to inhibit skin inflammation caused by microbes.

  2. 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. © The Author(s) 2015.

  3. Participation of the urokinase-type plasminogen activator receptor (uPAR) in neutrophil transendothelial migration.

    PubMed

    Pliyev, Boris K; Antonova, Olga A; Menshikov, Mikhail

    2011-05-01

    The mechanisms underlying migration of neutrophils across endothelium are not completely understood. The urokinase-type plasminogen activator receptor (uPAR) plays a key role in neutrophil adhesion and migration. In the present study, we addressed whether uPAR regulates neutrophil transendothelial migration. We first showed that siRNA-mediated knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) did not affect neutrophil migration across HUVEC monolayers indicating that endothelial uPAR does not regulate neutrophil transmigration. In contrast, the transmigration was significantly inhibited by Fab' fragment of anti-uPAR monoclonal antibody and proteolytically inactive urokinase (uPA), whereas inhibition of proteolytical activity of endogenous uPA (with amiloride or plasminogen activator inhibitor-1) did not affect the transmigration. Both the anti-uPAR Fab' fragment and proteolytically inactive uPA did not exert significant effects upon the transmigration conducted in the presence of F(ab')(2) fragment of blocking antibody to integrin Mac-1 indicating that uPAR regulates Mac-1-dependent transmigration. Mac-1-dependent, but not Mac-1-independent, transmigration was significantly reduced in the presence of N-acetyl-d-glucosamine and d-mannose, the saccharides that disrupt uPAR/Mac-1 association, but was unaffected in the presence of control saccharides (d-sorbitol and sucrose). We conclude that physical association of uPAR with Mac-1 mediates the regulatory effect of uPAR over the transmigration. Finally, we provide evidence that the functional cooperation between uPAR and Mac-1 is essential at both adhesion and diapedesis steps of neutrophil migration across endothelium. Thus, uPAR expressed on neutrophil plasma membrane regulates transendothelial migration independently of uPA proteolytical activity and acting as a cofactor for integrin Mac-1.

  4. Increased expression of the C3b receptor by neutrophils and complement activation during haemodialysis.

    PubMed Central

    Lee, J; Hakim, R M; Fearon, D T

    1984-01-01

    Activation of complement and the relative number of C3b receptors expressed by neutrophils was assessed in patients undergoing haemodialysis with new and reused cellulosic membranes, and with polymethylmethacrylate (PMMA) membranes. Activation of complement was assessed by radioimmunoassay of plasma C3adesArg, and neutrophil C3b receptors were measured by fluorescent flow cytometry of cells indirectly stained with F(ab')2 anti-C3b receptor. During first use of cellulosic dialysis membranes by four patients, the mean expression of C3b receptors by neutrophils in blood taken from the afferent line of the extra-corporeal system after 10, 20, 60 and 120 min of dialysis increased to 127, 189, 255 and 296%, respectively. The mean plasma C3adesArg concentrations in the corresponding samples of blood were 225, 320, 236 and 160% of the pre-dialysis levels. During third and fifth use of the same membranes by these patients, the mean C3b receptor expression by neutrophils did not exceed 150% of the predialysis determination, and correspondingly minimal increases in plasma C3adesArg were observed. Analysis of blood taken simultaneously from the afferent and efferent lines of the first use cellulosic dialysis system indicated that the increase in C3b receptor expression by neutrophils and generation of C3adesArg occurred when blood came in contact with the dialysis membrane. Haemodialysis of four additional patients with the non-complement activating PMMA membrane caused only modest or no increases in neutrophil C3b receptors. Thus, complement activation in vivo is associated with up-regulation of neutrophilic C3b receptors, indicating that this cellular response previously described only in model, in vitro systems, is a physiological mechanism by which this cell can augment its capacity for responding to C3b opsonized material. PMID:6232024

  5. Penetrating thorax injury leads to mild systemic activation of neutrophils without inflammatory complications.

    PubMed

    Groeneveld, Kathelijne M; Hietbrink, Falco; Hardcastle, Timothy C; Warren, Brian L; Koenderman, Leo; Leenen, Luke P H

    2014-03-01

    Trauma is one of the major causes of morbidity and mortality. Thoracic injuries are associated with inflammatory complications such as ARDS. The pathogenesis of this complication after pulmonary injury is incompletely understood, but neutrophils are thought to play a pivotal role. The aim of this project was to gain more insight in the role of thoracic injuries in the pathophysiological processes that link systemic neutrophil activation with inflammatory complications after trauma. In this prospective cohort study fifty-five patients with isolated penetrating thoracic injury were included at a level one Trauma Unit. Blood samples were analysed for neutrophil phenotype with the use of flowcytometry within 3 h of trauma and repeated six and 24 h after injury. The presence of inflammatory complications (e.g. ARDS or sepsis/septic shock) was assessed during admission, and this was related to the neutrophil phenotpe. The clinical follow-up of fifty-three patients was uneventful. Only two patients developed an inflammatory complication. Within 3 h after trauma, neutrophils showed a decreased expression of FcγRII (p=0.007) and FcγRIII (p=0.001) compared to healthy individuals. After 6 h, expression of active FcγRII (p=0.017), C5aR (p=0.004) and CAECAM8 (p=0.043) increased, whereas L-selectin (p=0.002) decreased. After 24 h also CXCR-2 (CD182) expression increased compared to healthy individuals (p=0.001). Penetrating thoracic trauma leads to a distinct primed activation status of circulating neutrophils within hours. In addition to activation of cells, both young and reverse migrated neutrophils are released into the circulation. This degree of systemic inflammation does not exceed a threshold of inflammation that is needed for the development of inflammatory complications like ARDS. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. 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.

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

    PubMed

    Antoine, Francis; Girard, Denis

    2015-01-01

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

  8. The effect of the bacterial product, succinic acid, on neutrophil bactericidal activity.

    PubMed

    Abdul-Majid, K B; Kenny, P A; Finlay-Jones, J J

    1997-02-01

    We investigated the effect of succinic acid on neutrophil bactericidal activity in a model of intra-abdominal abscess induced in mice by the peritoneal inoculation of 5 x 10(6) cfu ml-1 E. coli and 5 x 10(8) cfu ml-1 B. fragilis plus 1 mg of bran as faecal fibre analogue. The mean pH of the induced abscesses at week 1 was 6.7, higher than the pH associated with succinic acid inhibitory activity. We therefore determined the effect of succinic acid (0-100 mM) at pH 6.7 on the bactericidal activity of mouse bone marrow-derived neutrophils. Phagocytic killing of Proteus mirabilis by neutrophils was significantly inhibited by 30-100 mM succinic acid at pH 6.7 but there was no significant effect of succinic acid on engulfment of bacteria at this pH. However, significant inhibition of intracellular killing (assayed by adding succinic acid to suspensions of neutrophils which had engulfed bacteria in low serum concentrations but in the absence of succinic acid) was noted at 70 and 100 mM. These results indicate that succinic acid inhibits neutrophil bactericidal activity at a physiological pH, principally through inhibition of intracellular killing mechanisms and therefore contributing to bacterial persistence in this model of abscess formation.

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

    PubMed

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

    1994-02-01

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

  10. 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

  11. RNA-Seq Reveals Activation of Both Common and Cytokine-Specific Pathways following Neutrophil Priming

    PubMed Central

    Moots, Robert J.; Edwards, Steven W.

    2013-01-01

    Neutrophils are central to the pathology of inflammatory diseases, where they can damage host tissue through release of reactive oxygen metabolites and proteases, and drive inflammation via secretion of cytokines and chemokines. Many cytokines, such as those generated during inflammation, can induce a similar “primed” phenotype in neutrophils, but it is unknown if different cytokines utilise common or cytokine-specific pathways to induce these functional changes. Here, we describe the transcriptomic changes induced in control human neutrophils during priming in vitro with pro-inflammatory cytokines (TNF-α and GM-CSF) using RNA-seq. Priming led to the rapid expression of a common set of transcripts for cytokines, chemokines and cell surface receptors (CXCL1, CXCL2, IL1A, IL1B, IL1RA, ICAM1). However, 580 genes were differentially regulated by TNF-α and GM-CSF treatment, and of these 58 were directly implicated in the control of apoptosis. While these two cytokines both delayed apoptosis, they induced changes in expression of different pro- and anti-apoptotic genes. Bioinformatics analysis predicted that these genes were regulated via differential activation of transcription factors by TNF-α and GM-CSF and these predictions were confirmed using functional assays: inhibition of NF-κB signalling abrogated the protective effect of TNF-α (but not that of GM-CSF) on neutrophil apoptosis, whereas inhibition of JAK/STAT signalling abrogated the anti-apoptotic effect of GM-CSF, but not that of TNF-α (p<0.05). These data provide the first characterisation of the human neutrophil transcriptome following GM-CSF and TNF-α priming, and demonstrate the utility of this approach to define functional changes in neutrophils following cytokine exposure. This may provide an important, new approach to define the molecular properties of neutrophils after in vivo activation during inflammation. PMID:23554905

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Automated quantitation of circulating neutrophil and eosinophil activation in asthmatic patients

    PubMed Central

    Leckie, M.; Bryan, S.; Khan, J.; Dewar, A.; Aikman, S.; McGrath, J.; Okrongly, D.; Burman, J.; Barnes, P.; Hansel, T.

    2000-01-01

    BACKGROUND—Asthma has been associated with eosinophil activation, measured in serum, sputum, bronchoalveolar lavage (BAL) fluid, and urine. A whole blood automated method was developed to assess eosinophil and neutrophil activity in terms of peroxidase content and cell morphology using the Bayer haematology analyser. The method was applied to an in vitro stimulation model when fMLP was added to whole blood and the samples were then analysed for changes in granularity and shape. In addition, cells stimulated with interleukin (IL)-8 were examined by electron microscopy.
METHODS—A cross sectional analysis was performed on venous blood from non-atopic, non-asthmatic normal subjects (n = 37), mild (n= 46) and symptomatic (n = 22) asthmatic patients on inhaled β2 agonist only, and more severe asthmatic patients (n = 17) on inhaled and oral corticosteroid therapy. Samples were analysed by the haematology analyser and peroxidase leucograms gated using the WinMDI software program.
RESULTS—There were significant differences in the amount of light scatter by the neutrophil populations in the symptomatic (p = 0.007) and severe asthmatic (p = 0.0001) groups compared with the control group. However, abnormalities in eosinophil populations were not observed. In vitro activation of whole blood with fMLP caused similar changes in neutrophil light scatter, suggesting that neutrophil activation is present in peripheral blood of symptomatic asthmatic patients. IL-8 caused a change in shape of the neutrophils seen using transmission electron microscopy.
CONCLUSIONS—Evidence of neutrophil activation can be seen in whole blood from patients with asthma using a novel automated method. This may potentially be applied to other inflammatory diseases.

 PMID:10817795

  14. The role of activated neutrophils in the early stage of equine laminitis.

    PubMed

    de la Rebière de Pouyade, Geoffroy; Serteyn, Didier

    2011-07-01

    Despite ongoing research and a widening range of treatment options, laminitis remains a severely damaging condition with poorly understood pathophysiology. Results obtained from cytokine regulation studies during the last decade have highlighted the inflammatory nature of laminitis. This review will describe the role of systemic activation and local infiltration of neutrophils in laminar tissues in the induction of laminitis. Particular emphasis is placed on the role of neutrophil activation in subsequent vascular dysfunction and oxidative and proteolysis imbalances that are pathways previously implicated in laminitis. Neutrophils, by the way of their interdependent relationship with endothelial cells and keratinocytes, dramatically increase the inflammatory response culminating in the failure of the laminar dermal-epidermal interface.

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

    PubMed

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

    2010-01-15

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

  16. 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

  17. 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

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

    SciTech Connect

    Tao, W.

    1988-01-01

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

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

    PubMed

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

    2013-05-15

    The study goal was to evaluate responses in humans following decompression from open-water SCUBA diving with the hypothesis that exertion underwater and use of a breathing mixture containing more oxygen and less nitrogen (enriched air nitrox) would alter annexin V-positive microparticle (MP) production and size changes and neutrophil activation, as well as their relationships to intravascular bubble formation. Twenty-four divers followed a uniform dive profile to 18 m of sea water breathing air or 22.5 m breathing 32% oxygen/68% nitrogen for 47 min, either swimming with moderately heavy exertion underwater or remaining stationary at depth. Blood was obtained pre- and at 15 and 120 min postdive. Intravascular bubbles were quantified by transthoracic echocardiography postdive at 20-min intervals for 2 h. There were no significant differences in maximum bubble scores among the dives. MP number increased 2.7-fold, on average, within 15 min after each dive; only the air-exertion dive resulted in a significant further increase to 5-fold over baseline at 2 h postdive. Neutrophil activation occurred after all dives. For the enriched air nitrox stationary at depth dive, but not for other conditions, the numbers of postdive annexin V-positive particles above 1 μm in diameter were correlated with intravascular bubble scores (correlation coefficients ∼0.9, P < 0.05). We conclude that postdecompression relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation appear to exist, but more study is required to improve confidence in the associations.

  20. Invariant NKT cells modulate the suppressive activity of Serum Amyloid A-differentiated IL-10-secreting neutrophils

    PubMed Central

    De Santo, Carmela; Arscott, Ramon; Booth, Sarah; Karydis, Ioannis; Jones, Margaret; Asher, Ruth; Salio, Mariolina; Middleton, Mark; Cerundolo, Vincenzo

    2010-01-01

    Neutrophils are the primary effector cells during inflammation, but can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms modulating their plasticity remain unclear. We now show that systemic serum amyloid A-1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory IL-10-secreting neutrophils but also promoted invariant NKT (iNKT) cell interaction with these neutrophils, a process that limits their suppressive activity by reducing IL-10 and enhancing IL-12 production. Because SAA-1-producing melanomas promote differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by reducing the frequency of immunosuppressive neutrophils and restoring tumor specific immune responses. PMID:20890286

  1. Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils.

    PubMed

    Dent, Gordon; Loweth, Sam C; Hasan, Anwar Matar; Leslie, Fiona M

    2014-10-01

    The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines - CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) - were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P<0.005) and CXCL5 levels (P<0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P<0.05) and CXCL8 (P<0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel.

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

    PubMed

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

    2015-11-01

    Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13, <3%: n = 8). Eosinophils and neutrophils from blood and sputum were analysed for expression of activation and degranulation markers by flow cytometry. Data were analysed by both classical, non-parametric statistics and a multi-dimensional approach, using principal component analysis (PCA). Patients with sputum eosinophilia were characterized by increased asthma control questionnaire (ACQ) scores and blood eosinophil counts. Both sputum neutrophils and eosinophils displayed an activated and degranulated phenotype compared to cells obtained from blood. Specifically, degranulation of all granule types was detected in sputum cells, combined with an increased expression of the activation markers (activated) Mac-1 (CD11b), programmed death ligand 1 (PD-L1) (CD274) and a decreased expression of CD62L. CD69 expression was only increased on sputum eosinophils. Surface marker expression of neutrophils was similar in the presence or absence of eosinophilia, either by single or multi-dimensional analysis. Sputum neutrophils were highly activated and degranulated irrespective of sputum eosinophilia. Therefore, we conclude that differences in granulocyte activation in sputum and/or blood are not associated with clinical differences in the two groups of asthma patients. The finding of PD-L1 expression on sputum granulocytes suggests an immunomodulatory role of these cells in the tissue. © 2015 British Society for Immunology.

  3. Neutrophil activity in chronic venous leg ulcers—A target for therapy?

    PubMed Central

    McDaniel, Jodi C.; Roy, Sashwati; Wilgus, Traci A.

    2013-01-01

    Chronic venous leg ulcers (CVLUs) affect approximately 600,000 people annually in the United States and accrue yearly treatment costs of US$2.5–5 billion. As the population ages, demands on health care resources for CVLU treatments are predicted to drastically increase because the incidence of CVLUs is highest in those ≥65 years of age. Furthermore, regardless of current standards of care, healing complications and high recurrence rates prevail. Thus, it is critical that factors leading to or exacerbating CVLUs be discerned and more effective, adjuvant, evidence-based treatment strategies be utilized. Previous studies have suggested that CVLUs’ pathogenesis is related to the prolonged presence of high numbers of activated neutrophils secreting proteases in the wound bed that destroy growth factors, receptors, and the extracellular matrix that are essential for healing. These events are believed to contribute to a chronically inflamed wound that fails to heal. Therefore, the purpose of this project was to review studies from the past 15 years (1996–2011) that characterized neutrophil activity in the microenvironment of human CVLUs for new evidence that could explicate the proposed relationship between excessive, sustained neutrophil activity and CVLUs. We also appraised the strength of evidence for current and potential therapeutics that target excessive neutrophil activity. PMID:23551462

  4. Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils.

    PubMed Central

    Dekker, L V; Leitges, M; Altschuler, G; Mistry, N; McDermott, A; Roes, J; Segal, A W

    2000-01-01

    We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils. PMID:10727429

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

    PubMed

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

    2017-06-21

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

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

    PubMed Central

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

    2011-01-01

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

  7. Enterococcus faecalis Bearing Aggregation Substance Is Resistant to Killing by Human Neutrophils despite Phagocytosis and Neutrophil Activation

    PubMed Central

    Rakita, Robert M.; Vanek, Natalie N.; Jacques-Palaz, Karen; Mee, Mee; Mariscalco, M. Michele; Dunny, Gary M.; Snuggs, Mark; Van Winkle, W. Barry; Simon, Scott I.

    1999-01-01

    Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of l-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis. PMID:10531268

  8. HS1 deficiency impairs neutrophil recruitment in vivo and activation of the small GTPases Rac1 and Rap1.

    PubMed

    Latasiewicz, Joanna; Artz, Annette; Jing, Ding; Blanco, Mariana Pacheco; Currie, Silke M; Avila, Martha Velázquez; Schnoor, Michael; Vestweber, Dietmar

    2017-01-25

    Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the β2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.

  9. 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. © 2015 by The American Society of Hematology.

  10. Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages.

    PubMed

    Soehnlein, O; Kenne, E; Rotzius, P; Eriksson, E E; Lindbom, L

    2008-01-01

    Macrophages represent a multi-functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil-derived granule proteins in the anti-microbial activity of macrophages. Neutrophil secretion obtained by antibody cross-linking of the integrin subunit CD18 (X-link secretion) or by treatment with N-Formyl-Met-Leu-Phe (fMLP secretion) induced a several-fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca(2+). Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X-link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti-microbial therapy.

  11. Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

    PubMed Central

    Secundino, Ismael; Lizcano, Anel; Roupé, K. Markus; Wang, Xiaoxia; Cole, Jason N.; Olson, Joshua; Ali, S. Raza; Dahesh, Samira; Amayreh, Lenah K.; Henningham, Anna; Varki, Ajit; Nizet, Victor

    2015-01-01

    Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan “self-associated molecular patterns” to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism. PMID:26411873

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

    PubMed

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

    2010-10-15

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

  13. 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.

  14. Inhibition of kinases impairs neutrophil activation and killing of Staphylococcus aureus.

    PubMed Central

    Schnyder, B; Meunier, P C; Car, B D

    1998-01-01

    Intracellular phosphorylations polymorphonuclear neutrophils are mediated by kinases, including mitogen activated-protein (MAP) kinases and phosphatidylinositol 3-kinase. In the present study we demonstrate their effector functions upon both ligation of cell-surface seven-transmembrane-spanning receptors by bacterial peptide formylmethionyl-leucylphenylalanine as well as in the process of destruction of Staphylococcus aureus. To regulate neutrophil MAP kinases p38 and p44/42, specifically, we made use of their specific inhibitors 10 microM SK&F 86002 (for p38) and PD 098059 (for activating kinase of p44/42). SK&F 86002 was a potent inhibitor (by 70%) of induced antimicrobial oxygen-radical generation compared with PD 098059 (by 20%). SK&F 86002 and PD 098059 inhibited mobilization of a dominant neutrophil adhesion molecule, beta2 integrin, from cytoplasmic granules to the plasma membrane by 40 and 10% respectively, and the combination of the two drugs resulted in a 90% effect. The combined effect of both drugs was moderate inhibition of bacterial destruction, despite the fact that neither compound had detectable effect on bactericidal activity if applied individually. Bacterial destruction was also inhibited by wortmannin (0.1 microM), the specific inhibitor of phosphatidylinositol 3-kinase, which had previously been described to target various other activations of the neutrophil, including oxygen-radical generation. Although the relative contribution of p38 and p44/42 MAP kinases varied, the marked effects of the combined inhibition of the kinases revealed their concerted actions to be critical for normal neutrophil function. PMID:9531489

  15. Platelet, monocyte and neutrophil activation and glucose tolerance in South African Mixed Ancestry individuals

    PubMed Central

    Davison, Glenda M.; Nkambule, Bongani B.; Mkandla, Zibusiso; Hon, Gloudina M.; Kengne, Andre P.; Erasmus, Rajiv T.; Matsha, Tandi E.

    2017-01-01

    Platelet activation has been described in patients with chronic inflammation, however in type 2 diabetes mellitus it remains controversial. We compared levels of platelet leucocyte aggregates, monocyte and granulocyte activation across glucose tolerance statuses in mixed ancestry South Africans. Individuals (206) were recruited from Bellville-South, Cape Town, and included 66% with normal glucose tolerance, 18.7% pre-diabetes, 8.7% screen-detected diabetes and 6.3% known diabetes. Monocyte and neutrophil activation were measured by calculating the percentage of cells expressing CD142 and CD69 while platelet monocyte aggregates were defined as CD14++ CD42b+ events and platelet neutrophil aggregates as CD16++ CD42b+ events. The percentage of monocytes and neutrophils expressing CD69 and CD142 was significantly higher in known diabetes and prediabetes, but, lowest in screen-detected diabetes (both p ≤ 0.016). The pattern was similar for platelet monocyte and neutrophil aggregates (both p ≤ 0.003). In robust linear regressions adjusted for age and gender, known diabetes was significantly and positively associated with the percentage of monocytes expressing CD69 [beta 11.06 (p = 0.016)] and CD42b (PMAs) [19.51 (0.003)] as well as the percentage of neutrophils expressing CD69 [14.19 (<0.0001)] and CD42b [17.7 (0.001)]. We conclude that monitoring platelet activation in diagnosed diabetic patients may have a role in the management and risk stratification. PMID:28091589

  16. Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton

    PubMed Central

    He, Yuan; Li, Dong; Cook, Sara L.; Yoon, Mee-Sup; Kapoor, Ashish; Rao, Christopher V.; Kenis, Paul J. A.; Chen, Jie; Wang, Fei

    2013-01-01

    Chemotaxis allows neutrophils to seek out sites of infection and inflammation. The asymmetric accumulation of filamentous actin (F-actin) at the leading edge provides the driving force for protrusion and is essential for the development and maintenance of neutrophil polarity. The mechanism that governs actin cytoskeleton dynamics and assembly in neutrophils has been extensively explored and is still not fully understood. By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis. Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading-edge establishment, and directional migration in neutrophils stimulated with chemoattractants. Of interest, depletion of mSin1, an integral component of mTORC2, causes no detectable defects in neutrophil polarity and chemotaxis. In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis. Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils. Together our findings reveal an mTORC2- and mTOR kinase–independent function and mechanism of Rictor in the regulation of neutrophil chemotaxis. PMID:24006489

  17. Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8

    PubMed Central

    1992-01-01

    In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine- prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC. PMID:1315317

  18. Alkalinity of Neutrophil Phagocytic Vacuoles Is Modulated by HVCN1 and Has Consequences for Myeloperoxidase Activity

    PubMed Central

    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

  19. The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasm autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation

    PubMed Central

    Radford, D J; Lord, J M; Savage, C O S

    1999-01-01

    The ability of antineutrophil cytoplasm autoantibodies (ANCA) from patients with systemic vasculitis to stimulate protein kinase C (PKC) and tyrosine kinases was examined in human neutrophils. Using the superoxide dismutase-inhibitable reduction of ferricytochrome C, the kinetics of ANCA-induced superoxide (O2−) production were characterized and subsequently manipulated by specific inhibitors of PKC and tyrosine kinases. With this approach, ANCA IgG, but not normal IgG or ANCA F(ab′)2 fragments caused a time and dose dependent release of O2− from TNF-α primed neutrophils. The kinetics of ANCA-induced O2− production showed an initial 10–15 min lag phase compared to the N-formyl-l-methionyl-l-leucyl-l-phenylalanine response, suggesting differences in the signalling pathways recruited by these two stimuli. Inhibitor studies revealed that ANCA-activation involved members of both the Ca2+-dependent and -independent PKC isoforms and also tyrosine kinases. ANCA IgG resulted in the translocation of the βII isoform of PKC at a time corresponding to the end of the lag phase of O2− production, suggesting that PKC activity may be instrumental in processes regulating the activity of the NADPH oxidase in response to ANCA. Tyrosine phosphorylation of numerous proteins also peaked 10–15 min after stimulation with ANCA but not normal IgG. These data suggest that PKC and tyrosine kinases regulate O2− production from neutrophils stimulated with autoantibodies from patients with systemic vasculitis. PMID:10540175

  20. Do native and polymeric alpha1-antitrypsin activate human neutrophils in vitro?

    PubMed

    Persson, Caroline; Subramaniyam, Devipriya; Stevens, Tim; Janciauskiene, Sabina

    2006-06-01

    alpha(1)-Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT. The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro, were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (neutrophil chemotaxis, whereas N-formyl methionyl leucyl phenylalanine (fMLP), a positive control, increased chemotaxis fourfold. A small but nonsignificant increase in neutrophil chemotaxis, however, was observed with AAT preparations containing higher levels of endotoxin (>/= 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release. The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.

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

    PubMed

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

    2005-01-01

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

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

    PubMed

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

    2016-09-01

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

  3. Purification, primary structure, and antimicrobial activities of a guinea pig neutrophil defensin.

    PubMed Central

    Selsted, M E; Harwig, S S

    1987-01-01

    A broad-spectrum antimicrobial peptide present in guinea pig neutrophils was isolated, characterized biochemically, and assessed for microbicidal range and potency in vitro. The guinea pig neutrophil peptide (GPNP) was purified to homogeneity from a granule-rich subcellular fraction of peritoneal exudate neutrophils by gel filtration and reversed-phase high-performance liquid chromatography. GPNP was microbicidal for selected bacterial, fungal, and viral test organisms at concentrations in the microgram per milliliter range. Composition and primary structure analyses revealed that GPNP was homologous to a recently characterized family of antimicrobial peptides, termed defensins, isolated from rabbit and human neutrophils. The entire amino acid sequence of GPNP was determined, revealing that 8 of 31 residues were among those invariant in six rabbit and three human defensin peptides. The conserved sequence included six disulfide-linked cysteine residues, a common structural feature of defensins. The sequence of GPNP also included three nonconservative substitutions in positions otherwise invariant in the human and rabbit peptides. Characterization of GPNP provides new insight into structural features which may be essential for the broad-spectrum antimicrobial activities of defensins. Images PMID:3623703

  4. 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

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

    PubMed Central

    Nosal, Radomir; Svitekova, Klara; Drabikova, Katarina

    2013-01-01

    Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role) were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular) membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC)—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues. PMID:24288583

  6. 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

  7. CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells

    PubMed Central

    1992-01-01

    Neutrophil adherence to cytokine-activated endothelial cell (EC) monolayers depends on the expression of the endothelial leukocyte adhesion molecule-1 (ELAM-1). The ligand for ELAM-1 is the sialylated Lewis-x antigen (SLe(x)) structure. The selectin LAM-1 (or LECAM-1) has been described as one of the SLe(x)-presenting glycoproteins involved in neutrophil binding to ELAM-1. Other presenter molecules have not yet been described. Our data demonstrate that the carcinoembryonic antigen (CEA)-like surface molecules on neutrophils--known as the nonspecific cross-reacting antigens (NCAs)--are involved in neutrophil adherence to monolayers of IL-1-beta-activated EC. The NCAs are recognized by CD66 (NCA-160 and NCA-90) and CD67 (NCA-95). Because NCA-95 and NCA-90 have previously been found to be phosphatidylinositol (PI)-linked, paroxysmal nocturnal hemoglobinuria (PNH) neutrophils (which lack PI- linked surface proteins) were tested as well. PNH neutrophils showed a diminished binding to activated EC. CD66 (on PNH cells still recognizing the transmembrane NCA-160 form) still inhibited the adherence of PNH cells to IL-1-beta-activated EC, but to a limited extent. Soluble CEA(-related) antigens inhibited normal neutrophil adherence as well, whereas neutrophil transmigration was unaffected. Sialidase-treatment as well as CD66 preclearing abolished the inhibitory capacity of the CEA(-related) antigens. The binding of soluble CEA antigens to IL-1-beta-pretreated EC was blocked by anti- ELAM-1. These soluble antigens, as well as the neutrophil NCA-160 and NCA-90, both recognized by CD66 antibodies, presented the SLe(x) determinant. Together, these findings indicate that the CD66 antigens (i.e., NCA-160/NCA-90) function as presenter molecules of the SLe(x) oligosaccharide structures on neutrophils that bind to ELAM-1 on EC. PMID:1378450

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

    PubMed

    Rossi, Alessandra; Lord, Janet M

    2013-12-01

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

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

    PubMed Central

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

    2015-01-01

    Abstract Background and objective Respiratory viral infections are a major cause of asthma exacerbations. Neutrophils accumulate in the airways and the mechanisms that link neutrophilic inflammation, viral infections and exacerbations are unclear. This study aims to investigate anti‐viral responses in neutrophils from patients with and without asthma and to investigate if neutrophils can be directly activated by respiratory viruses. Methods Neutrophils from peripheral blood from asthmatic and non‐asthmatic individuals were isolated and stimulated with lipopolysaccharide (LPS) (1 μg/mL), f‐met‐leu‐phe (fMLP) (100 nM), imiquimod (3 μg/mL), R848 (1.5 μg/mL), poly I:C (10 μg/mL), RV16 (multiplicity of infection (MOI)1), respiratory syncytial virus (RSV) (MOI1) or influenza virus (MOI1). Cell‐free supernatants were collected after 1 h of neutrophil elastase (NE) and matrix metalloproteinase (MMP)‐9 release, or after 24 h for CXCL8 release. Results LPS, fMLP, imiquimod and R848 stimulated the release of CXCL8, NE and MMP‐9 whereas poly I:C selectively induced CXCL8 release only. R848‐induced CXCL8 release was enhanced in neutrophils from asthmatics compared with non‐asthmatic cells (P < 0.01). RSV triggered the release of CXCL8 and NE from neutrophils, whereas RV16 or influenza had no effect. Conclusion Neutrophils release CXCL8, NE and MMP‐9 in response to viral surrogates with R848‐induced CXCL8 release being specifically enhanced in asthmatic neutrophils. Toll‐like receptor (TLR7/8) dysregulation may play a role in neutrophilic inflammation in viral‐induced exacerbations. PMID:26477783

  10. 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.

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

    PubMed

    Binet, François; 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 eIF2alpha 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.

  12. NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL-1β secretion but not in inflammasome activation.

    PubMed

    Gabelloni, María Laura; Sabbione, Florencia; Jancic, Carolina; Fuxman Bass, Juan; Keitelman, Irene; Iula, Leonardo; Oleastro, Matías; Geffner, Jorge R; Trevani, Analía S

    2013-12-01

    Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role.

  13. D-lactic acid interferes with the effects of platelet activating factor on bovine neutrophils.

    PubMed

    Alarcón, P; Conejeros, I; Carretta, M D; Concha, C; Jara, E; Tadich, N; Hidalgo, M A; Burgos, R A

    2011-11-15

    D-lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of D-lactic acid and measured intracellular pH using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that D-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). D-lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease L-selectin expression, and induces late apoptosis. We concluded that D-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM D-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. An Intense and Short-Lasting Burst of Neutrophil Activation Differentiates Early Acute Myocardial Infarction from Systemic Inflammatory Syndromes

    PubMed Central

    Maugeri, Norma; Rovere-Querini, Patrizia; Evangelista, Virgilio; Godino, Cosmo; Demetrio, Monica; Baldini, Mattia; Figini, Filippo; Coppi, Giovanni; Slavich, Massimo; Camera, Marina; Bartorelli, Antonio; Marenzi, Giancarlo; Campana, Lara; Baldissera, Elena; Sabbadini, Maria Grazia; Cianflone, Domenico; Tremoli, Elena; D’Angelo, Armando; Manfredi, Angelo A.; Maseri, Attilio

    2012-01-01

    Background Neutrophils are involved in thrombus formation. We investigated whether specific features of neutrophil activation characterize patients with acute coronary syndromes (ACS) compared to stable angina and to systemic inflammatory diseases. Methods and Findings The myeloperoxidase (MPO) content of circulating neutrophils was determined by flow cytometry in 330 subjects: 69 consecutive patients with acute coronary syndromes (ACS), 69 with chronic stable angina (CSA), 50 with inflammation due to either non-infectious (acute bone fracture), infectious (sepsis) or autoimmune diseases (small and large vessel systemic vasculitis, rheumatoid arthritis). Four patients have also been studied before and after sterile acute injury of the myocardium (septal alcoholization). One hundred thirty-eight healthy donors were studied in parallel. Neutrophils with normal MPO content were 96% in controls, >92% in patients undergoing septal alcoholization, 91% in CSA patients, but only 35 and 30% in unstable angina and AMI (STEMI and NSTEMI) patients, compared to 80%, 75% and 2% of patients with giant cell arteritis, acute bone fracture and severe sepsis. In addition, in 32/33 STEMI and 9/21 NSTEMI patients respectively, 20% and 12% of neutrophils had complete MPO depletion during the first 4 hours after the onset of symptoms, a feature not observed in any other group of patients. MPO depletion was associated with platelet activation, indicated by P-selectin expression, activation and transactivation of leukocyte β2-integrins and formation of platelet neutrophil and -monocyte aggregates. The injection of activated platelets in mice produced transient, P-selectin dependent, complete MPO depletion in about 50% of neutrophils. Conclusions ACS are characterized by intense neutrophil activation, like other systemic inflammatory syndromes. In the very early phase of acute myocardial infarction only a subpopulation of neutrophils is massively activated, possibly via platelet-P selectin

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

    DTIC Science & Technology

    2007-11-02

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

  16. Abnormal neutrophil chemotactic activity in children with congenital insensitivity to pain with anhidrosis (CIPA): the role of nerve growth factor.

    PubMed

    Beigelman, Avraham; Levy, Jacov; Hadad, Nurit; Pinsk, Vered; Haim, Alon; Fruchtman, Yariv; Levy, Rachel

    2009-03-01

    A 1926-ins-T mutation in the TrkA gene encoding the tyrosine kinase receptor for nerve growth factor (NGF) was previously documented in patients with congenital insensitivity to pain with anhidrosis (CIPA). These patients suffer from skin lacerations which often evolve into deep tissue infections. Abnormality in neutrophil functions may explain this high rate of severe infections. In this study we show that chemotaxis was significantly (P<0.001) suppressed in patients' neutrophils, compared to healthy controls. Although NGF alone did not exert a chemotactic effect, its presence enhanced both migration toward fMLP and phosphorylation of MAP kinases (ERK and JNK) in neutrophils from healthy controls, but not in neutrophils from CIPA patients. The significantly impaired chemotactic activity of neutrophils from a CIPA patient, which has been attributed to the molecular defect in the TrkA receptor, may contribute to the high rate of infection.

  17. S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin

    PubMed Central

    Cohen, Taylor S.; Jones-Nelson, Omari; Hotz, Meghan; Cheng, Lily; Miller, Lloyd S.; Suzich, JoAnn; Stover, C. Kendall; Sellman, Bret R.

    2016-01-01

    Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by “friendly fire”, a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection. PMID:27739519

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

    PubMed

    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

  19. 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

  20. 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.

  1. p21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils

    PubMed Central

    Martyn, Kendra D.; Kim, Moon-Ju; Quinn, Mark T.; Dinauer, Mary C.; Knaus, Ulla G.

    2005-01-01

    The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays an instrumental role in host defense and contributes to microbicial killing by releasing highly reactive oxygen species. This multicomponent enzyme is composed of membrane and cytosolic components that assemble in the plasma membrane or phagolysosome. While the guanosine S′-triphosphatase (GTPase) Rac2 has been shown to be a critical regulator of NADPH oxidase activity and assembly, the role of its effector, p21-activated kinase (Pak), in oxidase function has not been well defined. Using HIV-1 Tat-mediated protein transduction of Pak inhibitory domain, we show here that Pak activity is indeed required for efficient superoxide generation in intact neutrophils. Furthermore, we show that Pak translocates to the plasma membrane upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation and colocalizes with translocated p47phox and with p22phox, a subunit of flavocytochrome b558. Although activated Pak phosphorylated several essential serine residues in the C-terminus of p47phox, direct binding to p47phox was not observed. In contrast, active Pak bound directly to p22phox, suggesting flavocytochrome b was the oxidase-associated membrane target of this kinase and this association may facilitate further phosphorylation of p47phox in the assembling NADPH oxidase complex. PMID:16099876

  2. The Pig: A Relevant Model for Evaluating the Neutrophil Serine Protease Activities during Acute Pseudomonas aeruginosa Lung Infection

    PubMed Central

    Bréa, Déborah; Vandebrouck, Clarisse; Barc, Céline; Pezant, Jérémy; Melo, Sandrine; Olivier, Michel; Delaunay, Rémy; Boulesteix, Olivier; Berthon, Patricia; Rossignol, Christelle; Burlaud Gaillard, Julien; Becq, Frédéric; Gauthier, Francis; Si-Tahar, Mustapha; Meurens, François; Berri, Mustapha; Caballero-Posadas, Ignacio; Attucci, Sylvie

    2016-01-01

    The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases. PMID:27992534

  3. 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.

  4. Granule Exocytosis Contributes to Priming and Activation of the Human Neutrophil Respiratory Burst

    PubMed Central

    Uriarte, Silvia M.; Rane, Madhavi J.; Luerman, Gregory C.; Barati, Michelle T.; Ward, Richard A.; Nauseef, William M.; McLeish, Kenneth R.

    2013-01-01

    The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT–SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT–SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT–SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT–SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT–SNAP-23 inhibited the increase in plasma membrane expression of gp91phox in TNF-α–primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase. PMID:21642540

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

    PubMed Central

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

    1987-01-01

    Sodium fluoride (20 mM) effected rapid hydrolysis of phosphatidylinositol bisphosphate (PIP2) in human neutrophils. Intracellular free Ca2+ levels increased after PIP2 hydrolysis but before respiratory burst activation. Both the increase in intracellular free Ca2+ levels and the extent of functional activation were dependent on the availability of extracellular Ca2+. The rate of F(-)-stimulated PIP2 hydrolysis, however, was not affected when the rise in cytosolic Ca2+ was severely limited by depletion of extracellular Ca2+. Fluoride caused the specific hydrolysis of PIP2 in isolated neutrophil plasma membranes. This effect occurred in the presence of low levels of available Ca2+ and was accompanied by the release of inositol phosphates. We conclude that PIP2 hydrolysis is an early event in the response of neutrophils to F-. This response is not Ca2+-regulated but may lead to an influx of Ca2+ from the extracellular medium. Activation of a PIP2-specific phospholipase independent of a change in cytosolic free Ca2+ levels may be the initial event in the stimulus-response pathway triggered by fluoride. PMID:3036911

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

    PubMed

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

    1987-07-01

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

  7. 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

  8. Chronic inflammation and neutrophil activation as possible causes of joint diseases in ballet dancers.

    PubMed

    Borges, Leandro da Silva; Bortolon, José Ricardo; 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. 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.

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

    PubMed

    Fu, Hua-Wen

    2014-05-14

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

  10. 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.

  11. Interaction between the respiratory burst activity of neutrophil leukocytes and experimentally induced Escherichia coli mastitis in cows.

    PubMed

    Heyneman, R; Burvenich, C; Vercauteren, R

    1990-04-01

    The respiratory burst activity of neutrophil leukocytes from bovine peripheral blood was studied before and during an experimentally induced Escherichia coli mastitis. The competence of neutrophils to generate reactive oxygen species following stimulation with opsonized particles prior to infection was negatively correlated with severity of subsequently induced E. coli mastitis. In the presence of the soluble activator, phorbol myristate acetate, no such correlation was obtained. However, combination of blood neutrophil numbers with phorbol myristate acetate induced respiratory burst competence, called reactive oxygen species-generating capacity, displayed a negative correlation with the intensity of a subsequent inflammation of the bovine mammary gland. At the onset of mastitis, a concomitant reduction in blood neutrophil numbers, a strong shift in cell types, and a substantial decrease in production of reactive oxygen species occurred. Reestablishment and even enhancement of the respiratory burst activity coincided with the reappearance of mature neutrophils. Possible stimulatory effects on neutrophil superoxide generation are discussed. Data suggest that generation of reactive oxygen species by mature neutrophils may be of primary importance for microbial killing during the onset and recovery from mastitis.

  12. Vesicles generated during storage of red blood cells enhance the generation of radical oxygen species in activated neutrophils.

    PubMed

    Jank, Herbert; Salzer, Ulrich

    2011-01-18

    Erythrocytes are known to shed vesicles in vivo, under various conditions in vitro, and, with impact for transfusion medicine, during storage of red blood cell concentrates (Vsto vesicles). Vsto vesicles of blood transfusions have been shown to deliver glycosylphosphatidylinositol-linked proteins to recipient erythrocytes, to display prothrombotic activity, and to have an inhibitory effect on macrophages. The interaction of Vsto vesicles with and their effect on neutrophilic granulocytes has not yet been studied in detail. Fluorescently labeled Vsto and calcium-induced vesicles were prepared in order to study the uptake of labeled vesicular components by neutrophils as compared to the process of phagocytosis of zymosan using flow cytometry and confocal microscopy. The activating effect of Vsto vesicles on neutrophils was addressed by a luminometric assay for stimulated radical oxygen species (ROS) generation.Coincubation of vesicles and neutrophils results in a transfer of vesicular components to the cells. This uptake is different from a phagocytotic process and is enhanced upon interference with the cellular actin cytoskeleton. Preincubation of neutrophils with Vsto vesicles results in an enhanced ROS generation by neutrophils,which is further increased upon fMLP stimulation and during zymosan phagocytosis. The activating effect of Vsto vesicles on neutrophils might be due to the specific accumulation of lysophospholipids in Vsto vesicles and should be considered as a possible contributor to the pathogenesis of transfusion-related acute lung injury.

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

    PubMed

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

    2014-08-25

    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.

  14. PGF2α, a Prostanoid Released by Endothelial Cells Activated by Hypoxia, Is a Chemoattractant Candidate for Neutrophil Recruitment

    PubMed Central

    Arnould, Thierry; Thibaut-Vercruyssen, Rose; Bouaziz, Najat; Dieu, Marc; Remacle, José; Michiels, Carine

    2001-01-01

    Despite increasing evidence supporting the involvement of neutrophils in ischemic and postischemic damages, the mechanisms underlying the early recruitment of these cells are not completely understood. In this report, the effects of conditioned media from hypoxic endothelial cells on neutrophil chemotaxis were investigated by biochemical and morphological studies. We showed that conditioned media collected from several endothelial cell origins submitted to hypoxia as well as ischemic rat liver perfusion liquids have a chemotactic activity for neutrophils. The role of various chemoattractant molecules like HETEs, platelet-activating factor, and cytokines such as interleukin-8 and interleukin-1 was examined in the same model. Chemotactic peptide contribution was ruled out as boiled conditioned media still trigger chemotaxis. However, cell treatment with cyclooxygenase inhibitors, neutralization of PGF2α biological activity with polyclonal antibodies, and the neutrophil preincubation with a specific PGF2α antagonist, all dramatically inhibited neutrophil chemotaxis. A strong chemoattractant effect of pure exogenous PGF2α or of a synthetic analog was also observed. The major effect of PGF2α on neutrophil chemotaxis was confirmed ex vivo in a rat liver perfusion ischemic model. These results suggest that PGF2α, a prostanoid abundantly released by the endothelium of hypoxic or ischemic tissues, is a chemoattractant molecule that might be involved in the early recruitment of neutrophils in ischemic organs. PMID:11438482

  15. The Essential Role of Type I Interferons in Differentiation and Activation of Tumor-Associated Neutrophils

    PubMed Central

    Pylaeva, Ekaterina; Lang, Stephan; Jablonska, Jadwiga

    2016-01-01

    Type I interferons (IFNs) were first characterized in the process of viral interference. However, since then, IFNs are found to be involved in a wide range of biological processes. In the mouse, type I IFNs comprise a large family of cytokines. At least 12 IFN-α and one IFN-β can be found and they all signal through the same receptor (IFNAR). A hierarchy of expression has been established for type I IFNs, where IFN-β is induced first and it activates in a paracrine and autocrine fashion a cascade of other type I IFNs. Besides its importance in the induction of the IFN cascade, IFN-β is also constitutively expressed in low amounts under normal non-inflammatory conditions, thus facilitating “primed” state of the immune system. In the context of cancer, type I IFNs show strong antitumor function as they play a key role in mounting antitumor immune responses through the modulation of neutrophil differentiation, activation, and migration. Owing to their plasticity, neutrophils play diverse roles during cancer development and metastasis since they possess both tumor-promoting (N2) and tumor-limiting (N1) properties. Notably, the differentiation into antitumor phenotype is strongly supported by type I IFNs. It could also be shown that these cytokines are critical for the suppression of neutrophil migration into tumor and metastasis site by regulating chemokine receptors, e.g., CXCR2 on these cells and by influencing their longevity. Type I IFNs limit the life span of neutrophils by influencing both, the extrinsic as well as the intrinsic apoptosis pathways. Such antitumor neutrophils efficiently suppress the pro-angiogenic factors expression, e.g., vascular endothelial growth factor and matrix metallopeptidase 9. This in turn restricts tumor vascularization and growth. Thus, type I IFNs appear to be the part of the natural tumor surveillance mechanism. Here we provide an up to date review of how type I IFNs influence the pro- and antitumor properties of

  16. 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).

  17. Oxidative stress-dependent conversion of hydrogen sulfide to sulfite by activated neutrophils.

    PubMed

    Mitsuhashi, Hideki; Yamashita, Shin; Ikeuchi, Hidekazu; Kuroiwa, Takashi; Kaneko, Yoriaki; Hiromura, Keiju; Ueki, Kazue; Nojima, Yoshihisa

    2005-12-01

    Sulfite, which is known as a major constituent of volcanic gas, is endogenously produced in mammals, and its concentration in serum is increased in patients with pneumonia. It has been reported that sulfite is produced by oxidation from hydrogen sulfide (H2S) as an intermediate in the mammalian body. The objective of this study was to investigate the ability of reactive oxygen species from neutrophils to produce sulfite from H2S. Sulfite production from activated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine gradually increased with an increased concentration of sodium hydrosulfide (NaHS) in the medium. The production of sulfite was markedly suppressed with an NADPH oxidase inhibitor, diphenyleneiodonium. When NaHS was added to the supernatant of activated neutrophils, a significant amount of sulfite was synthesized in the test tubes. Furthermore, when a medium containing NaHS was incubated with a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride, sulfite was formed in the solution and this increase was markedly suppressed by ascorbic acid. Finally, we determined serum concentrations of sulfite and H2S in an in vivo model of neutrophil activation induced by systemic injection of lipopolysaccharide (LPS) into rats. We found a significant increase in serum sulfite and H2S after LPS injection. Importantly, coadministration of ascorbic acid with LPS further increased serum H2S but suppressed sulfite levels. This finding implies that oxidative stress-dependent conversion of H2S to sulfite might occur in vivo. Thus, the oxidation of H2S is a novel sulfite production pathway in the inflammatory condition, and this chemical synthesis might be responsible for the upregulation of sulfite production in inflammatory conditions such as pneumonia.

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

    PubMed

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

    1986-07-01

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

  19. AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils

    PubMed Central

    Bae, Hong-Beom; Zmijewski, Jaroslaw W.; Deshane, Jessy S.; Tadie, Jean-Marc; Chaplin, David D.; Takashima, Seiji; Abraham, Edward

    2011-01-01

    Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46±7.8 or 85±26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21±1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.—Bae, H. -B., Zmijewski, J. W., Deshane, J. S., Tadie, J. -M., Chaplin, D. D., Takashima, S., Abraham, E. AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils. PMID:21885655

  20. 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.

  1. Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

    PubMed

    Parzy, Elodie; Bouchaud, Véronique; Massot, Philippe; Voisin, Pierre; Koonjoo, Neha; Moncelet, Damien; Franconi, Jean-Michel; Thiaudière, Eric; Mellet, Philippe

    2013-01-01

    Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations. These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

  2. Overhauser-Enhanced MRI of Elastase Activity from In Vitro Human Neutrophil Degranulation

    PubMed Central

    Parzy, Elodie; Bouchaud, Véronique; Massot, Philippe; Voisin, Pierre; Koonjoo, Neha; Moncelet, Damien; Franconi, Jean-Michel; Thiaudière, Eric; Mellet, Philippe

    2013-01-01

    Background Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. Methodology/Principal Findings We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×104 cells per milliliter, well under the physiological neutrophils concentrations. Conclusions/Significance These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research. PMID:23469112

  3. Volume-dependent regulation of the respiratory burst of activated human neutrophils.

    PubMed

    Kuchkina, N V; Orlov, S N; Pokudin, N I; Chuchalin, A G

    1993-11-15

    The effect of incubation medium osmolality on the respiratory burst of human neutrophils was studied using luminol-dependent chemiluminescence (CL) as an indicator of burst activity. Neutrophils were stimulated with N-formyl-Met-Leu-Phe (FMLP), phorbol-12-myristate-13-acetate (PMA), the calcium ionophore A23187, thermoaggregated IgG (IgGn), and opsonized zymosan (OZ). It was shown that increasing the osmolality of the incubation medium from 320 up to 420 mosM decreased the A23187- and OZ-induced CL responses by 90%. Under the same conditions PMA-, FMLP- and IgGn-induced CL responses were decreased by 40-60%. A decrease of osmolality to 200 mosM resulted in a 2-3 fold decrease of the A23187-, PMA- and FMLP-induced CL and in a 60-80% increase of OZ- and IgGn-induced CL. It is suggested that osmolality-mediated alteration of cell volume is an important mechanism for regulating neutrophil activity.

  4. mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis

    PubMed Central

    Bao, Yi; Ledderose, Carola; Graf, Amelie F.; Brix, Bianca; Birsak, Theresa; Lee, Albert; Zhang, Jingping

    2015-01-01

    Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca2+ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases. PMID:26416965

  5. mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis.

    PubMed

    Bao, Yi; Ledderose, Carola; Graf, Amelie F; Brix, Bianca; Birsak, Theresa; Lee, Albert; Zhang, Jingping; Junger, Wolfgang G

    2015-09-28

    Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca(2+) uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.

  6. Ischemia Activates Neutrophils But Inhibits Their Local and Remote Diapedesis.

    DTIC Science & Technology

    2007-11-02

    mediated polymorphonuclear leukocyte (PMN) activation and diapedesis . Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or...introduced into an abraded skin chamber or intratracheally induced diapedesis in non-ischemic animals. PMN accumulations in the+skin chamber were...exp 4) PMN/mm(exp 3) compared to 5 +/- 1 X 10(exp 4) PMN/mm(exp 3) with sham plasma (n = 4, pɘ.05). Diapedesis was completely prevented (0-3 PMN/mm(exp

  7. 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

  8. 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

  9. Activation of human neutrophil Mac-1 by anion substitution *

    PubMed Central

    Lomakina, Elena; Knauf, Philip A.; Schultz, Joanne B.; Law, Foon-Yee; McGraw, Matthew D.; Waugh, Richard E.

    2009-01-01

    Substituting the medium chloride with glucuronate or glutamate causes a rapid, 10 to 30 –fold, increase in the binding of the monoclonal antibody, CBRM1/5, which recognizes the high-affinity conformation of the Mac-1 integrin. This change is reflected in functional adhesion assays that show increased adhesion to ICAM-1 coated beads. Blocking antibodies indicate that the increased adhesion is almost entirely due to Mac-1. The inhibitor NPPB (100μM) reduces Cl- efflux into low Cl- medium by 75%, and blocks increased CBRM1/5 binding after stimulation with fMLP or TNF-α, but has no effect on the anion substitution induced increase in CBRM1/5 binding or adhesion to immobilized ICAM-1. Thus, changes in external anion composition, not internal chloride or increases in Cl- efflux, are responsible for Mac-1 activation. This effect is substantial. The percentage of Mac-1 in the high affinity state approaches 100% in glutamate and 50% in glucuronate, a far greater response than what is observed after stimulation with fMLP. PMID:19246218

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2012-10-01

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

  12. Amiloride (Am) dissociates human neutrophil (N) activation events

    SciTech Connect

    Berkow, R.L.; Dodson, R.; Kraft, A.S.

    1986-03-05

    Human N can be stimulated to release granule contents and superoxide anion (O/sub 2/sup -//). These events are associated with an Am sensitive Na/sup +//H/sup +/ exchange and N alkalinization. Am has been reported to inhibit protein kinase C (PKC) in HL-60 cells. Due to the central role of PKC in N activation they assessed the effect of prolonged exposure of N to Am. When N were treated with 10/sup -6/ to 10/sup -3/M Am at 37/sup 0/C for 15 min a dose dependent inhibition of O/sub 2/sup -// release was seen upon N stimulation with FMLP (10/sup -6/M), A23187 (10/sup -5/M), or serum treated Zymosan (Z) (2.5 mg/ml). Maximal inhibition depended on the time of exposure of N to Am prior to stimulation and remained after removal of Am by washing. N treated with 10/sup -3/M Am had a decreased influx of /sup 45/Ca/sup + +/ upon stimulation with FMLP. Phorbol myristate acetate induced release of N O/sub 2/sup -// was unaffected by pretreatment with Am. Similarly, Am did not inhibit stimulated N lysozyme release or the incorporation of /sup 32/P into proteins. Monensin (a Na/sup +//H/sup +/ ionophore) did not correct the Am induced inhibition of O/sub 2/sup -// suggesting that cell acidification alone can not explain the Am effect. In conclusion: (1) Na/sup +//H/sup +/ exchange modulates N O/sub 2/sup -// release upon stimulation with FMLP, A23187, and Z. PMA induced N responses are not affected by cell acidification; (2) N granule release is under separate cellular control than O/sub 2/sup -//; (3) Am does not inhibit PKC or protein phosphorylation in N; and (4) decreased /sup 45/Ca/sup + +/ influx may partially explain the Am effect on FMLP induced O/sub 2/sup -// release.

  13. 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.

  14. Critical behavior of subcellular density organization during neutrophil activation and migration.

    PubMed

    Baker-Groberg, Sandra M; Phillips, Kevin G; Healy, Laura D; Itakura, Asako; Porter, Juliana E; Newton, Paul K; Nan, Xiaolin; McCarty, Owen J T

    2015-12-01

    Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration.

  15. Critical behavior of subcellular density organization during neutrophil activation and migration

    PubMed Central

    Baker-Groberg, Sandra M.; Phillips, Kevin G.; Healy, Laura D.; Itakura, Asako; Porter, Juliana E.; Newton, Paul K.; Nan, Xiaolin; McCarty, Owen J.T.

    2015-01-01

    Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration. PMID:26640599

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

    PubMed

    Seper, Andrea; Hosseinzadeh, Ava; Gorkiewicz, Gregor; Lichtenegger, Sabine; Roier, Sandro; Leitner, Deborah R; Röhm, Marc; Grutsch, Andreas; Reidl, Joachim; Urban, Constantin F; Schild, Stefan

    2013-01-01

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

  17. Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

    PubMed Central

    Seper, Andrea; Hosseinzadeh, Ava; Gorkiewicz, Gregor; Lichtenegger, Sabine; Roier, Sandro; Leitner, Deborah R.; Röhm, Marc; Grutsch, Andreas; Reidl, Joachim; Urban, Constantin F.; Schild, Stefan

    2013-01-01

    The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation. PMID:24039581

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

    PubMed

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

    2003-04-01

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

  19. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

    PubMed Central

    Adkison, April M.; Raptis, Sofia Z.; Kelley, Diane G.; Pham, Christine T.N.

    2002-01-01

    Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation. PMID:11827996

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

    PubMed

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

    2014-09-01

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

  1. 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.

  2. 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.

  3. Neutrophil Dysfunction in Sepsis

    PubMed Central

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

    2016-01-01

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

  4. 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

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

    PubMed

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

    2014-12-01

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

  6. The in vitro effects of propranolol and atenolol on neutrophil motility and post-phagocytic metabolic activity.

    PubMed Central

    Anderson, R; van Rensburg, A J

    1979-01-01

    Propranolol at concentrations of 1 x 10(-6) to 1 x 10(-4) M consistently increased neutrophil motility as measured in Boyden chambers. The effects were not due solely to stimulation of random migration and chemokinesis but also of directional motility. Propranolol, over a similar concentration range, caused inhibition of post-phagocytic cell metabolic activity (hexose monophosphate shunt, nitro-blue tetrazolium reduction and protein iodination) without any detectable effect on the ingestion rate of Candida albicans. Atenolol had no effect on any of these neutrophil functions. Both drugs were without effect on glycolysis and intracellular cyclic AMP levels. Propranolol however, at concentrations which stimulated cell motility, caused increased intracellular cyclic GMP levels. It is suggested that propranolol may stimulate neutrophil motility by promoting increased intracellular cyclic GMP levels or by decreasing neutrophil superoxide production. PMID:223974

  7. Proteinase 3 and Serpin B1: a novel pathway in the regulation of caspase-3 activation, neutrophil spontaneous apoptosis, and inflammation.

    PubMed

    Loison, Fabien; Xu, Yuanfu; Luo, Hongbo R

    Neutrophils are the first responders of the inflammatory response. They are characterized by their potent cytotoxic content but also by their limited lifetime. This short half-life is thought to be a self-protecting mechanism for the host, as highlighted by the numerous pathologies associated with imbalanced neutrophil survival. Neutrophil spontaneous death is the prototype of programmed cell death, harboring all the phenotypic hallmarks of apoptosis and dependent on the activation of the effector caspase-3. However, the pathways regulating neutrophil spontaneous death remain ill-defined. In a recent publication, we determined that in aging neutrophils, the cleavage and activation of caspase-3 was mediated by the serine protease Proteinase 3 (PR3), and was independent of the canonical extrinsic and intrinsic apoptosis pathways. In mature neutrophils, PR3 was stored in granules and progressively released to the cytosol during neutrophil aging. The release of PR3 was dependent on lysosomal membrane permeabilization (LMP). Once in the cytosol, PR3 cleaved procaspase-3 at a site upstream of the caspase-9 cleavage site, leading to caspase-3 activation. Inhibition, knockdown or knockout of PR3 delayed neutrophil apoptosis in vitro and in vivo. The adoptive transfer of both WT and PR3-deficient neutrophils to WT mice revealed that the delayed death of neutrophils lacking PR3 in vivo was due to an altered intrinsic apoptosis/survival pathway and not to difference in the inflammatory microenvironment. The cytosolic inhibitor of serine proteases serpin b1 counterbalanced the activity of PR3 in the cytosol of neutrophils, and the deletion of serpinb1 in neutrophils accelerated their spontaneous death. In summary, our results reveal that PR3 and serpinB1 are part of a newly characterized apoptosis pathway, regulating caspase-3 activation and neutrophil spontaneous death and the survival of neutrophils during inflammation.

  8. Proteinase 3 and Serpin B1: a novel pathway in the regulation of caspase-3 activation, neutrophil spontaneous apoptosis, and inflammation

    PubMed Central

    Xu, Yuanfu; Luo, Hongbo R

    2015-01-01

    Neutrophils are the first responders of the inflammatory response. They are characterized by their potent cytotoxic content but also by their limited lifetime. This short half-life is thought to be a self-protecting mechanism for the host, as highlighted by the numerous pathologies associated with imbalanced neutrophil survival. Neutrophil spontaneous death is the prototype of programmed cell death, harboring all the phenotypic hallmarks of apoptosis and dependent on the activation of the effector caspase-3. However, the pathways regulating neutrophil spontaneous death remain ill-defined. In a recent publication, we determined that in aging neutrophils, the cleavage and activation of caspase-3 was mediated by the serine protease Proteinase 3 (PR3), and was independent of the canonical extrinsic and intrinsic apoptosis pathways. In mature neutrophils, PR3 was stored in granules and progressively released to the cytosol during neutrophil aging. The release of PR3 was dependent on lysosomal membrane permeabilization (LMP). Once in the cytosol, PR3 cleaved procaspase-3 at a site upstream of the caspase-9 cleavage site, leading to caspase-3 activation. Inhibition, knockdown or knockout of PR3 delayed neutrophil apoptosis in vitro and in vivo. The adoptive transfer of both WT and PR3-deficient neutrophils to WT mice revealed that the delayed death of neutrophils lacking PR3 in vivo was due to an altered intrinsic apoptosis/survival pathway and not to difference in the inflammatory microenvironment. The cytosolic inhibitor of serine proteases serpin b1 counterbalanced the activity of PR3 in the cytosol of neutrophils, and the deletion of serpinb1 in neutrophils accelerated their spontaneous death. In summary, our results reveal that PR3 and serpinB1 are part of a newly characterized apoptosis pathway, regulating caspase-3 activation and neutrophil spontaneous death and the survival of neutrophils during inflammation. PMID:26029732

  9. Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils.

    PubMed

    Gorudko, Irina V; Mukhortava, Ann V; Caraher, Brendan; Ren, Melody; Cherenkevich, Sergey N; Kelly, Gregory M; Timoshenko, Alexander V

    2011-12-15

    The gp91phox subunit of flavocytochrome b(558) is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b(558). gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin-gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H(2)O(2) generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H(2)O(2) production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. JAK2 V617F uses distinct signalling pathways to induce cell proliferation and neutrophil activation.

    PubMed

    Oku, Seido; Takenaka, Katsuto; Kuriyama, Takuro; Shide, Kotaro; Kumano, Takashi; Kikushige, Yoshikane; Urata, Shingo; Yamauchi, Takuji; Iwamoto, Chika; Shimoda, Haruko K; Miyamoto, Toshihiro; Nagafuji, Koji; Kishimoto, Junji; Shimoda, Kazuya; Akashi, Koichi

    2010-08-01

    The acquired JAK2 V617F mutation is observed in the majority of patients with BCR-ABL1 negative chronic myeloproliferative neoplasms (MPN). BCR-ABL1 negative MPN displays myeloproliferation with an elevated leucocyte alkaline phosphatase (LAP) activity, a neutrophil activation marker. We tried to separate the downstream signalling of JAK2 V617F to stimulate myeloproliferation and LAP activity. NB4, a myeloid lineage cell line, was transduced with Jak2 V617F mutation or wild-type Jak2. We found that Jak2 V617F mutation, but not wild-type Jak2 enhanced LAP expression in NB4-derived neutrophils and proliferation of NB4 cells. JAK2 V617F induces constitutive phosphorylation of STAT3 and STAT5, and uses signalling targets such as Ras/MEK/ERK and PI3K/Akt pathways. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, and STAT3 or STAT5 siRNAs, JAK2 V617F was found to specifically use the STAT3 pathway to enhance LAP expression, while STAT5, Ras/MEK/ERK and PI3K/Akt, but not STAT3 pathways, were able to stimulate cell proliferation. These data strongly suggest that JAK2 V617F uses distinct signalling pathways to induce typical pathological features of MPN, such as high LAP activity and enhanced cell proliferation.

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

    PubMed

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

    2016-11-01

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

  12. 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

  13. 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

    2015-12-14

    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+).

  14. Plane of nutrition during the preweaning period but not the grower phase influences the neutrophil activity of Holstein calves.

    PubMed

    Obeidat, B S; Cobb, C J; Sellers, M D; Pepper-Yowell, A R; Earleywine, T J; Ballou, M A

    2013-01-01

    .6 kg of DM of the same concentrate per head per day. All calves were fed alfalfa hay (16.2% CP; DM basis) ad libitum. Overall, average daily gain was greater in HPN calves than LPN calves. No differences were noticed for concentrations of plasma urea nitrogen, glucose, neutrophil L-selectin expression, percentage of neutrophils producing OB, and plasma haptoglobin concentration between the 2 planes of nutrition. In summary, intake and performance were improved in calves fed the HPN than calves fed the LPN in both experiments. The neutrophil responses of calves fed an LPN were more active during the preweaning period than calves fed an HPN; however, this response was not observed during the immediate postweaning period or the grower phase.

  15. 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.

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

    PubMed

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

    1988-07-01

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

  17. 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.

  18. Bryostatins trigger human polymorphonuclear neutrophil and monocyte oxidative metabolism: association with in vitro antineoplastic activity.

    PubMed

    Esa, A H; Warren, J T; Hess, A D; May, W S

    1995-01-01

    Bryostatin-1-but not bryostatin-13-a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, triggered human polymorphonuclear neutrophil (PMN) and monocyte release of reactive oxygen radicals, as measured by the generation of lucigenin chemiluminescence and by the ferricytochrome c reduction assay. The release of oxygen radicals by bryostatins was sensitive to inhibitors of protein kinases, but resistant to the inhibition of phospholipase A2 activity and arachidonic acid metabolism (prior treatment with mepacrine or indomethacin). Comparison of the effect of protein kinase (PK) inhibitors H-8, H-7 and staurosporine on bryostatin-1-induced neutrophil oxygen radical release further suggested a requirement for activation of phospholipid-dependent PKC, but not for cGMP- or cAMP-dependent PK. In cytostatic assays, PMNs treated with bryostatin-1 inhibited the growth of the erythroleukaemic cell line K562 in a concentration-dependent manner. These findings suggest that the reported antineoplastic effect of bryostatins may result at least in part from activation of PMNs and monocytes.

  19. Effect of sulphasalazine and its active metabolite, 5-amino-salicylic acid, on toxic oxygen metabolite production by neutrophils.

    PubMed Central

    Williams, J G; Hallett, M B

    1989-01-01

    The possibility that the mode of action of sulphasalazine and its active metabolite 5-amino-salicylic acid (5ASA) involves modification of toxic oxygen metabolite production by neutrophils has been investigated by measuring the effect of these drugs on luminol-dependent chemiluminescence, superoxide release and oxygen consumption by stimulated neutrophils in vitro. 5ASA, and to a lesser extent sulphasalazine, had profound inhibitory effects on the luminol dependent chemiluminescent response of neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (1 microM) + cytochalasin B (5 micrograms/ml). A concentration of 50 microM 5ASA or sulphasalazine produced 93.8 (2.3)% and 65.7 (3.7)% inhibition of control responses respectively. The concentration of 5ASA and sulphasalazine producing 50% inhibition of chemiluminescence were 3.6 (1.8) microM and 16.5 (6) microM respectively. Both drugs had little effect on the chemiluminescent response of neutrophils stimulated with phorbol myristate acetate (1 microgram/ml), producing only 11.4 (3.9)% and 34 (7)% inhibition respectively, at a concentration of 50 microM. Superoxide release from fMLP + CB stimulated neutrophils was also inhibited slightly by 5ASA (50 microM) by 35.6% and by sulphasalazine (50 microM) by 7.9%. Similarly, there was little inhibition in the rate of oxygen consumption by fMLP + CB stimulated neutrophils by either 5ASA or sulphasalazine at concentrations which produced near total abolition of luminol dependent chemiluminescence. These results show that sulphasalazine and 5ASA inhibit the reaction of toxic metabolites produced by stimulated neutrophils with luminol, without inhibition of the oxidase system producing these metabolites. The site of action of these drugs on neutrophils in vitro is thus extracellular, by scavenging a released metabolite, probably hypochlorite. This has important implications for their mode of action in vivo in inflammatory bowel disease. PMID:2574700

  20. Adaptation to Resistance Training Is Associated with Higher Phagocytic (but Not Oxidative) Activity in Neutrophils of Older Women.

    PubMed

    Bartholomeu-Neto, João; Brito, Ciro José; Nóbrega, Otávio Toledo; Sousa, Vinícius Carolino; Oliveira Toledo, Juliana; Silva Paula, Roberta; Alves, David Junger Fonseca; Ferreira, Aparecido Pimentel; Franco Moraes, Clayton; Córdova, Cláudio

    2015-01-01

    Failure in antimicrobial activity contributes to high morbidity and mortality in the geriatric population. Little is known about the potential effect of resistance training (RT) on the functional properties of the innate immunity. This study aimed to investigate the influence of long-term RT on the endocytic and oxidative activities of neutrophils and monocytes in healthy older women. Our results indicate that the phagocytosis index (PhI) of neutrophils (but not of monocytes) in the RT-adapted group was significantly higher (P < 0.001; effect size, (d) = 0.90, 95% CI: [0.75-1.04]) compared to that in sedentary subjects. In contrast, the oxidative activity of either neutrophils or monocytes was not significantly influenced by RT. Also, total energy and carbohydrate intake as well as serum IL6 levels had a significant influence on the phagocytic activity of neutrophils (P = 0.04), being considered in the model. Multivariate regression identified the physical condition of the subject (β = 0.425; P = 0.01) as a significant predictor of PhI. In conclusion, circulating neutrophils of older women adapted to a long-term RT program expressed higher phagocytic activity.

  1. Adaptation to Resistance Training Is Associated with Higher Phagocytic (but Not Oxidative) Activity in Neutrophils of Older Women

    PubMed Central

    Bartholomeu-Neto, João; Brito, Ciro José; Nóbrega, Otávio Toledo; Sousa, Vinícius Carolino; Oliveira Toledo, Juliana; Silva Paula, Roberta; Alves, David Junger Fonseca; Ferreira, Aparecido Pimentel; Franco Moraes, Clayton; Córdova, Cláudio

    2015-01-01

    Failure in antimicrobial activity contributes to high morbidity and mortality in the geriatric population. Little is known about the potential effect of resistance training (RT) on the functional properties of the innate immunity. This study aimed to investigate the influence of long-term RT on the endocytic and oxidative activities of neutrophils and monocytes in healthy older women. Our results indicate that the phagocytosis index (PhI) of neutrophils (but not of monocytes) in the RT-adapted group was significantly higher (P < 0.001; effect size, (d) = 0.90, 95% CI: [0.75–1.04]) compared to that in sedentary subjects. In contrast, the oxidative activity of either neutrophils or monocytes was not significantly influenced by RT. Also, total energy and carbohydrate intake as well as serum IL6 levels had a significant influence on the phagocytic activity of neutrophils (P = 0.04), being considered in the model. Multivariate regression identified the physical condition of the subject (β = 0.425; P = 0.01) as a significant predictor of PhI. In conclusion, circulating neutrophils of older women adapted to a long-term RT program expressed higher phagocytic activity. PMID:26524964

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

    PubMed Central

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

    1990-01-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections. Images PMID:2394841

  3. p47phox Molecular Activation for Assembly of the Neutrophil NADPH Oxidase Complex*

    PubMed Central

    Marcoux, Julien; Man, Petr; Petit-Haertlein, Isabelle; Vivès, Corinne; Forest, Eric; Fieschi, Franck

    2010-01-01

    The p47phox cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47phox activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22phox upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22phox, reflecting an intermediate conformation between the autoinhibited and activated forms. PMID:20592030

  4. 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.

  5. Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase.

    PubMed

    Zhang, Qin; Itagaki, Kiyoshi; Hauser, Carl J

    2010-07-01

    Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

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

    PubMed

    Wilhelmsen, Kevin; Farrar, Katherine; Hellman, Judith

    2013-08-23

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

  7. Expression and activity of N-myristoyltransferase in lung inflammation of cattle and its role in neutrophil apoptosis

    PubMed Central

    Shrivastav, Anuraag; Suri, Sarabjeet S.; Mohr, Ryan; Janardhan, Kyathanahalli S.; Sharma, Rajendra K.; Singh, Baljit

    2009-01-01

    N-myristoyltransferase (NMT) attaches a 14 carbon fatty acid, myristic acid, to the N-terminal glycine residue of proteins. NMT exists in two isoforms NMT1 and NMT2. Myristoylated proteins play critical roles in protein–protein interactions, cell signaling and oncogenesis. Although elevated expression of NMT1 has been described in colorectal carcinoma, its expression and roles in normal and inflamed lungs of the cattle are unknown. Therefore, we investigated the expression and activity of NMT in a bovine model of lung inflammation induced with Mannheimia hemolytica and in vitro in neutrophils and macrophages. Western blots revealed increased expression of NMT1 in lungs from infected animals compared to control animals. Total NMT activity was reduced in inflamed lungs compared to control animals (p < 0.05) along with increased expression of enolase, a putative inhibitor of NMT. NMT1 staining was observed in the septum, vascular endothelium and the epithelium in the lungs from control as well as infected calves. NMT1 expression was intense in neutrophils in the necrotic areas in the inflamed lungs. Immuno-electron microscopy localized NMT1 in cytoplasm and nuclei of endothelium, pulmonary intravascular macrophages and airway epithelium. Total NMT activity and NMT1 expression were increased in neutrophils and macrophages exposed to Escherichia coli LPS in vitro. NMT knockdown increased apoptosis in activated neutrophils. This is the first report demonstrating expression of NMT in normal and inflamed lungs and a novel role for NMT in regulation of neutrophil lifespan. PMID:19796608

  8. The phagocyte chemiluminescence paradox: luminol can act as an inhibitor of neutrophil NADPH-oxidase activity.

    PubMed

    Fäldt, J; Ridell, M; Karlsson, A; Dahlgren, C

    1999-01-01

    The chemiluminescence system amplified by luminol or isoluminol is a sensitive and widely used method for determination of respiratory burst products generated by the NADPH-oxidase in phagocytes. The present study shows that luminol, but not isoluminol, can inhibit the release of oxygen metabolites generated by human neutrophil NADPH-oxidase. The difference in structure between luminol and isoluminol (rendering luminol more lipophilic than isoluminol, and thereby membrane-permeable), is suggested to determine indirectly whether or not the molecule is inhibitory. Luminol was shown to have an increased inhibitory effect after preincubation of neutrophils on a surface of aggregated IgG, suggesting that the cells can be transferred from a 'luminol-insensitive' to a 'luminol-sensitive' state. Since luminol had no inhibitory effect in a cell-free NADPH-oxidase system, it is likely that it interferes with the signal transduction pathway, leading to assembly and/or activation of the oxidase. As a consequence of the present results, showing that luminol but not isoluminol can inhibit NADPH-oxidase activity, we suggest that isoluminol is used in future studies of superoxide anion release from phagocytes. Copyright 1999 John Wiley & Sons, Ltd.

  9. Neutrophil activation: an alternative to prostaglandin inhibition as the mechanism of action for NSAIDs.

    PubMed

    Altman, R D

    1990-02-01

    Experimental findings suggest that inhibition of neutrophil activation rather than suppression of prostaglandin formation may represent the principal mechanism of action of antiinflammatory drugs. This theory would account for the effectiveness of prostaglandin preserving agents, such as the nonacetylated salicylate salsalate, in the treatment of rheumatic disease. Results of the controlled clinical trials described in other papers contained in this supplement indicate that salsalate is equally effective as aspirin and the newer NSAID naproxen in relieving the signs and symptoms of rheumatoid arthritis. The damage to the gastric mucosa associated with NSAID use is believed to be attributable to impairment of mucosal defense mechanisms resulting from the inhibition of gastroprotective prostaglandins. Confirmation of neutrophil activation as the mechanism of action of NSAIDs would explain the efficacy of salsalate in light of its lower incidence of gastrointestinal side effects in controlled clinical trials with aspirin and naproxen. Establishment of such a mechanism would also suggest that the other adverse effects related to prostaglandin inhibition, such as hypersensitivity reactions, platelet dysfunction, and a reduction in renal function, are not necessary correlates of effective antiinflammatory therapy.

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

    PubMed Central

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

    2016-01-01

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

  11. Myeloperoxidase anti-neutrophil cytoplasmic antibody affinity is associated with the formation of neutrophil extracellular traps in the kidney and vasculitis activity in myeloperoxidase anti-neutrophil cytoplasmic antibody-associated microscopic polyangiitis.

    PubMed

    Yoshida, Masaharu; Yamada, Muneharu; Sudo, Yasuyo; Kojima, Tadasu; Tomiyasu, Tomohiro; Yoshikawa, Noriko; Oda, Takashi; Yamada, Michiyuki

    2016-07-01

    Anti-neutrophil cytoplasmic antibody (ANCA) is associated with small-vessel vasculitis particularly in the kidneys and can induce the formation of neutrophil extracellular traps (NETs) from primed neutrophils. Recently we have reported that the induction of NETs correlates with ANCA affinity for myeloperoxidase (MPO) and disease activity in patients with MPO-ANCA-associated microscopic polyangiitis. To investigate whether MPO-ANCA affinity is associated with the formation of NETs in vivo, we examined the occurrence of NETs in the renal tissues of patients with MPO-ANCA-associated microscopic polyangiitis and ANCA affinity by double immunofluorescence staining for NET components of citrullinated histone, MPO and PAD4 and by ELISA competition with MPO, respectively. We divided 30 MPO-ANCA-associated microscopic polyangiitis patients into 2 groups based on their ANCA affinity levels (IC50 for the high: 0.11 ± 0.04 µg/mL (Group1) and IC50 for the low: 0.66 ± 0.24 µg/mL (Group2)). Group1 showed a higher Birmingham vasculitis activity score (15.6 ± 5.7) and 73% of the patients presented clinically with rapidly progressive glomerulonephritis and histologically with focal/crescentic glomerulonephritis (GN). Group 2 showed a lower Birmingham vasculitis activity score (9.2 ± 4.9) and 73% of the patients presented clinically with chronic renal failure and histologically with mixed/sclerotic GN. Group 1 showed a much higher occurrence of NETs than Group 2. Our findings indicate that ANCA affinity was associated with the in vivo formation of NETs, which might be involved in the pathophysiology of patients with MPO-ANCA-associated microscopic polyangiitis. © 2016 The Authors Nephrology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Nephrology.

  12. 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.

  13. Activation of the A3 Adenosine Receptor Suppresses Superoxide Production and Chemotaxis of Mouse Bone Marrow Neutrophils

    PubMed Central

    van der Hoeven, Dharini; Wan, Tina C.; Auchampach, John A.

    2008-01-01

    Adenosine is formed in injured/ischemic tissues where it suppresses the actions of essentially all cells of the immune system. Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the Gs protein-coupled A2A adenosine receptor (AR). Here, we report that the A3AR is highly expressed in murine neutrophils isolated from bone marrow. Selective activation of the A3AR with CP-532,903 potently inhibited mouse bone marrow neutrophil superoxide generation and chemotaxis induced by various activating agents. The selectivity of CP-532,903 was confirmed in assays using neutrophils obtained from A2AAR and A3AR gene “knock-out” mice. In a model of thioglycollate-induced inflammation, treating mice with CP-532,903 inhibited recruitment of leukocytes into the peritoneum by specifically activating the A3AR. Collectively, our findings support the theory that the A3AR contributes to the anti-inflammatory actions of adenosine on neutrophils, and provide a potential mechanistic explanation for the efficacy of A3AR agonists in animal models of inflammation, i.e., inhibition of neutrophil-mediated tissue injury. PMID:18583455

  14. The Virulence Regulator Agr Controls the Staphylococcal Capacity to Activate Human Neutrophils via the Formyl Peptide Receptor 2

    PubMed Central

    Kretschmer, Dorothee; Nikola, Nele; Dürr, Manuela; Otto, Michael; Peschel, Andreas

    2012-01-01

    The Agr quorum-sensing system represents the master regulator for staphylococcal virulence factors and is known to have a strong impact on the release of pathogen-associated molecular pattern (PAMP) molecules. Among the various staphylococcal PAMPs, phenol-soluble modulin (PSM) peptides have attracted increasing interest because they are crucial for staphylococcal virulence and have neutrophil-recruiting properties. The latter depend on recognition of PSMs by the neutrophil formyl peptide receptor 2 (FPR2/ALX), for which PSMs are highly efficient agonists. We demonstrate that Agr inactivation in Staphylococcus aureus or S. epidermidis leads to strongly reduced neutrophil responses, which is in agreement with the previously reported strict control of PSM expression by Agr. Agr had a distinct and profound impact on activation of FPR2/ALX but not of the related FPR1 receptor that senses bacterial formylated peptides. S. epidermidis PSMs had similar FPR2/ALX-activating properties but differed in their dependence on N-terminal formylation compared to S. aureus PSMs. Moreover, S. aureus and S. epidermidis PSMs upregulated the neutrophil complement receptor CD11b via FPR2/ALX stimulation in an Agr-dependent fashion. Hence, Agr controls the capacity of staphylococcal pathogens to activate FPR2/ALX-dependent neutrophil responses, underscoring the crucial role of FPR2/ALX and PSMs in staphylococcus-host interaction. PMID:22067547

  15. The virulence regulator Agr controls the staphylococcal capacity to activate human neutrophils via the formyl peptide receptor 2.

    PubMed

    Kretschmer, Dorothee; Nikola, Nele; Dürr, Manuela; Otto, Michael; Peschel, Andreas

    2012-01-01

    The Agr quorum-sensing system represents the master regulator for staphylococcal virulence factors and is known to have a strong impact on the release of pathogen-associated molecular pattern (PAMP) molecules. Among the various staphylococcal PAMPs, phenol-soluble modulin (PSM) peptides have attracted increasing interest because they are crucial for staphylococcal virulence and have neutrophil-recruiting properties. The latter depend on recognition of PSMs by the neutrophil formyl peptide receptor 2 (FPR2/ALX), for which PSMs are highly efficient agonists. We demonstrate that Agr inactivation in Staphylococcus aureus or S. epidermidis leads to strongly reduced neutrophil responses, which is in agreement with the previously reported strict control of PSM expression by Agr. Agr had a distinct and profound impact on activation of FPR2/ALX but not of the related FPR1 receptor that senses bacterial formylated peptides. S. epidermidis PSMs had similar FPR2/ALX-activating properties but differed in their dependence on N-terminal formylation compared to S. aureus PSMs. Moreover, S. aureus and S. epidermidis PSMs upregulated the neutrophil complement receptor CD11b via FPR2/ALX stimulation in an Agr-dependent fashion. Hence, Agr controls the capacity of staphylococcal pathogens to activate FPR2/ALX-dependent neutrophil responses, underscoring the crucial role of FPR2/ALX and PSMs in staphylococcus-host interaction. Copyright © 2012 S. Karger AG, Basel.

  16. Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats

    PubMed Central

    Delbosc, Sandrine; Alsac, Jean-Marc; Journe, Clement; Louedec, Liliane; Castier, Yves; Bonnaure-Mallet, Martine; Ruimy, Raymond; Rossignol, Patrick; Bouchard, Philippe; Michel, Jean-Baptiste; Meilhac, Olivier

    2011-01-01

    Background Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. Methods and Findings Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. Conclusions Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression. PMID:21533243

  17. 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

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

    PubMed

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

    2015-01-01

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

  19. Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance

    PubMed Central

    Nguyen, Giang T.; Green, Erin R.; Mecsas, Joan

    2017-01-01

    Reactive oxygen species (ROS) generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs), toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs). Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation. Nonetheless, these

  20. Immunological Activation of Polymorphonuclear Neutrophils for Fungal Killing: Studies with Murine Cells and Blastomyces dermatitidis In Vitro,

    DTIC Science & Technology

    The interaction of elicited murine polymorphonuclear neutrophils (PMN) and the thermally dimorphic fungal pathogen Blastomyces dermatitidis in vitro...albicans compared to normal PMN. Fungicidal activity was abrogated in the presence of catalase , implicating hydrogen peroxide generation as the killing mechanism in the activated cells.

  1. Albumin oxidation leads to neutrophil activation in vitro and inaccurate measurement of serum albumin in patients with diabetic nephropathy.

    PubMed

    Michelis, Regina; Kristal, Batya; Zeitun, Teuta; Shapiro, Galina; Fridman, Yoav; Geron, Ronit; Sela, Shifra

    2013-07-01

    Previous studies suggest that oxidative modifications of serum albumin lead to underestimation of albumin concentrations using conventional assays. In addition, oxidation of serum albumin may cause neutrophil activation and further oxidation of albumin, which may result in a series of reciprocal cyclical processes. Because hypoalbuminemia, systemic inflammation, and oxidative stress are common in diabetic nephropathy patients, the aim of this study was to show that albumin modifications and neutrophil activation underlie these reciprocal systemic processes. Blood samples from a cohort of 19 patients with diabetic nephropathy and 15 healthy controls were used for albumin separation. An oxidation-dependent "albumin detection index," representing the detection efficacy of the universal bromocresol green assay, was determined for each subject. This index was correlated with serum albumin levels, various markers of oxidative stress or inflammation, and kidney function. Activation of separated neutrophils by glycoxidized albumin was assessed by the release of neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase (MPO). The albumin detection index of diabetic nephropathy patients was significantly lower compared to that of controls, correlating positively with serum levels of albumin and kidney function and negatively with albumin glycoxidation and inflammatory markers. Glycoxidized albumin had a direct role in neutrophil activation, resulting in NGAL and MPO release. The hypoalbuminemia observed in patients with diabetic nephropathy partially results from underestimation of modified/oxidized albumin using the bromocresol green assay. However, modified or oxidized albumin may lead to a cycle of accelerated oxidative stress and inflammation involving neutrophil activation. We suggest that the albumin detection index, a new marker of oxidative stress, may also serve as a biomarker of diabetic nephropathy severity and its progression.

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

    PubMed

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

    2015-08-14

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

  3. Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.

    PubMed

    Okamura, N; Sakai, T; Nishimura, Y; Sakai, M; Araki, S; Yamaguchi, M; Ishibashi, S

    1999-08-01

    NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

  4. Antithrombin reduces monocyte and neutrophil CD11b up regulation in addition to blocking platelet activation during extracorporeal circulation.

    PubMed

    Rinder, Christine S; Rinder, Henry M; Smith, Michael J; Fitch, Jane C K; Tracey, Jayne B; Chandler, Wayne L; Rollins, Scott A; Smith, Brian R

    2006-07-01

    Patients undergoing cardiac surgery requiring cardiopulmonary bypass develop a systemic inflammatory reaction. Antithrombin III (AT) has anticoagulant effects but also shows evidence of anti-inflammatory activity. The aim of this study was to examine whether exogenous AT could reduce white blood cell activation (CD11b up regulation or elastase release), in addition to inhibiting platelet (PLT) activation and fibrin generation, during simulated cardiopulmonary bypass (sCPB), undertaken in the absence of endothelium. sCPB was carried out with minimally heparinized (2 U/mL) human blood for 90 minutes in controls and with supplementation by low-dose (1 U/mL) and high-dose (5 U/mL) AT. High-dose AT blunted thrombin generation during sCPB (prothrombin fragment 1.2); both doses significantly inhibited thrombin activity (fibrinopeptide A). Complement activation (C3a and C5b-9) was unaffected by AT. High-dose AT inhibited PLT activation (P-selectin expression and P-selectin-dependent monocyte-PLT conjugate formation). AT supplementation at the higher dose significantly abrogated monocyte and neutrophil CD11b up regulation and neutrophil elastase release. In addition to anticoagulant and anti-PLT effects, pharmacologic AT doses significantly blunted monocyte and neutrophil CD11b up regulation and neutrophil elastase release during sCPB, independent of endothelial effects. These data provide evidence for the direct anti-inflammatory activity of AT that has clinical relevance for CPB complications.

  5. Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelling of the cytoskeleton.

    PubMed

    Anderson, Stephen I; Behrendt, Barbara; Machesky, Laura M; Insall, Robert H; Nash, Gerard B

    2003-02-01

    Neutrophils migrate rapidly by co-ordinating regulation of their beta2-integrin adhesion with turnover of filamentous F-actin. The seven-protein Arp2/3 complex regulates actin polymerisation upon activation by proteins of the WASP-family. To investigate links between actin polymerisation, adhesion, and migration, we used a novel osmotic-shock method to load neutrophils with peptides: (1). WASP-WA and Scar-WA (which incorporate the actin- and Arp2/3-binding regions of WASP and Scar1), to compete with endogenous WASP-family members; (2). proline rich motifs (PRM) from the ActA protein of L. monocytogenes or from vinculin, which bind vasodilator-stimulated phosphoprotein (VASP), a regulator of cytoskeleton assembly. In a flow system, rolling-adherent neutrophils were stimulated with formyl tri-peptide. This caused rapid immobilisation, followed by migration with increasing velocity, supported by activated beta2-integrin CD11b/CD18. Loading ActA PRM (but not vinculin PRM) caused concentration-dependent reduction in migration velocity. At the highest concentration, unstimulated neutrophils had elevated F-actin and were rigid, but could not change their F-actin content or shape upon stimulation. Scar-WA also caused marked reduction in migration rate, but WASP-WA had a lesser effect. Scar-WA did not modify activation-dependent formation of F-actin or change in shape. However, a reduction in rate of downregulation of integrin adhesion appeared to contribute to impaired migration. These studies show that interference in cytoskeletal reorganisation that follows activation in neutrophils, can impair regulation of integrin function as well as motility. They also suggest a role of the Arp2/3 complex and WASP-family in co-ordinating actin polymerisation and integrin function in migrating neutrophils. Copyright 2003 Wiley-Liss, Inc.

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

    PubMed Central

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

    2006-01-01

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

  7. Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis

    SciTech Connect

    Kato, Takayuki; Ikemoto, Masaru; Hato, Fumihiko; Kitagawa, Seiichi

    2009-04-10

    Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-{alpha} and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells. These findings suggest that granule membrane may become destabilized during neutrophil apoptosis, leading to rapid proteolysis of these molecules by granule-derived serine proteases during preparation of cell lysates with the conventional lysis buffer.

  8. Fever-range hyperthermia improves the anti-apoptotic effect induced by low pH on human neutrophils promoting a proangiogenic profile

    PubMed Central

    Díaz, Fernando Erra; Dantas, Ezequiel; Cabrera, Maia; Benítez, Constanza A; Delpino, María V; Duette, Gabriel; Rubione, Julia; Sanjuan, Norberto; Trevani, Analía S; Geffner, Jorge

    2016-01-01

    Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis, limiting their deleterious potential. However, this tightly regulated cell death program can be modulated by pathogen-associated molecular patterns (PAMPs), danger-associated molecular pattern (DAMPs), and inflammatory cytokines. We have previously reported that low pH, a hallmark of inflammatory processes and solid tumors, moderately delays neutrophil apoptosis. Here we show that fever-range hyperthermia accelerates the rate of neutrophil apoptosis at neutral pH but markedly increases neutrophil survival induced by low pH. Interestingly, an opposite effect was observed in lymphocytes; hyperthermia plus low pH prevents lymphocyte activation and promotes the death of lymphocytes and lymphoid cell lines. Analysis of the mechanisms through which hyperthermia plus low pH increased neutrophil survival revealed that hyperthermia further decreases cytosolic pH induced by extracellular acidosis. The fact that two Na+/H+ exchanger inhibitors, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and amiloride, reproduced the effects induced by hyperthermia suggested that it prolongs neutrophil survival by inhibiting the Na+/H+ antiporter. The neutrophil anti-apoptotic effect induced by PAMPs, DAMPs, and inflammatory cytokines usually leads to the preservation of the major neutrophil effector functions such as phagocytosis and reactive oxygen species (ROS) production. In contrast, our data revealed that the anti-apoptotic effect induced by low pH and hyperthermia induced a functional profile characterized by a low phagocytic activity, an impairment in ROS production and a high ability to suppress T-cell activation and to produce the angiogenic factors VEGF, IL-8, and the matrix metallopeptidase 9 (MMP-9). These results suggest that acting together fever and local acidosis might drive the differentiation of neutrophils into a profile able to promote both cancer progression and tissue repair during the

  9. Fever-range hyperthermia improves the anti-apoptotic effect induced by low pH on human neutrophils promoting a proangiogenic profile.

    PubMed

    Díaz, Fernando Erra; Dantas, Ezequiel; Cabrera, Maia; Benítez, Constanza A; Delpino, María V; Duette, Gabriel; Rubione, Julia; Sanjuan, Norberto; Trevani, Analía S; Geffner, Jorge

    2016-10-27

    Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis, limiting their deleterious potential. However, this tightly regulated cell death program can be modulated by pathogen-associated molecular patterns (PAMPs), danger-associated molecular pattern (DAMPs), and inflammatory cytokines. We have previously reported that low pH, a hallmark of inflammatory processes and solid tumors, moderately delays neutrophil apoptosis. Here we show that fever-range hyperthermia accelerates the rate of neutrophil apoptosis at neutral pH but markedly increases neutrophil survival induced by low pH. Interestingly, an opposite effect was observed in lymphocytes; hyperthermia plus low pH prevents lymphocyte activation and promotes the death of lymphocytes and lymphoid cell lines. Analysis of the mechanisms through which hyperthermia plus low pH increased neutrophil survival revealed that hyperthermia further decreases cytosolic pH induced by extracellular acidosis. The fact that two Na(+)/H(+) exchanger inhibitors, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and amiloride, reproduced the effects induced by hyperthermia suggested that it prolongs neutrophil survival by inhibiting the Na(+)/H(+) antiporter. The neutrophil anti-apoptotic effect induced by PAMPs, DAMPs, and inflammatory cytokines usually leads to the preservation of the major neutrophil effector functions such as phagocytosis and reactive oxygen species (ROS) production. In contrast, our data revealed that the anti-apoptotic effect induced by low pH and hyperthermia induced a functional profile characterized by a low phagocytic activity, an impairment in ROS production and a high ability to suppress T-cell activation and to produce the angiogenic factors VEGF, IL-8, and the matrix metallopeptidase 9 (MMP-9). These results suggest that acting together fever and local acidosis might drive the differentiation of neutrophils into a profile able to promote both cancer progression and tissue repair

  10. 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

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

    PubMed

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

    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.

  12. Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways.

    PubMed

    Fernández, Gabriela C; Ilarregui, Juan M; Rubel, Carolina J; Toscano, Marta A; Gómez, Sonia A; Beigier Bompadre, Macarena; Isturiz, Martín A; Rabinovich, Gabriel A; Palermo, Marina S

    2005-05-01

    Galectin-3 (Gal-3), a member of a family of highly conserved carbohydrate-binding proteins, has recently emerged as a novel cellular modulator at inflammatory foci. Here we investigated the effects of Gal-3 on central effector functions of human neutrophils, including phagocytosis, exocytosis of secretory granules, and survival. We examined the effects of Gal-3 alone or in combination with soluble fibrinogen (sFbg), an extracellular mediator that plays a key role during the early phase of the inflammatory response through binding to integrin receptors. In addition we evaluated the intracellular signals triggered by these mediators in human neutrophils. Human neutrophils incubated with recombinant Gal-3 alone increased their phagocytic activity and CD66 surface expression. In contrast to the known antiapoptotic effect of Gal-3 on many cellular types, Gal-3 enhanced PMN apoptotic rate. Preincubation with Gal-3 primed neutrophils to the effects of sFbg, resulting in a synergistic action on degranulation. On the other hand, Gal-3 and sFbg had opposite effects on PMN survival, and the simultaneous action of both agonists partially counteracted the proapoptotic effects of Gal-3. In addition, although sFbg induced its effects through the activation of the ERKs, Gal-3 led to p38 phosphorylation. Disruption of this signaling pathway abrogated Gal-3-mediated modulation of neutrophil degranulation, phagocytosis, and apoptosis. Together, our results support the notion that Gal-3 and sFbg are two physiological mediators present at inflammatory sites that activate different components of the MAPK pathway and could be acting in concert to modulate the functionality and life span of neutrophils.

  13. Interleukin-8 secretion and neutrophil recruitment accompanies induced sputum eosinophil activation in children with acute asthma.

    PubMed

    Norzila, M Z; Fakes, K; Henry, R L; Simpson, J; Gibson, P G

    2000-03-01

    Although airway inflammation is recognized as a key feature of asthma, the characteristics of airway inflammation in children with acute severe asthma are not well defined. The aim of this study was to describe the characteristics of airway inflammation in children with an acute exacerbation of asthma using sputum cell counts and fluid-phase measurements and to examine the changes in these parameters upon resolution of the exacerbation. Children (n = 38) presenting to the Emergency Department with acute asthma underwent successful sputum induction using ultrasonically nebulized normal saline (n = 22), or expectorated sputum spontaneously (n = 16). Sputum induction was repeated at least 2 wk later when the children had recovered (n = 28). Sputum portions were selected, dispersed and total and differential cell counts performed. Neutrophil elastase and EG2-positive eosinophils were assessed and fluid-phase eosinophil cationic protein (ECP), myeloperoxidase (MPO), interleukin-8 (IL-8), and IL-5 were measured. During the acute exacerbation the median (range) total cell count was 8.4 x 10(6)/ml (0.5 to 190.3), and fell significantly at resolution to 1.3 x 10(6)/ml (p < 0.01). The inflammatory cell infiltrate was mixed and included eosinophils (0.8 x 10(6)/ml), neutrophils (3.3 x 10(6)/ml), and mast cells. EG2(+) cells were high and correlated with the degree of airflow obstruction (r = -0.5, p = 0.02). They decreased significantly at resolution as did supernatant ECP (1,078 versus 272 ng/ml), suggesting that eosinophils were activated during the exacerbation. MPO was 220 ng/ ml at exacerbation and fell significantly to 1 ng/ml at resolution. Levels of IL-8 and IL-5 were elevated during the acute exacerbation and IL-8 concentrations decreased at resolution. In conclusion, airway inflammation can be studied in children with acute asthma by sputum induction. Airway inflammation is present during an acute exacerbation of asthma, and is characterized by infiltration and

  14. 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.

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

    PubMed Central

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

    2011-01-01

    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 was dependent upon 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 ICAM-1 is locally expressed and actively participates in this process. Injured gp91phox 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

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

    PubMed

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

    2014-03-01

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

  17. Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

    PubMed Central

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

    2014-01-01

    All cultivated ammonia-oxidizing archaea (AOA) within the Nitrososphaera cluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence of Nitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth of Nitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis of amoA genes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that 13CO2 assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both 13C-labeled amoA and 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strains Nitrososphaera viennensis EN76 and JG1 within the Nitrososphaera cluster. Our results provide strong evidence for the adaptive growth of Nitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated. PMID:24375137

  18. 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

    2011-10-09

    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

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

    PubMed

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

    2011-01-01

    OBJECTIVE. The aim of the study was to establish the dynamics of β-glucuronidase activity in subjects suffering from type 1 diabetes and chronic untreated generalized periodontitis, subjects suffering from chronic untreated generalized periodontitis only, and control subjects not suffering from generic diseases with healthy periodontal tissue. MATERIAL AND METHODS. The study involved 165 19-50-year-old subjects who were divided into three groups: healthy subjects (n=55), subjects with chronic untreated generalized periodontitis (n=55), and subjects with type 1 diabetes and chronic untreated generalized periodontitis (n=55). Neutrophilic leukocytes of peripheral venous blood were exposed to bacterial stimuli: opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan. The activity of β-glucuronidase was determined by the spectrofluorimetry method. RESULTS. The diagnostic value of changes in β-glucuronidase activity of neutrophilic leukocytes markedly increased in all study groups after stimulation of neutrophilic leukocytes by opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan as compared to control media not exposed to any stimulus (P<0.001). The strongest relationship (canonical correlation coefficient eta, 0.993) between the intensity of periodontal pathology markers and the activity of β-glucuronidase of neutrophilic leukocytes in incubated media in patients with type 1 diabetes mellitus and periodontitis was found under the effect of nonopsonized Staphylococcus aureus. CONCLUSIONS. If periodontal impairment is severe, diabetes mellitus possibly causes a faster destruction of the periodontal tissue and presents a higher risk of periodontitis for patients with diabetes.

  20. Histamine up-regulates phosphodiesterase 4 activity and reduces prostaglandin E2-inhibitory effects in human neutrophils.

    PubMed

    Dasí, F J; Ortiz, J L; Cortijo, J; Morcillo, E J

    2000-11-01

    To investigate whether histamine produces up-regulation of phosphodiesterase (PDE) activity with functional consequences in human peripheral blood neutrophils. PDE activity was studied by a radioisotopic method following anion-exchange chromatography. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of mRNA transcripts of PDE4 subtypes. Cyclic AMP (cAMP) levels were measured by enzyme-immunoassay, and superoxide generation by cytochrome c reduction. Neutrophils were incubated for 4 h with histamine (1 microM). PDE4 was the only isoenzyme activity increased in treated neutrophils. Kinetic analysis showed a approximately 1.5-fold increase in Vmax without alteration of Km values. cAMP content in treated cells was higher than resting values (0.52+/-0.07 vs. 2.75+/-0.31 pmol/10(6) cells). RT-PCR showed increased expression of mRNA transcripts for PDE4B in histamine-treated cells. Functionally, up-regulation of PDE4 reduced the inhibition by prostaglandin E2 of zymosan-induced superoxide generation. Histamine up-regulates PDE4 activity and produces heterologous desensitisation of human neutrophils.

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

    PubMed Central

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

    1990-01-01

    Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria. Images PMID:2254020

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

    PubMed Central

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

    2017-01-01

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

  3. 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.

  4. 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.

  5. Human neutrophil-mediated fungistasis against Histoplasma capsulatum. Localization of fungistatic activity to the azurophil granules.

    PubMed Central

    Newman, S L; Gootee, L; Gabay, J E

    1993-01-01

    Human neutrophils (PMN) demonstrated potent fungistatic activity against Histoplasma capsulatum (Hc) yeasts in a sensitive microassay that quantifies the growth of yeasts by the incorporation of [3H]leucine. At a PMN:yeast ratio of 1:2, PMN inhibited the growth of yeasts by 37%. Maximum inhibition of 85% to 95% was achieved at a PMN/yeast ratio of 10:1 to 50:1. Opsonization of the yeasts in fresh or heat-inactivated serum was required for PMN-mediated fungistasis, but ingestion of the yeasts was not required. Recognition and phagocytosis of opsonized yeasts was via PMN complement receptor (CR) type 1 (CR1), CR3, and FcRIII (CD16). PMN fungistatic activity was evident by 2 h, was maximum at 24 h, and persisted up to 5 d. In contrast, yeasts multiplied within monocytes to a greater extent than in culture medium alone. PMN from three patients with chronic granulomatous disease (CGD) inhibited the growth of Hc yeasts by an average of 97%, compared with 86% in three normal controls. Furthermore, preincubation of PMN with the lysosomotropic agent NH4Cl inhibited fungistatic activity in a concentration-dependent manner. Finally, experiments with subcellular fractions of PMN demonstrated that the principal component of the fungistatic activity of PMN was localized in the azurophil granules. These data demonstrate that human PMN possess potent fungistatic activity against Hc yeasts and further show that fungistasis is mediated by antimicrobial agents contained in the azurophil granules. PMID:8349801

  6. Poly(vinyl alcohol)-coated silver nanoparticles: activation of neutrophils and nanotoxicology effects in human hepatocarcinoma and mononuclear cells.

    PubMed

    Paino, Iêda Maria Martinez; Zucolotto, Valtencir

    2015-03-01

    Silver nanoparticles (AgNps) have been described as important for their excellent biocompatibility, biomedical applications. Nevertheless, AgNps can interact with the immune system which is essential to analyze human exposure to assess their potential risk to health and environment. In general, the primary site for accumulation of nanoparticles has been demonstrated to be the liver. Furthermore, the direct activation of neutrophils or oxidative burst by a given nanoparticle is poorly documented. In this paper, we investigated the cell uptake, apoptosis, necrosis, DNA damage in human hepatocarcinoma cells (HepG2), primary normal human peripheral blood mononuclear cells (PBMC) and the direct activation of primary isolated neutrophils through the oxidative burst on exposure to AgNps coated with Polyvinyl-alcohol (PVA). All cell types were incubated in the presence of 1.0 and 50.0 μM of AgNps-PVA for 24h. A significant cyto- and genotoxic-response and the activation of human neutrophils were induced by AgNps-PVA (p<0.05). Our results revealed that AgNps can interact with the normal isolated neutrophils, PBMC and HepG2 cells in vitro, which opens the way for further studies on the toxicological effects of AgNps in the human immune system response and cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Activated neutrophils injure the isolated, perfused rat liver by an oxygen radical-dependent mechanism.

    PubMed Central

    Dahm, L. J.; Schultze, A. E.; Roth, R. A.

    1991-01-01

    Under certain circumstances, segmented neutrophils (PMNs) injure extrahepatic tissue by releasing toxic oxygen species and degradative enzymes. The authors used an isolated, perfused rat liver preparation to determine whether PMNs might injure the liver. Livers from fasted rats were perfused with Krebs-Ringer bicarbonate buffer (pH 7.4) containing 3% bovine serum albumin (BSA) in a recirculating system. Rat peritoneal PMNs (4 x 10(8] or vehicle (Hank's balanced salt solution [HBSS], pH 7.35) were added, and liver injury was assessed 90 minutes later by release of alanine aminotransferase (ALT) into the perfusion medium and histopathologic analysis of liver sections. Perfusion of livers receiving only HBSS for 90 minutes resulted in a small increase in ALT activity in the perfusion medium but did not significantly alter histologic features of liver sections. Addition of unstimulated PMNs did not increase further the ALT activity and, with the exception of vascular neutrophilia, did not significantly change the histomorphology compared with controls. When PMNs activated with a combination of phorbol myristate acetate (PMA, 31 ng/ml) and lithocholate (100 mumol/l [micromolar]) were added to the perfusion system, however, livers released greater amounts of ALT than those perfused with PMA, lithocholate, and HBSS. Activated PMNs caused a transient reduction in flow of perfusion medium that lasted approximately 5 to 15 minutes. Liver sections had multifocal to coalescing foci of moderate to severe, acute hepatocellular necrosis associated with the areas of intense sinusoidal neutrophilia. In addition a second type of lesion was observed and was characterized by triangular foci of necrosis located adjacent to periportal regions of sinusoids or portal veins containing neutrophilic thrombi. These lesions were void of PMNs and were consistent with infarcts. A combination of superoxide dismutase and catalase added to the perfusion medium (500 U/ml each) prevented the

  8. TLR8, but not TLR7, induces the priming of the NADPH oxidase activation in human neutrophils.

    PubMed

    Makni-Maalej, Karama; Marzaioli, Viviana; Boussetta, Tarek; Belambri, Sahra Amel; Gougerot-Pocidalo, Marie-Anne; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

    2015-06-01

    Neutrophils play a key role in host defense against invading pathogens by releasing toxic agents, such as ROS and antimicrobial peptides. Human neutrophils express several TLRs that recognize a variety of microbial motifs. The interaction between TLR and their agonists is believed to help neutrophils to recognize and to kill pathogens efficiently by increasing their activation, a process called priming. However, excessive activation can induce tissue injury and thereby, contribute to inflammatory disorders. Agonists that activate TLR7 and TLR8 induce priming of neutrophil ROS production; however, which receptor is involved in this process has not been elucidated. In this study, we show that the selective TLR8 agonist, CL075 (3M002), induced a dramatic increase of fMLF-stimulated NOX2 activation, whereas the selective TLR7 agonist, loxoribine, failed to induce any priming effect. Interestingly, CL075, but not loxoribine, induced the phosphorylation of the NOX2 cytosolic component p47phox on several serines and the phosphorylation of p38MAPK and ERK1/2. The inhibitor of p38MAPK completely blocked CL075-induced phosphorylation of p47phox Ser345. Moreover, CL075, but not loxoribine, induced the activation of the proline isomerase Pin1, and juglone, a Pin1 inhibitor, prevented CL075-mediated priming of fMLF-induced superoxide production. These results indicate that TLR8, but not TLR7, is involved in priming of human neutrophil ROS production by inducing the phosphorylation of p47phox and p38MAPK and that Pin1 is also involved in this process. © Society for Leukocyte Biology.

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

    PubMed Central

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

    1998-01-01

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

  10. The impact of platelet-activating factor (PAF)-like mediators on the functional activity of neutrophils: anti-inflammatory effects of human PAF-acetylhydrolase

    PubMed Central

    Kuijpers, T W; Van Den Berg1, J M; Tool, A T J; Roos, D

    2001-01-01

    Platelet-activating factor (PAF) is a proinflammatory agent in infectious and inflammatory diseases, partly due to the activation of infiltrating phagocytes. PAF exerts its actions after binding to a monospecific PAF receptor (PAFR). The potent bioactivity is reflected by its ability to activate neutrophils at picomolar concentrations, as defined by changes in levels of intracellular Ca2+ ([Ca2+]i), and induction of chemotaxis and actin polymerization at nanomolar concentration. The role of PAF in neutrophil survival is, however, less well appreciated. In this study, the inhibitory effects of synthetic PAFR-antagonists on various neutrophil functions were compared with the effect of recombinant human plasma-derived PAF-acetylhydrolase (rPAF-AH), as an important enzyme for PAF degradation in blood and extracellular fluids. We found that endogenously produced PAF (–like) substances were involved in the spontaneous apoptosis of neutrophils. At concentrations of 8 µg/ml or higher than normal plasma levels, rPAF-AH prevented spontaneous neutrophil apoptosis (21 ± 4% of surviving cells (mean ± SD; control) versus 62 ± 12% of surviving cells (mean ± SD; rPAF-AH 20 µg/ml); P < 0·01), during overnight cultures of 15 h. This effect depended on intact enzymatic activity of rPAF-AH and was not due to the resulting product lyso-PAF. The anti-inflammatory activity of rPAF-AH toward neutrophils was substantiated by its inhibition of PAF-induced chemotaxis and changes in [Ca2+]i. In conclusion, the efficient and stable enzymatic activity of rPAF-AH over so many hours of coculture with neutrophils demonstrates the potential for its use in the many inflammatory processes in which PAF (–like) substances are believed to be involved. PMID:11298128

  11. In vitro model of intestinal crypt abscess. A novel neutrophil-derived secretagogue activity.

    PubMed Central

    Nash, S; Parkos, C; Nusrat, A; Delp, C; Madara, J L

    1991-01-01

    In order to model crypt abscesses, a histological finding which correlates with disease activity in intestinal inflammation, human polymorphonuclear leukocytes (PMN) were layered onto monolayers of the human intestinal epithelial cell line T84, a crypt-like epithelium which is capable of Cl- secretion. Such PMN-epithelial interaction had no substantial effect on monolayer integrity or function. However, when PMN were stimulated by conditions including those present naturally in the human colonic lumen, monolayers responded with a bumetanide-sensitive short circuit current (Isc) indicative of Cl- secretion, the basis of secretory diarrhea. This Isc response was induced by a neutrophil-derived secretagogue (NDS), which was only active when applied to the luminal surface of monolayers and did not require PMN-epithelial contact. NDS activity is resistant to boiling, acid, and trypsin and passes a 500 nominal mol wt cutoff filter. NDS activity is not secondary to the respiratory burst products O2- or H2O2 and does not appear to be a myeloperoxidase product. We speculate NDS elicited Cl- secretion may contribute to the secretory diarrhea seen in patients with intestinal inflammation and crypt abscesses. PMID:2010557

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

    PubMed

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

    2005-11-01

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

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

    PubMed Central

    Kangne, Harshada K.; Jijina, Farah F.; Italia, Yazdi M.; Jain, Dipti L.; Nadkarni, Anita H.; Gupta, Maya; Pradhan, Vandana; Mukesh, Rati D.; Ghosh, Kanjaksha K.; Colah, Roshan B.

    2013-01-01

    Objective. Sickle cell disease has variable clinical manifestations. Activation of neutrophils plays an important role in the initiation and propagation of vaso occlusive crises which can be analysed by determining the expression of neutrophil antigens such as CD16, CD32, and CD62L. The common FcγR polymorphisms (FcγRIIA and FcγRIIIB) are considered to influence clinical presentation. This study focuses on distribution of FcγR polymorphisms and their association with neutrophil activity among the patients from western India. Methods. In this paper 127 sickle cell anemia patients and 58 patients with sickle-β-thalassemia (median age 12 ± 8.58 years) with variable clinical phenotypes along with 175 normals were investigated. FcγRs polymorphisms were analysed by RFLP and AS-PCR. Activation of neutrophils was measured by flow cytometry. Results. The genotypic frequency of the H/R genotype of FcγRIIA and the NA1/NA1 genotype of FcγRIIIB was significantly decreased in patients compared to normals (P-0.0074, P-0.0471, resp.). We found a significant difference in the expression of CD32 and CD62L among the patients as against normals. A significantly higher expression of CD32 was seen in the milder patients with the H/H genotype (P-0.0231), whereas the expression of CD16 was higher in severe patients with the NA2/NA2 genotype (P-0.0312). Conclusion. The two FcγR polymorphisms had significant association with variable phenotypes of sickle cell disease. The expression of CD62L decreased in our patients indicating activation of neutrophils. PMID:24191245

  14. Inefficiency of C3H/HeN Mice to Control Chlamydial Lung Infection Correlates with Downregulation of Neutrophil Activation During the Late Stage of Infection

    PubMed Central

    Tang, Xiaofei; Bu, Xiaokun; Zhang, Naihong; Li, Xiaoxia; Huang, Huanjun; Bai, Hong; Yang, Xi

    2009-01-01

    We previously reported that massive infiltration of neutrophils in C3H/HeN (C3H) mice could not efficiently control Chlamydia muridarum (Cm) infection and might contribute to the high susceptibility of these mice to lung infection. To further define the nature of neutrophil responses in C3H mice during chlamydial infection, we examine the expression of adhesion molecules and CD11b related to neutrophils infiltration and activation, respectively, following intranasal Cm infection. The results showed that the expression of selectins (E-selectin, P-selectin and L-selectin), and intercellular cell adhesion molecule-1 (ICAM-1) in the lung of C3H mice increased more significantly than in C57BL/6 (B6) mice, the more resistant strain. These results correlated well with the massive neutrophils infiltration in C3H mice. In contrast, CD11b expression on peripheral blood and lung neutrophils in C3H mice exhibited a significant reduction compared with B6 mice during the late phage of infection (day 14). These findings suggest that the high-level expression of adhesion molecules in C3H mice may enhance neutrophils recruitment to the lung, but the decline of CD11b expression on neutrophils may attenuate neutrophil function. Therefore, CD11b down-regulation on neutrophils may contribute to the failure of C3H mice to control chlamydial lung infection. PMID:19728926

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. [The effect of infra-low frequency alternating magnetic field on the functional activity of blood neutrophils from rats with limited mobility].

    PubMed

    Temur'iants, N A; Mikhaĭlov, A V

    1988-01-01

    The influence of 8 Hz frequency alternating magnetic field has been investigated by 5 mcTl induction on the functional state of bacteroid systems, hydrolytic enzymes and phagocytic activity of rat blood neutrophils in different periods of hypokinesia. It was found that the alternating magnetic field of above mentioned parameters promotes normalization of rat neutrophils indicators during hypokinesia.

  17. Adhesive Dynamics Simulation of G-Protein-Mediated Chemokine-Activated Neutrophil Adhesion

    PubMed Central

    Caputo, Kelly E.; Hammer, Daniel A.

    2009-01-01

    Abstract To reach sites of inflammation, a blood-borne neutrophil first rolls over the vessel wall, becoming firmly adherent on activation, and then transmigrates through the endothelium. In this study, we simulate the transition to firm adhesion via chemokine-induced integrin activation. To recreate the transition from rolling to firm adhesion, we use an integrated signaling adhesive dynamics simulation that includes selectin, integrin, and chemokine interactions between the cell and an adhesive substrate. Integrin bonds are of low affinity until activated by chemokine binding to G-protein coupled receptors on the model cell. The signal propagates within the cell through probabilistic diffusion and reaction of the signaling elements to induce the high-affinity integrins required for firm adhesion. This model showed that integrins become progressively active as cells roll and interact with chemokines, leading to a slight slowing before firm adhesion on a timescale similar to that observed in experiments. Increasing the density of chemokine resulted in decreases in the rolling time before stopping, consistent with experimental observations. However, a limit is reached where further increases in chemokine density do not increase adhesion. We found that the timescale for integrin activation correlated with the time to stop. Further, altering parameters within the intracellular signaling cascade that changed the speed of integrin activation, such as effector activation and dissociation rates, correspondingly affected the time to firm adhesion. For all conditions tested, the number of active integrin bonds at the point of firm adhesion was relatively constant. The model predicts that the time to stop would be relatively independent of selectin or integrin density, but strongly dependent on the shear rate because higher shear rates limit the intrinsic activation rate of integrins and require more integrins for adhesion. PMID:19383446

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

    PubMed

    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

    2015-07-07

    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.

  19. 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

    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

    2015-02-15

    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 present 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 we showed that Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (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 (YVAD-FLICA 660), and bone marrow neutrophils stimulated with heat-killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks when stained for NLRP3, ASC, or Caspase-1. High-molecular mass 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. Taken together, these findings demonstrate an essential role for neutrophil-derived IL-1β in S. pneumoniae infection, and they elucidate the role of the NLRP3 inflammasome in cleavage and secretion of IL-1β in neutrophils. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases. Copyright © 2015 by The American Association of Immunologists, Inc.

  20. Mast Cell IL-6 Improves Survival From Klebsiella Pneumonia and Sepsis by Enhancing Neutrophil Killing

    PubMed Central

    Sutherland, Rachel E.; Olsen, Joanna S.; McKinstry, Andrew; Villalta, S. Armando; Wolters, Paul J.

    2008-01-01

    The pleiotropic cytokine interleukin 6 (IL-6) has favorable and harmful effects on survival from bacterial infections. While many innate immune cells produce IL-6, little is known about relevant sources in vivo and the nature of its contributions to host responses to severe bacterial infections. To examine these roles, we subjected mast cell-specific IL-6-deficient mice to the cecal ligation and puncture model of septic peritonitis, finding that survival in these mice is markedly worse than in controls. Following intranasal or intraperitoneal inoculation with Klebsiella pneumoniae, IL-6-/- mice are less likely to survive than wild-type controls and at the time of death have higher numbers of bacteria but not inflammatory cells in lungs and peritoneum. Similarly, mast cell-specific IL-6-deficient mice have diminished survival and higher numbers of K. pneumoniae following intraperitoneal infection. Neutrophils lacking IL-6 have greater numbers of live intracellular K. pneumonia, suggesting impaired intracellular killing contributes to reduced clearance in IL-6-/- mice. These results establish that mast cell IL-6 is a critical mediator of survival following K. pneumoniae infection and sepsis and suggest that IL-6 protects from death by augmenting neutrophil killing of bacteria. PMID:18832718

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

    PubMed

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

    2015-09-01

    The cell nucleus plays a key role in differentiation processes in eukaryotic cells. It is not the nucleus in particular, but the organization of the genes and their remodeling that provides the data for the adjustments to be made according to the medium. The neutrophil nucleus has a different morphology. It is a multi-lobed nucleus where some researchers argue no longer function. However, studies indicate that it is very probable the occurrence of chromatin remodeling during activation steps. It may be that the human neutrophil nucleus also contributes to the mobility of neutrophils through thin tissue spaces. Questions like these will be discussed in this small review. The topics include morphology of human neutrophil nucleus, maturation process and modifications of the neutrophil nucleus, neutrophil activation and chromatin modifications, causes and consequences of multi-lobulated segmented morphology, and importance of the nucleus in the formation of neutrophil extracellular traps (NETs). © 2015 Wiley Periodicals, Inc.

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

    PubMed

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

    2009-10-01

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

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

    SciTech Connect

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

    1986-03-05

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

  4. High frequency oscillatory ventilation attenuates the activation of alveolar macrophages and neutrophils in lung injury.

    PubMed

    Shimaoka; Fujino; Taenaka; Hiroi; Kiyono; Yoshiya

    1998-01-01

    BACKGROUND: Recent investigations have shown that leukocyte activation is involved in the pathogenesis of ventilator-associated lung injury. This study was designed to investigate whether the inflammatory responses and deterioration of oxygenation in ventilator-associated lung injury are attenuated by high-frequency oscillatory ventilation (HFO). We analyzed the effects of HFO compared with conventional mechanical ventilation (CMV) on the activation of pulmonary macrophages and neutrophils in 10 female rabbits. RESULTS: After surfactant depletion, the rabbits were ventilated by CMV or HFO at the same mean airway pressure. Surfactant-depletion followed by 4 h mechanical ventilation hindered pulmonary oxygenation in both groups. Impairment of oxygenation was less severe in the HFO group than in the CMV group. In the HFO group the infiltration of granulocytes into alveolar spaces occurred more readily than in the CMV group. Compared with CMV, HFO resulted in greater attenuation of beta2-integrin expression, not only on granulocytes, but also on macrophages. CONCLUSIONS: In the surfactant-depleted lung, the activation of leukocytes was attenuated by HFO. Reduced inflammatory response correlated with decreased impairment of oxygenation. HFO may reduce lung injury via the attenuation of pulmonary inflammation.

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

    PubMed

    Shenkar, R; Abraham, E

    1999-07-15

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

  6. 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

  7. Hypochlorous acid generated by neutrophils inactivates ADAMTS13: an oxidative mechanism for regulating ADAMTS13 proteolytic activity during inflammation.

    PubMed

    Wang, Yi; Chen, Junmei; Ling, Minhua; López, José A; Chung, Dominic W; Fu, Xiaoyun

    2015-01-16

    ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H2O2-Cl(-) system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H2O2 reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met(249), Met(331), and Met(496) in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.

  8. 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.

  9. 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

  10. Exacerbation of experimental autoimmune encephalomyelitis in ceramide synthase 6 knockout mice is associated with enhanced activation/migration of neutrophils.

    PubMed

    Eberle, Max; Ebel, Philipp; Mayer, Christoph A; Barthelmes, Julia; Tafferner, Nadja; Ferreiros, Nerea; Ulshöfer, Thomas; Henke, Marina; Foerch, Christian; de Bazo, Anika Männer; Grösch, Sabine; Geisslinger, Gerd; Willecke, Klaus; Schiffmann, Susanne

    2015-10-01

    Ceramides are mediators of inflammatory processes. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that CerS6 mRNA expression was upregulated 15-fold in peripheral blood leukocytes before the onset of EAE symptoms. In peripheral blood leukocytes from MS patients, a 3.9-fold upregulation was found. Total genetic deletion of CerS6 and the selective deletion of CerS6 in peripheral blood leucocytes exacerbated the progression of clinical symptoms in EAE mice. This was associated with enhanced leukocyte, predominantly neutrophil infiltration and enhanced demyelination in the lumbar spinal cord of EAE mice. Interferon-gamma/tumor necrosis factor alpha (IFN-γ/TNF-α) and granulocyte colony-stimulating factor (G-CSF) both drive EAE development and induce expression of the integrin CD11b and the chemokine receptor C-X-C motif chemokine receptor 2 (CXCR2), and we found they also induce CerS6 expression. In vivo, the genetic deletion of CerS6 enhanced the activation/migration of neutrophils, as reflected by an enhanced upregulation of CD11b and CXCR2. In vitro, the genetic deletion of CerS6 enhanced the activation status of IFN-γ/TNF-α-stimulated neutrophils, as shown by increased expression of nitric oxide and CD11b and an increased adhesion capacity. In G-CSF-stimulated neutrophils, the migration status was enhanced, as reflected by an elevated level of CXCR2 and an increased migration capacity. These data suggest that CerS6/C16-Cer mediates feedback regulation by inhibiting the formation of CD11b and CXCR2, which are induced either by IFN-γ/TNF-α or by G-CSF, respectively. We conclude that CerS6/C16-Cer mediates anti-inflammatory effects during the development of EAE and MS possibly by suppressing the migration and deactivation of neutrophils.

  11. 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.

  12. 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.

  13. Pegfilgrastim administered in an abbreviated schedule, significantly improved neutrophil recovery after high-dose radiation-induced myelosuppression in rhesus macaques.

    PubMed

    Farese, A M; Cohen, M V; Stead, R B; Jackson, W; Macvittie, T J

    2012-11-01

    Conventional daily administration of filgrastim is effective in reducing the duration of severe neutropenia and enhancing survival following lethal radiation, myelosuppressive cytotoxic therapy or myeloablation and stem cell transplantation. A sustained-duration form of filgrastim, pegfilgrastim has significantly simplified scheduling protocols after chemotherapy-induced neutropenia to a single injection while maintaining the therapeutic effectiveness of daily administration of filgrastim. We examined the ability of a single or double (weekly) administration of pegfilgrastim to significantly improve neutrophil recovery in a rhesus macaque model of severe radiation-induced myelosuppression. Animals were exposed to potentially lethal 6 Gy total-body X radiation. After irradiation all animals received supportive care and were administered either pegfilgrastim at 300 μg/kg on day 1 or day 1 and day 7 post exposure, or filgrastim at 10 μg/kg/day initiated on day 1 post exposure and continued daily through neutrophil recovery. Pharmacokinetic parameters and neutrophil-related values for duration of neutropenia, neutrophil nadir, time to recovery to an absolute neutrophil count ≥500/μL or ≥2000/μL, and days of antibiotic support were determined. Effective plasma concentrations of pegfilgrastim were maintained in neutropenic animals until after the onset of hematopoietic recovery, which is consistent with neutrophil-dependent properties of elimination. Administration of pegfilgrastim at day 1 and day 7 was most effective at improving neutrophil recovery compared to daily administration of filgrastim or a single injection of pegfilgrastim on day 1, after severe, radiation-induced myelosuppression in rhesus macaques.

  14. Effects of recombinant bactericidal/permeability-increasing protein (rBPI23) on neutrophil activity in burned rats.

    PubMed

    Hansbrough, J; Tenenhaus, M; Wikstrom, T; Braide, M; Rennekampff, O H; Kiessig, V; Bjursten, L M

    1996-06-01

    Bactericidal/permeability-increasing protein (BPI) is a neutrophil granule protein with potent bactericidal and lipopolysaccharide (LPS)-neutralizing activities. The purpose of this study was to determine if a human recombinant BPI product, rBPI23, would influence neutrophil (PMN) sequestration into various tissues in a rat burn injury model. Leukosequestration may produce local tissue injury from proteases and high-energy oxygen species released from PMNs. Rats received tracheostomy and venous cannulation, then received 17 to 20% total body surface area full-thickness contact burns and resuscitation with 20 ml, of intraperitoneal saline. Ten mg/kg body weight rBPI23 in saline was given by intravenous injection immediately after burn injury, followed by intravenous doses of 2 mg/kg at 2 and 4 hours. Control animals received intravenous saline only. PMN retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (before burn injury) and differentially radiolabeling PMNs (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hours after burn injury, and measuring tissue radioactivity 30 minutes later. Edema was estimated by measuring extravasated 125I-labeled albumin in the various tissues, 30 minutes after injection. Peripheral blood PMNS were analyzed for intracellular H2O2 content by flow cytometry using a fluorescent dye that reacts with H2O2. Radioisotope studies demonstrated significant (p < 0.05) leukosequestration into lung, liver, gut, kidney, and skin tissues at 5 hours after burn injury. Tissue edema, manifested by radiolabeled albumin retention, was not observed in any tissues. Postburn PMN deposition in lungs and skin was decreased (p < 0.05) by the immediate administration of rBPI23 after burn injury. Flow cytometry showed increased intracellular H2O2 content in peripheral blood PMNs 5 hours after burn injury (p < 0.05), which was unaffected by administration of rBPI23. Since sequestration of metabolically

  15. Lung sequestration in vivo of activated, tritium-labeled rabbit neutrophils

    SciTech Connect

    Lafuze, J.; Baker, M.; Oakes, A.; Landes, C.; Gasta, T.P.

    1986-03-01

    Lung endothelium may be damaged as it interacts with blood neutrophils (PMN) activated by certain bacterial filtrates. N-formyl-methionyl-leucyl-phenylalanine (FMLP) synthetic analog of E. coli endotoxin, causes transient neutropenia and respiratory distress when infused i.v. in rabbits. In this study the authors determined lung, spleen, liver and kidney sequestration of activated PMN. The authors transfused blood from a donor pre-treated with 3 mCi of (/sup 3/H)-thymidine into test and control (CON) rabbits. PMN were activated with 0.2 ug/Kg of FMLP i.v. Lungs, spleen, liver and kidneys were removed at 2.5 min. and were homogenized. Counts of 100 mg (wet weight) of each organ minus dpm of 100 mg CON were compared. Results were: lung 1242, spleen 448, kidney 235 and liver 209 (n = 10). Values for lung were significantly different from spleen (p < .03), kidney (p < .01) and liver (p < .01). Using the same model, the authors infused FMLP or vehicle and withdrew 6 ml venous blood at intervals. Test and CON values were compared. Each was calculated as percent change from time zero. Results were: at 2.5 min (+ FMLP) - 95% vs (CON) - 1% (p < .0001), at 20 min -69 vs -24 (p < .01), at 40 min -52 vs -19 (p < .01), and at 60 min -27 vs -25 (n.s.). These results indicate that on a per unit wt basis the lung sequestration of FMLP-activated PMN in vivo is significantly greater than that of spleen, kidney or liver and that the same cells sequestered are returned to the peripheral circulating pool.

  16. 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

  17. 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

  18. Role of platelet-activating factor in polymorphonuclear neutrophil recruitment in reperfused ischemic rabbit heart.

    PubMed Central

    Montrucchio, G.; Alloatti, G.; Mariano, F.; Comino, A.; Cacace, G.; Polloni, R.; De Filippi, P. G.; Emanuelli, G.; Camussi, G.

    1993-01-01

    This study investigated the role of platelet-activating factor in the recruitment of polymorphonuclear neutrophils (PMN) in a rabbit model of cardiac ischemia and reperfusion. The accumulation of PMN was evaluated 2 and 24 hours after removal of 40 minutes of coronary occlusion by morphometric analysis and 111In-labeled PMN infiltration. The administration of two structurally unrelated platelet-activating factor-receptor antagonists (SDZ 63-675, 5 mg/kg body weight, and WEB 2170, 5 mg/kg body weight) before reperfusion significantly reduced the accumulation of PMN, as well as the hemodynamic alterations and the size of necrotic area. Two hours after reperfusion, the percentage of increase of 111In-labeled PMN in transmural central ischemic zone was significantly reduced in rabbits pretreated with SDZ 63-675 (51.4 +/- 7.9) or WEB 2170 (32.4 +/- 8.8) with respect to untreated rabbits (107.6 +/- 13.5). The morphometric analysis of myocardial sections confirmed the reduction of PMN infiltration at 2 hours and demonstrated that at 24 hours the phenomenon was even more significant. In addition, SDZ 63-675 and WEB 2170 prevented early transient bradycardia and hypotension and reduced the infarct size, judged by staining with tetrazolium at 2 and 24 hours after reperfusion, and by histological examination at 24 hours. These results suggest that platelet-activating factor is involved in the accumulation of PMN in the reperfused ischemic myocardium and contributes to the evolution of myocardial injury. Images Figure 5 Figure 6 PMID:8434642

  19. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells.

    PubMed

    Blomgran, Robert; Desvignes, Ludovic; Briken, Volker; Ernst, Joel D

    2012-01-19

    Mycobacterium tuberculosis promotes its replication by inhibiting the apoptosis of infected macrophages. A proapoptotic M. tuberculosis mutant lacking nuoG, a subunit of the type I NADH dehydrogenase complex, exhibits attenuated growth in vivo, indicating that this virulence mechanism is essential. We show that M. tuberculosis also suppresses neutrophil apoptosis. Compared to wild-type, the nuoG mutant spread to a larger number of lung phagocytic cells. Consistent with the shorter lifespan of infected neutrophils, infection with the nuoG mutant resulted in fewer bacteria per infected neutrophil, accelerated bacterial acquisition by dendritic cells, earlier trafficking of these dendritic cells to lymph nodes, and faster CD4 T cell priming. Neutrophil depletion abrogated accelerated CD4 T cell priming by the nuoG mutant, suggesting that inhibiting neutrophil apoptosis delays adaptive immunity in tuberculosis. Thus, pathogen modulation of apoptosis is beneficial at multiple levels, and enhancing phagocyte apoptosis promotes CD4 as well as CD8 T cell responses.

  20. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells

    PubMed Central

    Blomgran, Robert; Desvignes, Ludovic; Briken, Volker; Ernst, Joel D.

    2012-01-01

    Summary Mycobacterium tuberculosis promotes its replication by inhibiting the apoptosis of infected macrophages. A proapoptotic M. tuberculosis mutant lacking nuoG, a subunit of the type I NADH dehydrogenase complex, exhibits attenuated growth in vivo, indicating that this virulence mechanism is essential. We show that M. tuberculosis also suppresses neutrophil apoptosis. Compared to wild-type, the nuoG mutant spread to a larger number of lung phagocytic cells. Consistent with the shorter lifespan of infected neutrophils, infection with the nuoG mutant resulted in fewer bacteria per infected neutrophil, accelerated bacterial acquisition by dendritic cells, earlier trafficking of these dendritic cells to lymph nodes, and faster CD4 T cell priming. Neutrophil depletion abrogated accelerated CD4 T cell priming by the nuoG mutant, suggesting that inhibiting neutrophil apoptosis delays adaptive immunity in tuberculosis. Thus, pathogen modulation of apoptosis is beneficial at multiple levels, and enhancing phagocyte apoptosis promotes CD4 as well as CD8 T cell responses. PMID:22264515

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

    Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits. PMID:27907124

  3. [Chemotactic activity of neutrophils from atopic and non-atopic subjects--effect of sodium cromoglycate (DSCG)].

    PubMed

    Szkudlińska, B; Kowalski, M L; Grzegorczyk, J; Pierzchała, A

    1996-01-01

    It has been well documented, that sodium cromoglycate (DSCG) is capable in inhibiting activity of several inflammatory cells putatively involved in allergic and non-allergic asthmatic inflammation. The goal of this study was to compare the effect of DSCG on random locomotion and chemotaxis of neutrophils to several stimuli in atopic and non-atopic subjects. In 10 seasonal asthmatic (SA), and 10 healthy subjects (HS) chemotactic responses of neutrophils were examined using modified Boyden's microchamber (Neuroprobe) technique. Neutrophils isolated from both HS and SA demonstrated similar spontaneous migration and dose dependent chemotactic responses to FMLP (10(-12) - 10(-5M)), PAF (10(-7) - 10(-5M)) and ZAS (2.5% - 50%). DSCG in concentration range 10(-7) - 10(-9M) expressed a dose-dependent inhibition of both random migration and chemotactic responses to all stimuli tested, with maximal inhibition ranging from 58%-89% and 67%-75% for HS and SA, respectively. Our results confirm potent anti-inflammatory activity of DSCG in vitro, and demonstrate, that this activity is similar in atopic asthmatics, and in healthy subjects.

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

    PubMed

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

    2012-10-01

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

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

    PubMed Central

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

    2012-01-01

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

  6. 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

  7. 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

  8. Apoptosis Is Essential for Neutrophil Functional Shutdown and Determines Tissue Damage in Experimental Pneumococcal Meningitis

    PubMed Central

    Kirschnek, Susanne; Obermaier, Bianca; Häcker, Hans; Paul, Robert; Häcker, Georg

    2009-01-01

    During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1β and G-CSF as well as reduced levels of anti-inflammatory TGF-β. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. PMID:19478887

  9. Activation of the neutrophil myeloperoxidase-H2O2 system by synovial fluid isolated from patients with rheumatoid arthritis.

    PubMed Central

    Nurcombe, H L; Bucknall, R C; Edwards, S W

    1991-01-01

    Synovial fluid isolated from 16 patients with rheumatoid arthritis activated luminol dependent chemiluminescence in bloodstream neutrophils, and the maximal activity stimulated varied over a 50-fold range. In contrast, these same fluids only activated a much lower range (two- to threefold) of maximal rates of lucigenin dependent chemiluminescence and cytochrome c reduction, two assays which only measure oxidant secretion which is independent of myeloperoxidase. Over 95% of the luminol dependent chemiluminescence activated by all samples was inhibited by azide (indicating its dependence upon myeloperoxidase), but anti-(myeloperoxidase) IgG (which specifically inhibits only the extracellular activity of this enzyme) only inhibited the response stimulated by some samples: those fluids which activated the highest luminol dependent chemiluminescence also stimulated the greatest activity of an extracellular myeloperoxidase-H2O2 system. A clear correlation was shown to exist between the activity of myeloperoxidase already present in the fluids (after its secretion from neutrophils in situ within the rheumatoid joint) and the ability of the fluid to activate luminol dependent chemiluminescence. It is concluded, therefore, that all synovial fluid samples tested possess almost equivalent levels of a factor(s) which activated O2-/H2O2 secretion and that the variations in the measured activity of the extracellular myeloperoxidase-H2O2 system are dependent upon the level of degranulation which had occurred within the joint. PMID:1851410

  10. 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.

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

    PubMed

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

    2014-07-01

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

  12. 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

  13. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions

    PubMed Central

    Akenhead, Michael L.; Horrall, Nolan M.; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y.

    2015-01-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s−1 shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia). PMID:26065495

  14. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions.

    PubMed

    Akenhead, Michael L; Horrall, Nolan M; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y

    2015-09-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s(-1) shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).

  15. Epithelial Neutrophil-Activating Peptide (ENA-78), Acute Coronary Syndrome Prognosis, and Modulatory Effect of Statins

    PubMed Central

    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-1β 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-1β-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

  16. 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

    2008-09-03

    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.

  17. Accumulation of phosphatidic acid mass and increased de novo synthesis of glycerolipids in platelet-activating-factor-activated human neutrophils.

    PubMed Central

    Tou, J; Jeter, J R; Dola, C P; Venkatesh, S

    1991-01-01

    Incubation of human neutrophils with 100 nM-platelet-activating factor (PAF) but without cytochalasin B resulted in a rapid (5 s) accumulation (1.6-fold) of phosphatidic acid (PtdOH) mass. The increased PtdOH mass reached a maximum (2.8-fold) at 1 min and remained elevated (1.7-fold) at 10 min. No methylamine-stable lyso-PtdOH was detectable in the total lipid extract from control or from PAF-activated cells, suggesting that diacyl-PtdOH was the predominant species. In PAF-activated cells, changes in 1,2-diacylglycerol (DG) mass were not detectable at 5 or 15 s. Increased DG mass (1.7-fold) was detected between 30 s and 2 min, but then it declined to basal levels by 10 min. PAF enhanced [3H]glycerol incorporation into PtdOH and DG by 2- and 3-fold respectively during 1-10 min incubations. PAF also increased the radioactivity but not the mass of phosphatidylinositol and of choline glycerophospholipid by 8-fold and 4-fold respectively at 10 min. In addition, PAF-activated cells showed increased (2-fold) glycerol incorporation into triacylglycerol. These results demonstrate that PAF enhances rapid accumulation of diacyl-PtdOH mass, and that increased de novo synthesis may contribute to PtdOH mass accumulation. Images Fig. 2. PMID:1662484

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

    PubMed Central

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

    1989-01-01

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

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

    PubMed

    Xu, Najia; Hossain, Mokarram; Liu, Lixin

    2013-01-01

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

  20. Proteinase-Activated Receptor-2 Agonist Activates Anti-Influenza Mechanisms and Modulates IFNγ-Induced Antiviral Pathways in Human Neutrophils

    PubMed Central

    Shpacovitch, Victoria; Ehrhardt, Christina; Fastrich, Michaela; Goerge, Tobias; Ludwig, Stephan; Steinhoff, Martin

    2013-01-01

    Proteinase-activated receptor-2 (PAR2) is expressed by human leukocytes and participates in the development of inflammatory diseases. Recent studies demonstrated an ability of PAR2 agonist to enhance IFNγ-induced antiviral responses of human leukocytes. However, the precise cellular antiviral defense mechanisms triggered in leukocytes after stimulation with IFNγ and/or PAR2 agonist remain elusive. Therefore, we aimed to identify neutrophil defense mechanisms involved in antiviral resistance. Here we demonstrated that PAR2 agonist enhanced IFNγ-related reduction of influenza A virus (IAV) replication in human neutrophils. PAR2-mediated decrease in IAV replication was associated with reduced NS-1 transcription. Moreover, PAR2-dependent neutrophil activation resulted in enhanced myeloperoxidase degranulation and extracellular myeloperoxidase disrupted IAV. The production of ROS was elevated in response to PAR2 activation. Interestingly, IFNγ did not influence both effects: PAR2 agonist-triggered myeloperoxidase (MPO) release and reactive oxygen species (ROS) production, which are known to limit IAV infections. In contrast, orthomyxovirus resistance gene A (MxA) protein expression was synergistically elevated through PAR2 agonist and IFNγ in neutrophils. Altogether, these findings emphasize two PAR2-controlled antiviral mechanisms that are independent of or modulated by IFNγ. PMID:24171176

  1. Neutrophil Toxicity of Amphotericin B

    PubMed Central

    Chunn, C. John; Starr, P. R.; Gilbert, David N.

    1977-01-01

    The toxicity of amphotericin B (AmB) for neutrophils and the protective effect of serum cholesterol were investigated. Neutrophils were exposed in vitro to varying concentrations of AmB. As judged by trypan blue exclusion, neutrophil viability decreased by 40% (P < 0.001) within 30 min of incubation in sterol-free buffer containing 5 μg of AmB per ml. In the presence of 4 mg of cholesterol per 100 ml in buffer, the AmB concentration could be increased to 50 μg/ml before significant (P < 0.01) neutrophil toxicity occurred. Hexose monophosphate shunt activity of neutrophils incubated in serum or cholesterol-containing buffer with 10 μg of AmB per ml was normal. These results suggest that serum contains a protective factor, probably cholesterol, which protects neutrophils in vitro from the toxic effects of AmB. PMID:900919

  2. 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.

  3. A dietary supplement improves facial photoaging and skin sebum, hydration and tonicity modulating serum fibronectin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins.

    PubMed

    Di Cerbo, Alessandro; Laurino, Carmen; Palmieri, Beniamino; Iannitti, Tommaso

    2015-03-01

    treatment. Serum levels of fibronectin, elastin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins were measured by enzyme-linked immunosorbent assay in the first cohort of patients affected by facial photoaging and healthy controls and, at baseline and 2 weeks after the end of treatment, in the second cohort of patients who underwent treatment with VISCODERM Pearls and placebo. VAS photoaging score was higher in patients affected by photoaging, if compared with healthy controls (p < 0.0001). pH and sebum were increased in patients affected by photoaging, if compared with healthy controls (both p < 0.0001), while elasticity, hydration and tonicity were decreased in patients affected by photoaging, if compared with healthy controls (all p < 0.0001). Serum fibronectin and hyaluronic acid concentrations were lower in patients affected by photoaging, if compared with healthy controls (both p < 0.0001). Serum neutrophil elastase 2, elastin and carbonylated protein concentrations were higher in patients affected by photoaging, if compared with healthy controls (p < 0.01, p < 0.01 and p < 0.0001, respectively). Dietary supplement administration resulted in an improvement in VAS photoaging score, if compared with placebo (p < 0.0001), as observed 2 weeks after the end of treatment. Facial sebum, hydration and tonicity were increased in the active treatment group vs. placebo (p < 0.0001, p < 0.0001 and p < 0.05, respectively) 2 weeks after the end of treatment. Serum fibronectin and hyaluronic acid concentrations were increased in the dietary supplement group, if compared with placebo (p < 0.01 and p < 0.001) 2 weeks after the end of treatment, while no statistical difference in serum elastin concentration was observed between the two groups. Serum neutrophil elastase 2 and carbonylated protein concentrations were decreased in the dietary supplement group 2 weeks after the end of treatment, if compared with placebo (p < 0.001 and p < 0.0001). We found significantly

  4. 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.

  5. Isolation and Functional Analysis of Human Neutrophils

    PubMed Central

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

    2015-01-01

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

  6. Combined activity of post-exercise concentrations of NA and eHsp72 on human neutrophil function: role of cAMP.

    PubMed

    Giraldo, Esther; Hinchado, María D; Ortega, Eduardo

    2013-09-01

    Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as "danger signals" during exercise-induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post-exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise-induced stress. Given this context, the aim of the present investigation was to study the combined activity of post-exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects. Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post-exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP. These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise-induced stress. Copyright © 2013 Wiley Periodicals, Inc.

  7. Effect of aromatic nitroso-compounds on superoxide-generating activity in neutrophils.

    PubMed

    Nakata, M; Nasuda-Kouyama, A; Isogai, Y; Kanegasaki, S; Iizuka, T

    1997-07-01

    Aromatic nitroso-compounds such as nitrosobenzene inhibited the respiratory burst of intact neutrophils induced by various stimulants, including phorbol 12-myristate 13-acetate and a chemotactic peptide. The compounds also inhibited NADPH-dependent oxygen consumption by cell-free preparations of neutrophils. This indicates that nitroso-compounds act directly on the NADPH-oxidase system. The inhibitory effects induced by several nitroso-compounds, 2-nitrosotoluene, nitrosobenzene, 4-nitrosophenol, and 1-nitrosopyrrolidine, were examined and their inhibition constants, the concentrations causing 50% reduction of oxygen consumption, were found to be 0.043, 0.173, 0.672, and 32.1 mM, respectively. These values correlated well with the hydrophobicity of the compounds: a more hydrophobic compound was a more potent inhibitor against NADPH oxidase, suggesting that the oxidase has a hydrophobic site(s) for interaction with the inhibitors.

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

    PubMed Central

    Tsumbu, Cesar N.; Deby-Dupont, Ginette; Tits, Monique; Angenot, Luc; Frederich, Michel; Kohnen, Stephane; Mouithys-Mickalad, Ange; Serteyn, Didier; Franck, Thierry

    2012-01-01

    Young leaves of Manihot esculenta Crantz (Euphorbiaceae), Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae) and Pteridium aquilinum (Dennstaedtiaceae) are currently consumed as green vegetables by peoples in sub-Saharan Africa, Latin America, Asia and their migrants living in Western Europe. Sub-Saharan peoples use Manihot, Abelmoschus and Hibiscus also in the folk medicine to alleviate fever and pain, in the treatment of conjunctivitis, rheumatism, hemorrhoid, abscesses, ... The present study investigates the effects of aqueous extracts of those plants on the production of reactive oxygen species (ROS) and the release of myeloperoxidase (MPO) by equine neutrophils activated with phorbol 12-myristate 13-acetate (PMA). The ROS production was measured by lucigenin-enhanced chemiluminescence (CL), and the release of total MPO by an ELISA method. The study also investigates the effect of the extracts on the activity of MPO by studying its nitration activity on tyrosine and by using a new technique called SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection) that allows studying the direct interaction of compounds with the enzyme. In all experiments, the aqueous extracts of the plants developed concentration-dependent inhibitory effects. A moderate heat treatment did not significantly modify the inhibitory capacity of the extracts in comparison to not heated ones. Total polyphenol and flavonoid contents were determined with an HPLC-UV/DAD analysis and a spectroscopic method using Folin-Ciocalteu reagent. Some polyphenols with well-known antioxidant activities (caffeic acid, chlorogenic acid, hyperoside, rosmarinic acid and rutin) were found in the extracts and may partly explain the inhibitory activities observed. The role of those dietary and medicinal plants in the treatment of ROS-dependent inflammatory diseases could have new considerations for health. PMID:22312276

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

    PubMed

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

    2013-11-14

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

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

    PubMed Central

    Aarestrup, F M; Scott, N L; Sordillo, L M

    1994-01-01

    This study investigated the functional capabilities of neutrophils against different Staphylococcus aureus genotypes isolated from cows with mastitis. Six strains of S. aureus were chosen for use in the study, two with a common genotype, two with an intermediate genotype, and two with a rare genotype. The interaction between bacteria and neutrophils was measured by phagocytosis and bactericidal effect. The average percent killing of bacteria was lowest (40.0%) with strains belonging to the most common genotype, medium (50%) with strains belonging to the intermediate type, and highest (64.2%) with strains belonging to the rare type (P < or = 0.001). Statistically significant differences (P < or = 0.001) in the numbers of phagocytized bacteria were also found between the most prevalent type (6.27 bacteria per cell) and the other two types (intermediate type, 9.26/cell; rare type, 10.5/cell). These findings suggest that one of the reasons for the variation in prevalence of different genotypes of S. aureus in the mammary gland is due to the superior ability of some types to resist phagocytosis and/or killing by bovine neutrophils. PMID:7960153

  11. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  12. Granulocyte colony-stimulating factor improves host defense to resuscitated shock and polymicrobial sepsis without provoking generalized neutrophil-mediated damage.

    PubMed

    Patton, J H; Lyden, S P; Ragsdale, D N; Croce, M A; Fabian, T C; Proctor, K G

    1998-05-01

    Granulocyte colony-stimulating factor (G-CSF) increases production and release of neutrophil precursors and activates multiple functions of circulating polymorphonuclear neutrophils (PMNs). G-CSF has therapeutic effects in many experimental models of sepsis; its actions with superimposed reperfusion insults are unknown. In traumatic conditions, G-CSF could exacerbate unregulated, PMN-dependent injury to otherwise normal host tissue or, it could partially reverse trauma-induced immune suppression, which may improve long-term outcome. This study tested whether stimulating PMN proliferation and function with G-CSF during recovery from trauma+sepsis potentiated reperfusion injury or whether it improved host defense. Anesthetized swine were subjected to cecal ligation and incision, 35% hemorrhage, and 1 hr of hypotension. Resuscitation consisted of intravenous G-CSF (5 microg/kg) or placebo followed by shed blood and 40 mL/kg of lactated Ringer's solution. The control group received laparotomy only. G-CSF or placebo was given daily. Animals were killed at 4 days. Observers, blind to the protocol, graded autopsy samples for localization of infection and quality of abscess wall formation. Data included complete blood count, granulocyte oxidative burst after phorbol myristate acetate stimulation in vitro (GO2B), bronchoalveolar lavage (BAL) cell count, BAL noncellular protein, lipopolysaccharide-stimulated tumor necrosis factor production in whole blood in vitro (lipopolysaccharide-tumor necrosis factor), and lung tissue myeloperoxidase (MPO). Neutrophilia and localization of infection, were significantly improved by G-CSF. Variables altered by G-CSF, though not significantly, showed GO2B potential increased by 50%, lipopolysaccharide-tumor necrosis factor decreased by 50%, and improved survival versus placebo (100% vs. 70%). G-CSF did not increase lung MPO, BAL cell count, or BAL protein. Both arterial and venous O2 saturations were unaltered. Our data show that G

  13. The impact of trauma on neutrophil function.

    PubMed

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

    2014-12-01

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

  14. The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques

    PubMed Central

    Montecucco, Fabrizio; Di Marzo, Vincenzo; da Silva, Rafaela F.; Vuilleumier, Nicolas; Capettini, Luciano; Lenglet, Sébastien; Pagano, Sabrina; Piscitelli, Fabiana; Quintao, Silvia; Bertolotto, Maria; Pelli, Graziano; Galan, Katia; Pilet, Lucie; Kuzmanovic, Kristina; Burger, Fabienne; Pane, Bianca; Spinella, Giovanni; Braunersreuther, Vincent; Gayet-Ageron, Angèle; Pende, Aldo; Viviani, Giorgio Luciano; Palombo, Domenico; Dallegri, Franco; Roux-Lombard, Pascale; Santos, Robson A.S.; Stergiopulos, Nikos; Steffens, Sabine; Mach, François

    2012-01-01

    Aims The activation of cannabinoid receptor type 2 (CB2)-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB2 pharmacological activation on markers of plaque vulnerability in vivo and in vitro. Methods and results The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB1 (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB2 protein expression was reduced when compared with asymptomatic patients. In these portions, CB2 levels were inversely correlated (r = −0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB2 co-localized with neutrophils and MMP-9. Treatment with the selective CB2 agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. Conclusion Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB2 activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans. PMID:22112961

  15. The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques.

    PubMed

    Montecucco, Fabrizio; Di Marzo, Vincenzo; da Silva, Rafaela F; Vuilleumier, Nicolas; Capettini, Luciano; Lenglet, Sébastien; Pagano, Sabrina; Piscitelli, Fabiana; Quintao, Silvia; Bertolotto, Maria; Pelli, Graziano; Galan, Katia; Pilet, Lucie; Kuzmanovic, Kristina; Burger, Fabienne; Pane, Bianca; Spinella, Giovanni; Braunersreuther, Vincent; Gayet-Ageron, Angèle; Pende, Aldo; Viviani, Giorgio Luciano; Palombo, Domenico; Dallegri, Franco; Roux-Lombard, Pascale; Santos, Robson A S; Stergiopulos, Nikos; Steffens, Sabine; Mach, François

    2012-04-01

    The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

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

    PubMed

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

    2012-10-01

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

  17. Protective effect of platelet activating factor antagonists on cultured endothelial cell lysis induced by elastase or activated neutrophils.

    PubMed Central

    Renesto, P.; Vicart, P.; Paulin, D.; Chignard, M.

    1996-01-01

    1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates. PMID:8851508

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

    PubMed Central

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

    2014-01-01

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

  19. Assessment of antioxidant activity of spray dried extracts of Psidium guajava leaves by DPPH and chemiluminescence inhibition in human neutrophils.

    PubMed

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

    2014-01-01

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

  20. MFG-E8 inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation

    PubMed Central

    AZIZ, MONOWAR; YANG, WENG-LANG; CORBO, LANA M; CHAUNG, WAYNE W; MATSUO, SHINGO; WANG, PING

    2015-01-01

    We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG-E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG-E8 attenuates neutrophil migration. Recombinant human MFG-E8 (rhMFG-E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL-60, was treated with rhMFG-E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin-8 (IL-8) as the chemoattractant. Surface CXCR2 and intracellular G protein-coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen-activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG-E8 resulted in a significant inhibition of dHL-60 cell migration in a dose-dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG-E8-treated dHL-60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL-60 cells, treatment with rhMFG-E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10–30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL-60 cell migration which was significantly inhibited treatment with rhMFG-E8. Furthermore, blocking the MFG-E8 receptors, αvβ3/αvβ5-integrins, by anti-αv-integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG-E8-induced inhibition of dHL-60 cell migration. Finally, treatment of the dHL-60 cells with SB203580 and PD98059 neutralized the rhMFG-E8-induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG-E8 through which it inhibits neutrophil migration through αvβ3-integrin

  1. The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils

    PubMed Central

    Henríquez, C.; Riquelme, T. T.; Vera, D.; Julio-Kalajzić, F.; Ehrenfeld, P.; Melvin, J. E.; Figueroa, C. D.; Sarmiento, J.; Flores, C. A.

    2017-01-01

    Aim Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. Methods Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. Results We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1−/− mice are significantly less effective at recruiting neutrophils into the site of inflammation. Conclusions These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions. PMID:26138196

  2. The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

    PubMed

    Henríquez, C; Riquelme, T T; Vera, D; Julio-Kalajzić, F; Ehrenfeld, P; Melvin, J E; Figueroa, C D; Sarmiento, J; Flores, C A

    2016-01-01

    Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

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

    PubMed

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

    2011-09-15

    The antioxidant activity of methanol extracts from Passiflora edulis and Passiflora alata pulp, and P. edulis rinds, healthy or infected with the passion fruit woodiness virus (PWV), was investigated using the oxidant activities of the neutrophil and the neutrophil granule enzyme myeloperoxidase (MPO), both playing key roles in inflammation. The reactive oxygen species produced by stimulated neutrophils were evaluated by lucigenin-enhanced chemiluminescence (CL) and the activity of purified MPO was measured by SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection), a technique for studying the direct interaction of a compound with the enzyme. The rind extracts of P. edulis possessed higher and dose-dependent inhibitory effects on CL response and on the peroxidase activity of MPO than total pulp extracts from both passion fruit species. The quantification of isoorientin in the extracts showed a correlation with their antioxidant activity, suggesting the potential of P. edulis rinds as functional food or as a possible source of natural flavonoids. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Activation of Adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome

    PubMed Central

    Liu, Yang-Wuyue; Yang, Ting; Zhao, Li; Ni, Zhenhong; Yang, Nan; He, Fengtian; Dai, Shuang-Shuang

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is an overwhelming whole body inflammation caused by infectious diseases or sterile insults. Neutrophils are the dominant participants during inflammation, and their survival and death determine the initiation as well as resolution of SIRS. Apoptosis and autophagy are two fundamental cellular processes that modulating cell fate, but their correlation and regulators in neutrophils under SIRS condition have not been elucidated. In this study, we demonstrated that high dose of LPS induced both apoptosis and autophagy of neutrophils in a mouse SIRS model and LPS-stimulated neutrophils in vitro. Moreover, we found that the adenosine 2A receptor (A2AR), a known anti-inflammatory G protein-coupled receptor (GPCR), could inhibit LPS-induced neutrophil apoptosis by suppressing the LPS-induced autophagy. Activation of A2AR suppressed LPS-induced autophagy by inhibiting the ROS-JNK pathway as well as promoting GPCR βϒ subunit–AKT signaling. The A2AR-inhibited autophagy suppressed apoptosis of neutrophils by blocking caspase8, caspase3 and PARP signaling. These findings not only increase our understandings of neutrophils’ fate and function in response to systemic inflammation, but also identify a novel anti-inflammatory role of A2AR in modulating neutrophils’ survival during inflammation. PMID:27647162

  5. APPLICATION OF PROTEOMICS TO NEUTROPHIL BIOLOGY

    PubMed Central

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

    2009-01-01

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

  6. Perflubron emulsion prevents PMN activation and improves myocardial functional recovery after cold ischemia and reperfusion.

    PubMed

    Gale, Stephen C; Gorman, Grace D; Copeland, Jack G; McDonagh, Paul F

    2007-03-01

    In cardiopulmonary bypass, extracorporeal circulation activates neutrophils, which contribute to ischemia reperfusion injury and postoperative myocardial dysfunction. Perfluorocarbons (PFCs) are compounds that dissolve oxygen and have anti-inflammatory and neutrophil-stabilizing properties. We hypothesized that perflubron emulsion (PFE), a PFC, would attenuate neutrophil activation during simulated extracorporeal circulation (SECC) and would preserve myocardial functional recovery during reperfusion after cold ischemia. In a SECC, diluted blood was circulated for 120 min and subsequently used to reperfuse isolated rat hearts after 2 h of cold (12 degrees C) ischemia. Three groups were studied: noncirculated control; SECC/no additive; and SECC/PFE added. In control and SECC/no additive groups, whole blood was diluted 1:1 with plasmalyte. SECC/PFE blood was diluted 1:1 with plasmalyte and PFE (0.075 mL/mL diluted whole blood). Blood counts and neutrophil activation experiments were performed before and after 120 min of SECC. Reperfusion was accomplished using a modified Langendorff preparation. Left ventricular developed pressure, dP/dt, and coronary flow were measured at 10, 15, and 20 min of reperfusion. After 120 min SECC, neutrophil activation was significantly reduced in the SECC/PFE group compared to the SECC/no additive group (38.1 +/- 2.3% versus 51.7 +/- 1.0%, P < 0.05). Compared to cold ischemic hearts reperfused with fresh, non-recirculated blood, left ventricular developed pressure and dP/dt were significantly impaired in the cold ischemic hearts reperfused with SECC/no additive blood (P < 0.05). In contrast, myocardial functional recovery was not impaired in the hearts reperfused with SECC/PFE blood. SECC-induced neutrophil activation was attenuated with Perflubron treatment. In addition, the progressive impairment in myocardial functional recovery after cold ischemia was significantly improved with treatment. PFE has clinical potential to limit

  7. Endogenous Acute Phase Serum Amyloid A Lacks Pro-Inflammatory Activity, Contrasting the Two Recombinant Variants That Activate Human Neutrophils through Different Receptors

    PubMed Central

    Christenson, Karin; Björkman, Lena; Ahlin, Sofie; Olsson, Maja; Sjöholm, Kajsa; Karlsson, Anna; Bylund, Johan

    2013-01-01

    Most notable among the acute phase proteins is serum amyloid A (SAA), levels of which can increase 1000-fold during infections, aseptic inflammation, and/or trauma. Chronically elevated SAA levels are associated with a wide variety of pathological conditions, including obesity and rheumatic diseases. Using a recombinant hybrid of the two human SAA isoforms (SAA1 and 2) that does not exist in vivo, numerous in vitro studies have given rise to the notion that acute phase SAA is a pro-inflammatory molecule with cytokine-like properties. It is however unclear whether endogenous acute phase SAA per se mediates pro-inflammatory effects. We tested this in samples from patients with inflammatory arthritis and in a transgenic mouse model that expresses human SAA1. Endogenous human SAA did not drive production of pro-inflammatory IL-8/KC in either of these settings. Human neutrophils derived from arthritis patients displayed no signs of activation, despite being exposed to severely elevated SAA levels in circulation, and SAA-rich sera also failed to activate cells in vitro. In contrast, two recombinant SAA variants (the hybrid SAA and SAA1) both activated human neutrophils, inducing L-selectin shedding, production of reactive oxygen species, and production of IL-8. The hybrid SAA was approximately 100-fold more potent than recombinant SAA1. Recombinant hybrid SAA and SAA1 activated neutrophils through different receptors, with recombinant SAA1 being a ligand for formyl peptide receptor 2 (FPR2). We conclude that even though recombinant SAAs can be valuable tools for studying neutrophil activation, they do not reflect the nature of the endogenous protein. PMID:23626589

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-07-01

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

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

    PubMed Central

    Solís, Camila J.; Poblete-Morales, Matías; Cabral, Sergio; Valdés, Juan A.; Reyes, Ariel E.; Avendaño-Herrera, Ruben; Feijóo, Carmen G.

    2015-01-01

    Flavobacterium psychrophilum is a Gram-negative bacterium, responsible for the bacterial cold-water disease and the rainbow trout fry syndrome in freshwater salmonid fish. At present, there is only one commercial vaccine in Chile, made with two Chilean F. psychrophilum isolates and another licensed in Europe. The present study analyzed neutrophil migration, as a marker of innate immune activation, in zebrafish (Danio rerio) in response to different F. psychrophilum bath vaccines, which is the first step in evaluating vaccine effectiveness and efficiency in fish. Results indicated that bacterins of the LM-02-Fp isolate were more immunogenic than those from the LM-13-Fp isolate. However, no differences were observed between the same bacteria inactivated by either formaldehyde or heat. Importantly, the same vaccine formulation without an adjuvant only triggered a mild neutrophil migration compared to the complete vaccine. Observations also found that, after a year of storage at 4°C, the activation of the innate immune system by the different vaccines was considerably decreased. Finally, new vaccine formulations prepared with heat and formaldehyde inactivated LM-02-Fp were significantly more efficient than the available commercial vaccine in regard to stimulating the innate immune system. PMID:25815347

  11. Epithelial neutrophil-activating peptide 78 concentrations are elevated in the peritoneal fluid of women with endometriosis.

    PubMed

    Mueller, Michael D; Mazzucchelli, Luca; Buri, Caroline; Lebovic, Dan I; Dreher, Ekkehard; Taylor, Robert N

    2003-03-01

    To investigate the presence of epithelial neutrophil-activating peptide 78 (ENA-78) in peritoneal fluid of women with and without endometriosis and to identify the cells that produce this inflammatory protein. Case-control study. University hospital. Eighteen women with and 9 women without endometriosis. ENA-78 protein and mRNA levels were compared among women with and without endometriosis in samples of peritoneal fluid, samples of endometriotic lesions obtained by biopsy during laparoscopy, and peritoneal macrophages. Enzyme-linked immunosorbent assay, reverse transcription polymerase chain reaction, and in situ hybridization methods were used. Secretion of ENA-78 protein by interleukin-1beta-stimulated endometriotic stromal cells and in the media of lipopolysaccharide-stimulated peritoneal macrophages were compared to that in unstimulated cell cultures. Peritoneal fluid concentrations of ENA-78 were significantly higher in affected women than in controls. Ectopic epithelial and stromal cells and peritoneal macrophages express ENA-78 messenger RNA. Interleukin-1beta stimulation of stromal cell cultures resulted in a 23-fold increase in ENA-78 concentration, and lipopolysaccharide stimulation of peritoneal macrophages increased concentrations by 8-fold. Levels of ENA-78 are elevated in the peritoneal fluid of women with endometriosis. Ectopic glandular cells, ectopic stromal cells, and peritoneal macrophages express this inflammatory chemokine. Epithelial neutrophil-activating peptide 78 may play an important role in the pathogenesis of endometriosis.

  12. Hypochlorous Acid Converts the γ-Glutamyl Group of Glutathione Disulfide to 5-Hydroxybutyrolactam, a Potential Marker for Neutrophil Activation*

    PubMed Central

    Yuan, Wei; Wang, Yi; Heinecke, Jay W.; Fu, Xiaoyun

    2009-01-01

    In healthy cells, glutathione disulfide (GSSG) is rapidly reduced back to glutathione (GSH) by glutathione reductase to maintain redox status. The ratio of GSH/GSSG has been used as an indicator of oxidative stress. However, hypochlorous acid (HOCl) generated by the myeloperoxidase-H2O2-Cl− system of neutrophils converts GSH to irreversible oxidation products. Although several such products have been identified, yields of these compounds are very low in biological systems, and they cannot account quantitatively for thiol loss. In the current studies, we use liquid chromatography-mass spectrometry (LC-MS) to demonstrate that HOCl and chloramines oxidize GSSG to two irreversible products in high yield. The products, termed M-45 and M-90, are, respectively, 45 or 90 atomic mass units lighter than GSSG. The reaction pathway involves chloramine and aldehyde intermediates, and converts the γ-glutamyl residues of GSSG to 5-hydroxybutyrolactam. Importantly, M-45 and M-90 were resistant to reduction by glutathione reductase. Moreover, the monohydroxylbutyrolactam M-45 accounted for >90% of the endogenous GSH oxidation products generated by activated neutrophils. Because the reaction pathway involves chlorinating intermediates, hydroxylbutyrolactams are likely to be specific products of HOCl, which is generated only by myeloperoxidase. Therefore, our observations implicate M-45 as a potential biomarker for myeloperoxidase activity in vivo. PMID:19584048

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

    PubMed

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

    2013-06-15

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

  14. Hevein, an allergenic lectin from rubber latex, activates human neutrophils' oxidative burst.

    PubMed

    Rojas, E; Llinas, P; Rodríguez-Romero, A; Hernández, C; Linares, M; Zenteno, E; Lascurain, R

    2001-04-01

    Hevein is an N-acetyl-D-glucosamine (GlcNAc) specific lectin that has been hypothesized to participate in the IgE-mediated allergic reactions in patients with latex allergy. In this work we assessed the specificity and biological effect of hevein purified from rubber latex on human leukocytes, using epifluorescence microscopy and flow cytometry. Purified human granulocytes were stimulated in vitro with hevein, and production of oxidative radicals was measured by reduction of nitroblue tetrazolium formazan. Histochemical staining and flow cytometry showed that hevein recognizes specifically monocytes (CD14+) and neutrophils (CD16+), but not lymphoid cells. Hevein induced oxidative response in purified granulocytes; this effect was 1.3-1.5-fold higher than the effect observed with the lectin WGA (wheat germ agglutinin), or other lectins with different sugar specificity. The induced reactions and cellular recognition by hevein were inhibited with GlcNAc and its oligomers; as well as by glycoproteins containing tri-and tetra-antennary N-glycosydically linked glycans. Our findings suggest that neutrophils are the main target for latex hevein; this lectin induces production of oxidative radicals, which seem to play an important role in tissue damage during latex allergy.

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

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

    2013-01-01

    We examined the qualitative polymorphonuclear neutrophil (PMN)-associated immune impairment in patients with chronic lymphocytic leukemia (CLL) by characterizing phagocytic killing of key nonopsonized bacterial (Staphylococcus aureus and Pseudomonas aeruginosa) and fungal (Candida albicans and Aspergillus fumigatus) pathogens. Neutrophils were collected from 47 nonneutropenic CLL patients (PMN count > 1000/mm3), and age-matched and young healthy controls (five each). A subset of patients (13%) had prior or subsequent infections. We found that the CLL patients had diminished PMN microbicidal response against bacteria but not against fungi than did the controls. Compared to patients with effective PMN responses, we did not identify differences of basal PMN pathogen-associated molecular pattern receptor gene expression, soluble pathogen-associated molecular pattern gene expression, or inflammatory cytokine signatures in patients with impaired PMN responses when PMNs were analyzed in multiplex real-time polymerase chain reaction assays. However, differences in PMN microbicidal response against A. fumigatus in CLL patients were associated with the degree of hypogammaglobulinemia. PMID:23163595

  17. Hemoadsorption corrects hyperresistinemia and restores anti-bacterial neutrophil function.

    PubMed

    Bonavia, Anthony; Miller, Lauren; Kellum, John A; Singbartl, Kai

    2017-12-01

    Mounting evidence suggests that sepsis-induced morbidity and mortality are due to both immune activation and immunosuppression. Resistin is an inflammatory cytokine and uremic toxin. Septic hyperresistinemia (plasma resistin >20 ng/ml) has been associated with greater disease severity and worse outcomes, and it is further exacerbated by concomitant acute kidney injury (AKI). Septic hyperresistinemia disturbs actin polymerization in neutrophils leading to impaired neutrophil migration, a crucial first-line mechanism in host defense to bacterial infection. Our experimental objective was to study the effects of hyperresistinemia on other F-actin-dependent neutrophil defense mechanisms, in particular intracellular bacterial clearance and generation of reactive oxygen species (ROS). We also sought to examine the effects of hemoadsorption on hyperresistinemia and neutrophil dysfunction. Thirteen patients with septic shock and six control patients were analyzed for serum resistin levels and their effects on neutrophil migration. In vitro, following incubation with resistin-spiked serum samples, Pseudomonas aeruginosa clearance and ROS generation in neutrophils were measured. Phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDPK1) was assessed using flow cytometry. In vitro hemoadsorption with both Amberchrome™ columns (AC) and CytoSorb® cartridges (CC) were used to test correction of hyperresistinemia. We further tested AC for their effect on cell migration and ROS generation and CC for their effect on bacterial clearance. Patients with septic shock had higher serum resistin levels than control ICU patients and showed a strong, negative correlation between hyperresistinemia and neutrophil transwell migration (ρ= - 0.915, p < 0.001). In vitro, neutrophils exposed to hyperresistinemia exhibited twofold lower intracellular bacterial clearance rates compared to controls. Resistin impaired intracellular signaling and ROS production in a dose

  18. TLR9 and NF-κB Are Partially Involved in Activation of Human Neutrophils by Helicobacter pylori and Its Purified DNA

    PubMed Central

    Alvarez-Arellano, Lourdes; Cortés-Reynosa, Pedro; Sánchez-Zauco, Norma; Salazar, Eduardo; Torres, Javier; Maldonado-Bernal, Carmen

    2014-01-01

    Helicobacter pylori infection represents one of the most common bacterial infections worldwide. The inflammatory response to this bacterium involves a large influx of neutrophils to the lamina propria of the gastric mucosa. However, little is known about the receptors and molecular mechanisms involved in activation of these neutrophils. In this study, we aimed to determine the role of toll-like receptor 9 (TLR9) in the response of human neutrophils to H. pylori and purified H. pylori DNA (Hp-DNA). Neutrophils were isolated from the blood of adult volunteers and challenged with either H. pylori or Hp-DNA. We found that both, H. pylori and Hp-DNA induced increased expression and release of IL-8. Furthermore, we showed that TLR9 is involved in the induction of IL-8 production by H. pylori and Hp-DNA. IL-8 production induced by H. pylori but not by Hp-DNA was partially mediated by NF-κB. In conclusion, this study showed for first time that both, H. pylori and Hp-DNA activate TLR9 and induce a different inflammatory response that leads to activation of neutrophils. PMID:24987851

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

    PubMed Central

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

    2015-01-01

    Rheumatoid arthritis (RA) is a highly disabling disease that affects all structures of the joint and significantly impacts on morbidity and mortality in RA patients. RA is characterized by persistent inflammation of the synovial membrane lining the joint associated with infiltration of immune cells. Eighty to 90% of the leukocytes infiltrating the synovia are neutrophils. The specific role that neutrophils play in the onset of RA is not clear, but recent studies have evidenced that they have an important participation in joint damage and disease progression through the release of proteolytic enzymes, reactive oxygen species (ROS), cytokines, and neutrophil extracellular traps, in particular during frustrated phagocytosis of immune complexes (ICs). In addition, the local and systemic activation of the complement system contributes to the pathogenesis of RA and other IC-mediated diseases. This review discusses (i) the participation of Fcγ and complement receptors in mediating the effector functions of neutrophils in RA; (ii) the contribution of the complement system and ROS-dependent and ROS-independent mechanisms to joint damage in RA; and (iii) the use of plant extracts, dietary compounds, and isolated natural compounds in the treatment of RA, focusing on modulation of the effector functions of neutrophils and the complement system activity and/or activation. PMID:26346244

  20. TLR9 and NF-κB are partially involved in activation of human neutrophils by Helicobacter pylori and its purified DNA.

    PubMed

    Alvarez-Arellano, Lourdes; Cortés-Reynosa, Pedro; Sánchez-Zauco, Norma; Salazar, Eduardo; Torres, Javier; Maldonado-Bernal, Carmen

    2014-01-01

    Helicobacter pylori infection represents one of the most common bacterial infections worldwide. The inflammatory response to this bacterium involves a large influx of neutrophils to the lamina propria of the gastric mucosa. However, little is known about the receptors and molecular mechanisms involved in activation of these neutrophils. In this study, we aimed to determine the role of toll-like receptor 9 (TLR9) in the response of human neutrophils to H. pylori and purified H. pylori DNA (Hp-DNA). Neutrophils were isolated from the blood of adult volunteers and challenged with either H. pylori or Hp-DNA. We found that both, H. pylori and Hp-DNA induced increased expression and release of IL-8. Furthermore, we showed that TLR9 is involved in the induction of IL-8 production by H. pylori and Hp-DNA. IL-8 production induced by H. pylori but not by Hp-DNA was partially mediated by NF-κB. In conclusion, this study showed for first time that both, H. pylori and Hp-DNA activate TLR9 and induce a different inflammatory response that leads to activation of neutrophils.

  1. Influence of low molecular (below 5 KD) fraction from cord blood and actovegin on phagocytic activity of frozen-thawed neutrophils.

    PubMed

    Gulevsky, A K; Moiseyeva, N N; Gorina, O L

    2011-01-01

    The influence of the cattle cord blood low-molecular fraction (CBF below 5 kDa) as part of the rehabilitating medium in comparison with Actovegin on the functional activity of neutrophils after cryopreservation was studied. Incubation of frozen-thawed neutrophils in the rehabilitating media containing the low-molecular fraction or Actovegin stimulates their phagocytic function, in particular engulfing and digesting ability. After incubation of frozen-thawed neutrophils in the media containing 0.15 mg per ml CBF or 1.5 mg per ml Actovegin, their oxygen-dependent metabolism was activated, since the number of NBT-positive neutrophils increased significantly in comparison with the control. Gel-penetrating chromatography of CBF and Actovegin revealed differences between their chromatograms reflecting differences between the compositions compared. The recovery of the functional activity of frozen-thawed neutrophils was possible in the media containing the cattle cord blood low-molecular fraction (below 5 kDa) or Actovegin at 0.15 mg per ml and 1.5 mg per ml, respectively.

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

    PubMed Central

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

    2017-01-01

    Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. Objective: To determine the role of the AMPKα2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral arter