scholarly journals Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation

2020 ◽  
Vol 222 (10) ◽  
pp. 1702-1712 ◽  
Author(s):  
Fabian Cuypers ◽  
Björn Klabunde ◽  
Manuela Gesell Salazar ◽  
Surabhi Surabhi ◽  
Sebastian B Skorka ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Extracellular Space ◽  
Cytolytic Activity ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Pneumococcal Infections ◽  
Pneumococcal Strain ◽  
Neutrophil Degranulation ◽  
Neutrophil Response

Abstract Background In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and are recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyperinflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. Methods Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin-deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. Results Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. Conclusions Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin-induced neutrophil activation.

Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3206-3206
Author(s):  
Carlos E Vazquez ◽  
Gregory N Prado ◽  
Enrique R Maldonado ◽  
Gabriela Saca ◽  
Iren M Ortiz ◽  
...  
Keyword(s):  
Western Blot ◽  
Respiratory Burst ◽  
Mineralocorticoid Receptor ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Dead Cell ◽  
Cardiovascular Morbidity And Mortality

Abstract Abstract 3206 Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo. Disclosures: No relevant conflicts of interest to declare.

Neutrophil activation in response to monomeric myeloperoxidase

2018 ◽  
Vol 96 (5) ◽  
pp. 592-601 ◽  
Author(s):  
Irina V. Gorudko ◽  
Daria V. Grigorieva ◽  
Alexey V. Sokolov ◽  
Ekaterina V. Shamova ◽  
Valeria A. Kostevich ◽  
...  
Keyword(s):  
Acute Inflammation ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Calcium Stores ◽  
Cellular Functions ◽  
Leukocyte Accumulation ◽  
Cytoskeleton Reorganization ◽  
Neutrophil Degranulation ◽  
Intracellular Ca ◽  
Normal Human

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Iohexol-Induced Neutrophil Myeloperoxidase Release and Activation upon Contact with Vascular Stent-Graft Material: A Mechanism Contributing to the Postimplantation Syndrome?

2003 ◽  
Vol 10 (5) ◽  
pp. 958-967 ◽  
Author(s):  
Vibeke Videm ◽  
Asbjørn Ødegård ◽  
Hans Olav Myhre
Keyword(s):  
Stent Graft ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Maximal Response ◽  
Graft Material ◽  
Vascular Stent ◽  
Neutrophil Degranulation ◽  
Considerable Individual Variation ◽  
Vitro Effect

Purpose: To investigate whether the contrast medium iohexol alone or in combination with vascular stent-graft material induces neutrophil degranulation. Methods: Human whole blood or isolated human neutrophils were incubated with or without iohexol and vascular stent-graft material. Samples were also drawn from 5 patients undergoing diagnostic angiography using iohexol. Myeloperoxidase and lactoferrin concentrations were determined by enzyme immunoassays. Results: Both in vitro and in the patients, iohexol induced neutrophil degranulation with considerable individual variation in dose sensitivity and timing. The in vitro effect was independent of the type of anticoagulant used (ethylenediamine tetra-acetic acid, heparin, lepirudin). Experiments using isolated neutrophils showed that degranulation was independent of complement activation or platelet-derived mediators. The dose for maximal response varied from 5 to 50 mg I/mL (10.7–107.6 mg/mL iohexol). In vitro, vascular stent-graft material alone did not induce neutrophil degranulation. As compared to iohexol alone, incubation with iohexol and vascular stent-graft material in combination substantially increased the release of myeloperoxidase. Conclusions: Iohexol induces neutrophil degranulation, which is greatly enhanced when combined with vascular stent-graft material. Thus, iohexol-induced neutrophil activation may contribute to an inflammatory response following stent-graft implantation. We speculate that neutrophil activation during other procedures combining catheters and iohexol (e.g., angiography) may induce inflammation, which might have detrimental effects.

scholarly journals Neutrophil activation by anti-proteinase 3 antibodies in Wegener's granulomatosis: role of exogenous arachidonic acid and leukotriene B4 generation.

1996 ◽  
Vol 184 (4) ◽  
pp. 1567-1572 ◽  
Author(s):  
F Grimminger ◽  
K Hattar ◽  
C Papavassilis ◽  
B Temmesfeld ◽  
E Csernok ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Wegener’S Granulomatosis ◽  
Wegener's Granulomatosis ◽  
Neutrophil Activation ◽  
Tnf Alpha ◽  
Lipid Mediator ◽  
Human Neutrophils ◽  
Proteinase 3 ◽  
Lipoxygenase Pathway

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.

scholarly journals Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

1992 ◽  
Vol 116 (3) ◽  
pp. 695-706 ◽  
Author(s):  
G P Downey ◽  
C K Chan ◽  
P Lea ◽  
A Takai ◽  
S Grinstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Protein Phosphorylation ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Actin Assembly ◽  
Membrane Ruffling ◽  
Kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

scholarly journals Early and Late Processes Driving NET Formation, and the Autocrine/Paracrine Role of Endogenous RAGE Ligands

2021 ◽  
Vol 12 ◽  
Author(s):  
Olga Tatsiy ◽  
Vanessa de Carvalho Oliveira ◽  
Hugo Tshivuadi Mosha ◽  
Patrick P. McDonald
Keyword(s):  
Signaling Pathways ◽  
Neutrophil Extracellular Trap ◽  
Net Generation ◽  
Human Neutrophils ◽  
Chromatin Decondensation ◽  
Cellular Processes ◽  
Activated Neutrophils ◽  
Arginine Residues ◽  
Neutrophil Response

Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2574-2583 ◽  
Author(s):  
Belén Martı́n-Martı́n ◽  
Svetlana M. Nabokina ◽  
Juan Blasi ◽  
Pedro A. Lazo ◽  
Faustino Mollinedo
Keyword(s):  
Human Neutrophil ◽  
Coiled Coil ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Binding Studies ◽  
Specific Granules ◽  
Neutrophil Degranulation ◽  
Carboxy Terminal

Abstract To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca++ and GTP-γ-S–induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti–syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE– t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2574-2583 ◽  
Author(s):  
Belén Martı́n-Martı́n ◽  
Svetlana M. Nabokina ◽  
Juan Blasi ◽  
Pedro A. Lazo ◽  
Faustino Mollinedo
Keyword(s):  
Human Neutrophil ◽  
Coiled Coil ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Binding Studies ◽  
Specific Granules ◽  
Neutrophil Degranulation ◽  
Carboxy Terminal

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca++ and GTP-γ-S–induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti–syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE– t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

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