Calcium ionophore-activated platelets induce eosinophil extracellular trap formation

BackgroundPlatelets play a modulatory role in inflammatory response by secreting a vast array of granules and disintegrating into membrane-bound microparticles upon activation. The interplay between eosinophils and platelets is postulated to be implicated in the pathology of allergic airway inflammation. In this study, we investigated whether activated platelets can induce eosinophil extracellular trap (EET) formation, a cellular process by which activated eosinophils release net-like DNA fibers.MethodsPlatelets were stimulated with the calcium ionophore, A23187, and the platelet agonists, thrombin and adenosine diphosphate (ADP). Platelet cultures were fractionated into conditioned medium (CM) and pellet, which were then overlaid on eosinophils to examine EET formation.ResultsThe CM and pellet from A23187-activated platelets stimulated eosinophils to generate EET, whereas those from thrombin- or ADP-activated platelets failed to induce such generation. The EET-inducing activity of the A23187-activated platelet culture was linearly proportional to the number of activated platelets. Interestingly, while EET formation induced by the direct stimulation of eosinophils with A23187 was NADPH oxidase (NOX)-dependent, EET formation induced by A23187-activated platelets was NOX-independent and significantly inhibited by necroptosis pathway inhibitors.ConclusionsActivated platelets and their products may induce EET formation, thereby potentiating their role in eosinophilic airway inflammation.     

Elevated extracellular trap formation and contact system activation in acute leukemia

Leukemic cells release their nuclear contents into the extracellular space upon activation. The released nuclear contents, called extracellular traps, can activate the contact system of coagulation. This study accessed the extent of contact system activation, the levels of extracellular traps, and coagulation activation in hematologic malignancies including acute leukemia. In 154 patients with hematologic malignancies (acute leukemia, n?=?29; myelodysplastic syndrome, n?=?20; myeloproliferative neoplasms, n?=?69; plasma cell myeloma, n?=?36) and 48 normal controls, the levels of coagulation factors (fibrinogen and factor VII, VIII, IX, and XII), D-dimer, thrombin generation, extracellular trap markers (histone–DNA complex, cell-free dsDNA, leukocyte elastase), and contact system markers (activated factor XII [XIIa], high-molecular-weight kininogen, prekallikrein, bradykinin) were measured. Patients with acute leukemia showed the highest levels of peak thrombin, extracellular trap markers, and factor XIIa. Factor XIIa level was significantly associated with the presence of acute leukemia. The histone–DNA complex and cell-free dsDNA were revealed as significant associated factors with the factor XIIa level. Three markers of extracellular traps and two markers of thrombin generation significantly contributed to the hemostatic abnormalities in hematologic malignancies. Contact system was activated in acute leukemia and its activation was significantly associated with the extent of extracellular trap formation. This finding suggests that extracellular traps might be a major source of contact system activation and therapeutic strategies targeting extracellular trap formation or contact system activation may be beneficial in acute leukemia.     

Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity

The granule enzyme myeloperoxidase (MPO) plays an important role in neutrophil antimicrobial responses. However, the severity of immunodeficiency in patients carrying mutations in MPO is variable. Serious microbial infections, especially with Candida species, have been observed in a subset of completely MPO-deficient patients. Here we show that neutrophils from donors who are completely deficient in MPO fail to form neutrophil extracellular traps (NETs), indicating that MPO is required for NET formation. In contrast, neutrophils from partially MPO-deficient donors make NETs, and pharmacological inhibition of MPO only delays and reduces NET formation. Extracellular products of MPO do not rescue NET formation, suggesting that MPO acts cell-autonomously. Finally, NET-dependent inhibition of Candida albicans growth is compromised in MPO-deficient neutrophils. The inability to form NETs may contribute in part to the host defense defects observed in completely MPO-deficient individuals.     

miR-146a is a pivotal regulator of neutrophil extracellular trap formation promoting thrombosis

Neutrophil extracellular traps (NET) induce a procoagulant response linking inflammation and thrombosis. Low levels of miR-146a, a brake of inflammatory response, are involved in higher risk of cardiovascular events, but the mechanisms explaining how miR-146a exerts its function remain largely undefined. The aim of this study was to explore the impact of miR-146a deficiency in NETosis both in sterile and non-sterile models in vivo, and to investigate the underlying mechanism. Two models of inflammation were used: (i) Ldlr^-/- mice transplanted with bone marrow from miR-146a^-/- or wild-type mice were fed a high-fat diet, generating an atherosclerosis model; and (ii) an acute inflammation model was generated by injecting lipopolysaccharide (1 mg/kg) into miR-146a^-/- and wildtype mice. miR-146a deficiency increased NETosis in both models. Accordingly, miR-146a^-/- mice showed significantly reduced carotid occlusion time and elevated levels of NET in thrombi following FeCl3-induced thrombosis. Infusion of DNAse I abolished arterial thrombosis in both WT and miR-146a^-/- mice. Interestingly, miR-146a-deficient mice have aged, hyperreactive and pro-inflammatory neutrophils in their circulation which are more prone to form NET independently of the stimulus. Furthermore, we demonstrated that patients with community-acquired pneumonia with reduced miR-146a levels associated with the T variant of the functional rs2431697 had an increased risk of cardiovascular events due, in part, to an increased generation of NET.     

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts.

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.     

Leukotriene C4 production by murine mast cells: evidence of a role for extracellular leukotriene A4.

The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell-bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes.     

Modulation of myocardial activity by extracellular ATP

Extracellular ATP, at micromolar concentration, induces multiple functional changes in cardiac cells. Stimulation of P(2) purinoceptors is associated with Ca current increase and positive inotropic effect. On rapid application, ATP triggers membrane depolarization by activating a Cl conductance and by inducing an acidification following Cl-HCO(3) exchanger stimulation. Both effects might lead to cardiac arrhythmias following ATP release under pathophysiologic conditions.     

Inhibition of phenolic glycolipid-I synthesis in extracellular Mycobacterium leprae as an indicator of antimicrobial activity

The effects of 22 antimicrobial agents on the incorporation of [U14C] palmitic acid ([U14C] PA) into the unique phenolic glycolipid-I (PGL-I) antigen of Mycobacterium leprae were studied. Nude-mouse-propagated M. leprae were incubated in a modified Dubos medium in the presence of antimicrobial agents for 4 days. [U14C] PA was then added and incubation was continued for 8 days. The antileprosy agents dapsone, rifampin, and clofazimine (2 micrograms/ml each) caused a significant reduction in [U14C] PA incorporation into PGL-I. Among other agents, the most active were erythromycin, chloramphenicol, and cerulenin. Low concentrations of ethionamide, tetracycline, and minocycline stimulated label incorporation. This system may prove useful in the evaluation of antileprosy agents.     

Activation of mast cells by bile acids.

To test whether bile acids interact with mast cells, dilute, aqueous solutions of five pure unconjugated natural bile acids and their corresponding glycine or taurine conjugates were incubated with murine PT-18 cells (a mast cell line functionally and cytochemically similar to mucosal mast cells) or with freshly isolated rat peritoneal mast cells. Bile acid solutions ranged in concentration from 0.3 to 10 mmol/L; histamine release was assessed by a fluorimetric assay, and cell lysis by cytosolic enzyme (lactate dehydrogenase) release. Lipophilic, dihydroxy bile acids (chenodeoxycholic acid and deoxycholic acid as well as their glycine and taurine conjugates) caused histamine release in a dose-related manner; cholic acid and its conjugates caused much less or no histamine release. Two hydrophilic bile acids (ursodeoxycholic acid and ursocholic acid and their conjugates) were virtually devoid of activity. Histamine release, which was independent of extracellular Ca2+, occurred at 0.3 mmol/L, well below the critical micellization concentration. For a given concentration, unconjugated bile acids and glycine-conjugated bile acids induced more histamine release than taurine-conjugated bile acids; maximal release was observed at 3 mmol/L for lipophilic, dihydroxy bile acids. To test whether bile acids could also cause histamine release from cutaneous mast cells in vivo, rats were injected intradermally with bile acid solutions and histamine release assessed by capillary leakage of Evan's blue dye. Cutaneous blueing was greater with cytotoxic bile acids, chenodeoxycholyglycine or deoxycholylglycine, than with ursodeoxycholylglycine and was inhibited by prior antihistamine treatment. Histamine release correlated highly and positively with lipophilicity and with bile acid surface activity. It was concluded that lipophilic but not hydrophilic bile acids possess concentration-dependent cytotoxicity toward mast cells causing histamine release, that unconjugated and glycine-conjugated bile acids are more potent than taurine-conjugated bile acids, and that mast cell histamine release is highly correlated with lipophilicity of bile acids as well as their surface activity.     

Degranulating mast cells secrete an endoglycosidase that degrades heparan sulfate in subendothelial extracellular matrix.

Mast cells are widely distributed in perivascular connective tissues, especially in areas of active tumor growth and vascular reactivity. Incubation of metabolically [35S]O4 = -labeled subendothelial extracellular matrix (ECM) with lysates of bone marrow-derived mouse mast cells (BMMC) resulted in extensive degradation of heparan sulfate (HS) into fragments 5 to 6 times smaller than intact HS side chains. A much lower activity (seven- to eightfold) was expressed by intact BMMC incubated in contact with the ECM. These fragments were not produced in the presence of heparin, were sensitive to deamination with nitrous acid, and resistant to further degradation with papain or chondroitinase ABC. These results indicate that an endoglycosidase (heparanase) is involved in BMMC-mediated degradation of HS in the subendothelial ECM. Heparanase activity was not detected in medium conditioned by cultured BMMC, or in lysates of Ableson transformed BMMC and rat basophilic leukemic (RBL) cells. Both heparanase and beta-hexosaminidase, a mast cell granule enzyme, were released on degranulation of BMMC induced by the calcium ionophore A23187, or by exposure to IgE-Ag, suggesting that heparanase is localized in the cell granules. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase were released. Degradation of the ECM-HS by the mast cell heparanase and the associated release of HS-bound endothelial cell growth factors that are stored in ECM (Vlodavsky et al, Proc Natl Acad Sci USA 84:2292, 1987; Bashkin et al, Biochemistry 28:1737, 1989) may play a role in the proposed mast cell-mediated stimulation of neovascularization.     

Identification of aminopeptidase activity in the secretory granules of mouse mast cells.

Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme beta-hexosaminidase, thereby localizing approximately 60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 +/- 0.06 mM (mean +/- SD; n = 3) for leucine-beta-naphthylamide. When various amino acid beta-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala greater than Leu greater than Phe much greater than Arg much greater than Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of greater than 1 x 10(7) kDa as defined by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the amino-peptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.     

Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients’ sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: (i) the presence of DNase1 inhibitors or (ii) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.     

Impact of transplantation on neutrophil extracellular trap formation in patients with end-stage renal disease: A single-center,prospective cohort study

Increased neutrophil extracellular trap (NET) formation associates with high cardiovascular risk and mortality in patients with end-stage renal disease (ESRD). However, the effect of transplantation on NETs and its associated markers remains unclear. This study aimed to characterize circulating citrullinated Histone H3 (H3cit) and Peptidyl Arginase Deiminase 4 (PAD4) in ESRD patients undergoing transplantation and evaluate the ability of their neutrophils to release NETs.This prospective cohort study included 80 healthy donors and 105 ESRD patients, out of which 95 received a transplant. H3cit and PAD4 circulating concentration was determined by enzyme-linked immunosorbent assay in healthy donors and ESRD patients at the time of enrollment. An additional measurement was carried out within the first 6 months after transplant surgery. In vitro NET formation assays were performed in neutrophils isolated from healthy donors, ESRD patients, and transplant recipients.H3cit and PAD4 levels were significantly higher in ESRD patients (H3cit, 14.38 ng/mL [5.78–27.13]; PAD4, 3.22 ng/mL [1.21–6.82]) than healthy donors (H3cit, 6.45 ng/mL [3.30–11.65], P < .0001; PAD4, 2.0 ng/mL [0.90–3.18], P = .0076). H3cit, but not PAD4, increased after transplantation, with 44.2% of post-transplant patients exhibiting high levels (≥ 27.1 ng/mL). In contrast, NET release triggered by phorbol 12-myristate 13-acetate was higher in neutrophils from ESRD patients (70.0% [52.7–94.6]) than healthy donors (32.2% [24.9–54.9], P < .001) and transplant recipients (19.5% [3.5–65.7], P < .05).The restoration of renal function due to transplantation could not reduce circulating levels of H3cit and PAD4 in ESRD patients. Furthermore, circulating H3cit levels were significantly increased after transplantation. Neutrophils from transplant recipients exhibit a reduced ability to form NETs.     

IL-33 exacerbates antigen-induced arthritis by activating mast cells

IL-33, a cytokine of the IL-1 family, is closely associated with type II T cell responses. Here, we report an unexpected proinflammatory role of IL-33 in inflammatory arthritis. IL-33 was expressed in synovial fibroblasts from patients with rheumatoid arthritis (RA). Expression was markedly elevated in vitro by inflammatory cytokines. Mice lacking ST2, the IL-33 receptor α-chain, developed attenuated collagen-induced arthritis (CIA) and reduced ex vivo collagen-specific induction of proinflammatory cytokines (IL-17, TNFα, and IFNγ), and antibody production. Conversely, treatment of wild-type (WT) but not ST2^−/− mice with IL-33 exacerbated CIA and elevated production of both proinflammatory cytokines and anti-collagen antibodies. Mast cells expressed high levels of ST2 and responded directly to IL-33 to produce a spectrum of inflammatory cytokines and chemokines in vitro. In vivo, IL-33 treatment exacerbated CIA in ST2^−/− mice engrafted with mast cells from WT but not from ST2^−/− mice. Disease exacerbation was accompanied by elevated expression levels of proinflammatory cytokines. Our results demonstrate that IL-33 is a critical proinflammatory cytokine for inflammatory joint disease that integrates fibroblast activation with downstream immune activation mainly via an IL-33-driven, mast-cell-dependent pathway. Thus, this IL-1 superfamily member represents a therapeutic target for RA.     

Regulation of type 2 immunity to helminths by mast cells

Recently, we demonstrated a novel role for gastrointestinal mast cells (MCs) in the early events that lead to the generation of Th2 immunity to helminth infection. ( 1) Mice lacking MCs (Kit (W) /Kit (W-v) and Kit (W-Sh) ) showed a significant inhibition of Th2 cell priming following infection with the parasitic helminth Heligmosomoides polygyrus bakeri (Hp). We showed that MCs degranulate during the early stages of infection when the helminth larvae invade the small intestinal tissue. Furthermore, MC degranulation was required for the enhanced expression and production of the tissue-derived cytokines IL-25, IL-33 and TSLP, which are required for the optimal orchestration and priming of type 2 immunity. In this addendum we aim to address several questions raised by our findings - in particular, the mechanisms through which MCs may recognize helminth exposure in the early stages of infection and by which they may enhance expression of critical tissue cytokines thus, enabling Th2 priming. Furthermore, we will discuss these findings in the context of recently described novel innate immune cells, such as type 2 hematopoietic progenitors and type 2 innate lymphoid cells.     

Regulation of type 2 immunity to helminths by mast cells

Recently, we demonstrated a novel role for gastrointestinal mast cells (MCs) in the early events that lead to the generation of Th2 immunity to helminth infection.¹ Hepworth MR, Dani?owicz-Luebert E, Rausch S, Metz M, Klotz C, Maurer M, et al. Mast cells orchestrate type 2 immunity to helminths through regulation of tissue-derived cytokines. Proc Natl Acad Sci U S A 2012; 109:6644 - 9; http://dx.doi.org/10.1073/pnas.1112268109; PMID: 22493240 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Mice lacking MCs (Kit^W/Kit^W-v and Kit^W-Sh) showed a significant inhibition of Th2 cell priming following infection with the parasitic helminth Heligmosomoides polygyrus bakeri (Hp). We showed that MCs degranulate during the early stages of infection when the helminth larvae invade the small intestinal tissue. Furthermore, MC degranulation was required for the enhanced expression and production of the tissue-derived cytokines IL-25, IL-33 and TSLP, which are required for the optimal orchestration and priming of type 2 immunity. In this addendum we aim to address several questions raised by our findings — in particular, the mechanisms through which MCs may recognize helminth exposure in the early stages of infection and by which they may enhance expression of critical tissue cytokines thus, enabling Th2 priming. Furthermore, we will discuss these findings in the context of recently described novel innate immune cells, such as type 2 hematopoietic progenitors and type 2 innate lymphoid cells.