IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis

Interleukin 9 (IL-9) has been implicated in mast cell-related inflammatory diseases, such as asthma, where vascular endothelial growth factor (VEGF) is involved. Here we report that IL-9 (10-20 ng/ml) induces gene expression and secretion of VEGF from human LAD2. IL-9 does not induce mast cell degranulation or the release of other mediators (IL-1, IL-8, or TNF). VEGF production in response to IL-9 involves STAT-3 activation. The effect is inhibited (about 80%) by the STAT-3 inhibitor, Stattic. Gene-expression of IL-9 and IL-9 receptor is significantly increased in lesional skin areas of atopic dermatitis (AD) patients as compared to normal control skin, while serum IL-9 is not different from controls. These results imply that functional interactions between IL-9 and mast cells leading to VEGF release contribute to the initiation/propagation of the pathogenesis of AD, a skin inflammatory disease.     

Substance P induces granulocyte infiltration through degranulation of mast cells

Substance P, a potent vasodilatory neuropeptide, is released from peripheral nerve endings of sensory neurons by various stimuli. Although in vitro incubation of rat and human mast cells with substance P causes their degranulation, it is not known whether inflammatory changes induced by substance P are mediated by degranulation of mast cells. We investigated this point by using genetically mast cell-deficient WBB6F1-W/Wv and WCB6F1-Sl/Sld mice. The s.c. injection of substance P induced degranulation of mast cells in the skin of WBB6F1-+/+ mice, and then a marked eosinophil infiltration around the degranulated mast cells. However, WBB6F1-W/Wv and WCB6F1-Sl/Sld mice showed little or no eosinophil infiltration in the skin after the injection of substance P. When the mast cell deficiency of WBB6F1-W/Wv mice was rescued either systemically by bone marrow transplantation or locally by injection of cultured mast cells, injection of substance P induced the infiltration of eosinophils, suggesting that substance P-induced eosinophil infiltration was mediated through degranulation of mast cells.     

Nerve growth factor induces mast cell degranulation without changing intracellular calcium levels

Nerve growth factor (NGF) induces degranulation of rat peritoneal mast cells (RPMC) in a dose-dependent manner, providing direct evidence for its action on non-neuronal tissues. Activation of RPMC by NGF depends on lysophosphatidylserine and extracellular calcium. NGF-mediated RPMC degranulation is not coupled to a transient increase in intracellular free calcium ([Ca2+]i). It is suggested that NGF has a unique mode of action independent of [Ca2+]i and presumably also without involving protein kinase C activation as indicated by the effects of phorbol esters and NGF on antigen-evoked [Ca2+]i rise.     

Thrombin induces IL-6 but not TNFalpha secretion by mouse mast cells: threshold-level thrombin receptor and very low level FcepsilonRI signaling synergistically enhance IL-6 secretion

Mast cells become activated in multiple diseases wherein thrombin generation is often clinically apparent, but the effect of thrombin on cytokine release by mast cells remains unexplored. Thus, we examined IL-6 and TNFalpha release by thrombin-challenged mast cells. Thrombin and the protease-activated receptor (PAR)-1 peptide TRAP(14) induced these cells to secrete IL-6 in a dose-dependent fashion. Mast cells secreted > or =2800 pg IL-6/10(6) cells over 24 h, but only low levels of serotonin and no significant TNFalpha. Furthermore, at near-background levels of allergen, threshold doses of alpha-thrombin synergistically enhanced the IL-6 response (by up to 100-fold), but high-dose costimulation led to a simple additive response. Both the PI(3)- and sphingosine-kinase signaling pathways contributed importantly to the thrombin response. Our data thus clearly demonstrate that low-level thrombin and FcepsilonRI signaling can synergize to augment mast cell IL-6 responses, and that thrombin also differentially induces cytokine secretion by mast cells.     

IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells

We examined whether interleukin (IL)-6 exerts the stimulatory effects on the secretion of histamine from human mast cells triggered by crosslinking of the high affinity IgE receptor (FcepsilonRI) with IgE and anti-IgE. As target cells, we used peripheral blood-derived cultured mast cells grown with SCF, because they were superior in FcepsilonRIalpha expression to cord blood-derived mast cells. Incubation with SCF+IL-6 for 1 week increased the IgE-dependent release as well as intracellular content of histamine in the cultured mast cells, as compared with the values obtained by incubation with SCF alone. The magnitude of these increases was higher than that for priming with SCF+IL-4. A striking difference was also found in the expression of FcepsilonRIalpha between the two-factor combinations. The addition of IL-6 during FcepsilonRI crosslinking with IgE/anti-IgE in the presence of SCF did not influence histamine secretion. When SCF, IL-6 and IL-4 were used together, a further increase was observed in the anti-IgE-dependent liberation of histamine from the cultured mast cells, compared with the two-factor combinations. These results suggest that IL-6 functions as a secretagogue for the inflammatory mediator of human mast cells in the presence of SCF.     

B cell lymphoma 10 is essential for FcepsilonR-mediated degranulation and IL-6 production in mast cells

The adaptor protein B cell lymphoma 10 (Bcl10) plays an essential role in the functions of the AgRs in T and B cells. In this study, we report that Bcl10 also plays an important role in mast cells. Bcl10 is expressed in mast cells. Although Bcl10-deficient mast cells undergo normal development, we demonstrate that Bcl10 is essential for specific functions of FcepsilonR. Although Bcl10-deficient mast cells have normal de novo synthesis and release of the lipid mediator arachidonic acid, the mutant cells possess impaired FcepsilonR-mediated degranulation, indicated by decreased serotonin release, and impaired cytokine production, measured by release of IL-6. In addition, Bcl10-deficient mice display impaired IgE-mediated passive cutaneous anaphylaxis. Moreover, although Bcl10-deficient mast cells have normal FcepsilonR-mediated Ca(2+) flux, activation of PI3K, and activation of the three types of MAPKs (ERKs, JNK, and p38), the mutant cells have markedly diminished FcepsilonR-mediated activation of NF-kappaB and decreased activation of AP-1. Thus, Bcl10 is essential for FcepsilonR-induced activation of AP-1, NF-kappaB, degranulation, and cytokine production in mast cells.     

Cryptococcus neoformans induces IL-8 secretion and CXCL1 expression by human bronchial epithelial cells

Background

Smoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD. The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.

Methods

Mice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.

Results

Smoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4⁺CD25⁺ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4⁺CD25⁺ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.

Conclusion

These results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca²⁺ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10⁻⁴–10⁻⁵ M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

Endothelin-1 induces mucosal mast cell degranulation in the rat small intestine

The enhanced production of endothelial cell-derived vasoactive mediators and the activation of mast cells (MCs) have been implicated in the pathogenesis of mucosal damage during ischemia and reperfusion injuries. The first objective of our study was to define the in vivo relation between endothelin-1 (ET-1) and the MC system. Secondly, we determined whether pretreatment with ET receptor antagonists would attenuate MC responses to exogenous ET-1. In the first series of experiments, increasing doses of ET-1 (0. 1, 1 and 3 nmol/kg i.v.) were administered to anesthetized rats. In the second series, the animals were pretreated with equimolar doses of the ET-A receptor antagonist BQ-610 or ETR-P1/fl peptide, and the ET-B receptor antagonist IRL-1038. Intestinal perfusion changes and macrohemodynamics were recorded, and the proportion of degranulated MCs was determined in ileal biopsies. The average mucosal thickness was recorded with an image analysis system. ET-1 induced dose-dependent alterations in the hemodynamic and morphological parameters and caused pronounced mucosal injury, with a significant reduction in villus height. The ratio of degranulated MCs was similar in all ET-treated groups (77%, 82% and 86%) to that observed in animals subjected to 15-min ischemia and 60-min reperfusion (85% degranulation). Pretreatment with BQ-610 and ETR-P1/fl peptide attenuated the ET-1 induced alterations in the hemodynamic parameters and decreased structural injury to the mucosa. ET-induced MC degranulation was significantly inhibited by the ET-A receptor antagonists, but not by IRL-1038. These results indicate that elevated levels of circulating ET-1 might induce intestinal mucosal tissue injury and MC degranulation via activation of ET-A receptors, and raise the possibility that ET-A receptor antagonist administration could exert a potentially beneficial effect through a mechanism other than the blockade of vasoconstriction in pathologies associated with an increased ET-1 release.     

The vanilloid capsaicin induces IL-6 secretion in prostate PC-3 cancer cells

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a constituent of green and red peppers, has been linked with suppression of tumorigenesis through a mechanism that is not well understood. In the present study, we examined the effects of capsaicin on the production of the cytokine interleukin (IL)-6 by PC-3 cells at both protein and mRNA levels which were evaluated by ELISA and real-time PCR, respectively. Capsaicin-treated PC-3 cells increased the synthesis and secretion of IL-6 which was abrogated by the transient receptor potential vanilloid receptor subtype 1 (TRPV1) antagonist capsazepine, as well as by inhibitors of PKC-α, phosphoinositol-3 phosphate kinase (PI-3K), Akt and extracellular signal-regulated protein kinase (ERK). We analyzed the role of capsaicin in the tumor necrosis factor (TNF)-α secretion by PC-3 cells which was increased at shorter times than IL-6 production. Furthermore, incubation of PC-3 cells with an anti-TNF-α antibody blocked the capsaicin-induced IL-6 secretion. These results raise the possibility that capsaicin-mediated IL-6 increase in prostate cancer PC-3 cells is regulated at least in part by TNF-α secretion and signaling pathway involving Akt, ERK and PKC-α activation.     

IL-4 induces the proteolytic processing of mast cell STAT6

IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells.     

SNARE complex-mediated degranulation in mast cells

Mast cell function and dysregulation is important in the development and progression of allergic and autoimmune disease. Identifying novel proteins involved in mast cell function and disease progression is the first step in the design of new therapeutic strategies. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins demonstrated to mediate the transport and fusion of secretory vesicles to the membrane in mast cells, leading to the subsequent release of the vesicle cargo through an exocytotic mechanism. The functional role[s] of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we review recent and historical data on the expression, formation and localization of various SNARE proteins and their complexes in murine and human mast cells. We summarize the functional data identifying the key SNARE family members that appear to participate in mast cell degranulation. Furthermore, we discuss the utilization of RNA interference (RNAi) methods to validate SNARE function and the use of siRNA as a therapeutic approach to the treatment of inflammatory disease. These studies provide an overview of the specific SNARE proteins and complexes that serve as novel targets for the development of new therapies to treat allergic and autoimmune disease.     

Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.     

Activation of normal human B cells through their antigen receptor induces membrane expression of IL-1 alpha and secretion of IL-1 beta

We studied whether normal human B cells would express IL-1 activity and transcribe IL-1 genes before or after activation through their Ag receptor. Anti IgM antibody-activated B cells expressed IL-1 alpha on the cell surface and secreted IL-1 beta. Optimal induction of IL-1 occurred within 16 h of anti-IgM activation. mRNA specific for both IL-1 alpha and IL-1 beta was also induced upon activation. It is likely that the expression of m-IL-1 alpha on activated B cells together with the secretion of IL-1 beta represent important contributions in the efficient Ag-presenting capacity of B cells to T cells.     

Role of Ca(2+) on TNF-alpha and IL-6 secretion from RBL-2H3 mast cells

Ca2+ acts as an important second messenger in mast cells. However, the mechanisms involved in the secretion of inflammatory cytokines from activated mast cells are unknown. In this study, we examined the signaling pathway involved in calcium-related cytokine secretion in a mast cell line, RBL-2H3 cells. We report that treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a chelator of intracellular calcium, can inhibit IgE-stimulated TNF-alpha and IL-6 secretion in a concentration-dependent manner with IC50 values of 0.41 and 0.014 microM, respectively. Maximal inhibition of TNFalpha- and IL-6 secretion was 58.5 +/- 3% and 87 +/- 8% in BAPTA-AM, respectively. BAPTA-AM also completely inhibited the IgE-induced TNF-alpha and IL-6 mRNA levels. In activated RBL-2H3 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus. However, the level of NF-kappaB/Rel A in nucleus was decreased by treatment of BAPTA-AM. In addition, BAPTA-AM completely inhibited the IgE-induced IkappaB kinase beta (IKKbeta) activation and IkappaBalpha phosphorylation. These observations demonstrate that the intracellular Ca2+ may play an important role in IgE-induced TNF-alpha and IL-6 secretion from mast cells via IKKbeta activation.     

P2X7 receptors induce degranulation in human mast cells

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.