Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.
ABSTRACT: Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.
Project description:Roxatidine is an active metabolite of roxatidine acetate hydrochloride which is a histamine H2-receptor antagonist that is used to treat gastric and duodenal ulcers. In this study, we investigated the anti-allergic inflammatory effects and the underlying molecular mechanism of roxatidine in phorbol 12-myristate 13-acetate and calcium ionophore (PMACI)-stimulated human mast cells-1 (HMC-1), compound 48/80-induced anaphylactic animal model and chemical allergen-induced contact hypersensitivity (CHS) models. Roxatidine suppressed the mRNA and protein expression of inflammatory cytokines such as TNF-?, IL-6, and IL-1? in PMACI-stimulated HMC-1 and compound 48/80-induced anaphylactic mice. In addition, roxatidine attenuated PMACI-induced nuclear translocation of NF-?B and the phosphorylation of MKK3/6 and MK2, which are both involved in the p38 MAPK pathway. Furthermore, we observed that roxatidine suppressed the activation of caspase-1, an IL-1? converting enzyme, in PMACI-stimulated HMC-1 and compound 48/80-induced anaphylactic mice. In CHS model, roxatidine significantly reduced ear swelling, increased number of mast cells, production levels of cytokines and migration of dendritic cells. Our findings provide evidence that the anti-allergic inflammatory properties of roxatidine are mediated by the inhibition of NF-?B and caspase-1 activation, p38 MAPK pathway and mast cell-derived cytokine production. Taken together, the in vitro and in vivo anti-allergic inflammatory effects suggest a possible therapeutic application of roxatidine in allergic inflammatory diseases.
Project description:Peimine is a major biologically active component of Fritillaria ussuriensis. Peimine was investigated in chronic inflammation response, but it has not been studied in mast cell-related immediate allergic reaction. The present study aimed to evaluate anti-allergic effect of peimine in human mast cell (HMC-1).The effect of peimine on cell viability was measured by MTS assay in HMC-1. Histamine release was investigated in rat peritoneal mast cells (RPMCs). Interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) expressions were measured by ELISA assay and reverse transcription-polymerase chain reaction. Mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB) were examined by Western blot. Passive cutaneous anaphylaxis (PCA) reactions were evaluated using Sprague-Dawley (SD) rats.Peimine inhibited the production of pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-α. Moreover, peimine reduced MAPKs phosphorylation and the nuclear NF-κB expression in PMACI-induced HMC-1. Peimine decreased PCA reactions in rats as well.Our study proved that peimine might be suitable for the treatment of mast cell-derived allergic inflammatory reactions.Peimine inhibited the production of pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-αPeimine reduced MAPKs phosphorylation and the nuclear NF-κB expression in PMACI-induced HMC-1Peimine decreased PCA reactions in ratsPeimine has anti-allergic effect through regulation of pro-inflammatory mechanism on mast cell. Abbreviations used: HMC-1: Human mast cell, MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, RPMCs: Rat peritoneal mast cells. IL-6: Interleukin 6, IL-8: Interleukin 8, TNF-α: Tumor necrosis factor-α, MAPKs: Mitogen-activated protein kinases; NF-κB: Nuclear factor-kappaB, PCA: Passive cutaneous anaphylaxis reactions, SD: Sprague-Dawley.
Project description:Mast cells play a critical role in the development of the allergic response. Upon activation by allergens and IgE via the high affinity receptor for IgE (Fc?RI), these cells release histamine and other functional mediators that initiate and propagate immediate hypersensitivity reactions. Mast cells also secrete cytokines that can regulate immune activity. These processes are controlled, in whole or part, by increases in intracellular Ca(2+) induced by the Fc?RI. We show here that N-(4-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), a pyrazole derivative, inhibits activation-induced Ca(2+) influx in the rat basophil cell line RBL-2H3 and in bone marrow-derived mast cells (BMMCs), without affecting global tyrosine phosphorylation of cellular proteins or phosphorylation of the mitogen-activated protein kinases Erk1/2, JNK and p38. BTP2 also inhibits activation-induced degranulation and secretion of interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, tumor necrosis factor (TNF)-?, and granulocyte macrophage-colony stimulating factor (GM-CSF) by BMMCs, which correlates with the inhibition of Nuclear Factor of Activated T cells (NFAT) translocation. In vivo, BTP2 inhibits antigen-induced histamine release. Structure-activity relationship analysis indicates that substitution at the C3 or C5 position of the pyrazole moiety on BTP2 (5-trifluoromethyl-3-methyl-pyrazole or 3-trifluoromethyl-5-methyl-pyrazole, respectively) affected its activity, with the trifluoromethyl group at the C3 position being critical to its activity. We conclude that BTP2 and related compounds may be potent modulators of mast cell responses and potentially useful for the treatment of symptoms of allergic inflammation.
Project description:Background:BIOGF1K, a fraction of Panax ginseng, has desirable antimelanogenic, anti-inflammatory, and antiphotoaging properties that could be useful for treating skin conditions. Because its potential positive effects on allergic reactions in skin have not yet been described in detail, this study's main objective was to determine its efficacy in the treatment of atopic dermatitis (AD). Methods:High-performance liquid chromatography was used to verify the compounds in BIOGF1K, and we used the (3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide method to determine its cytotoxicity in RBL-2H3 and HMC-1 cell lines. RBL-2H3 cells were induced using both anti-DNP-IgE/DNP-BSA and calcium ionophore (A2187) treatments, whereas HMC-1 cells were induced using A2187 alone. To measure mast cell degranulation, we performed histamine (enzyme-linked immunosorbent assay) and ?-hexosaminidase assays. To quantify interleukin (IL)-4, IL-5, and IL-13 levels in RBL-2H3 cells, we performed quantitative polymerase chain reaction (PCR); to quantify expression levels of IL-4 and IL-13 in HMC-1 cells, we used semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Finally, we detected the total and phosphorylated forms of extracellular signal-regulated kinase, p-38, and c-Jun N-terminal kinase proteins by immunoblotting. Results:BIOGF1K decreased the AD response by reducing both histamine and ?-hexosaminidase release as well as reducing the secretion levels of IL-4, IL-5, and IL-13 in RBL-2H3 cells and IL-4 and IL-13 in HMC-1 cells. In addition, BIOGF1K decreased MAPK pathway activation in RBL-2H3 and HMC-1 cells. Conclusions:BIOGF1K attenuated the AD response, hence supporting its use as a promising and natural approach for treating AD.
Project description:We previously reported the anti-inflammatory and anti-asthmatic activities of the extract of the Inula japonica Thunb. Aiming for discovery of a novel anti-inflammatory compound, we isolated spinacetin from the extract and investigated its in vitro and in vivo anti-inflammatory effect and the related mechanism. Effect of spinacetin on the Syk signaling pathway was studied in bone marrow-derived mast cells (BMMCs), and that on the nuclear factor-?B (NF-?B) and mitogen-activated protein kinases (MAPKs) was investigated in Rat basophilic leukemia (RBL)-2H3 cells and human mast cell line (HMC-1). The in vivo anti-inflammatory activity was assessed with passive cutaneous anaphylaxis (PCA) reaction assay. Spinacetin significantly inhibited the release of histamine, and production of inflammatory mediators such as leukotriene C4 (LTC4) and interlukin-6 (IL-6) in IgE/Ag stimulated BMMCs. Analysis of the signaling pathways demonstrated that spinacetin inhibited activation of Syk, linker of activated T cells (LAT), phospholipase C? (PLC?), cytosolic phospholipase A2 (cPLA2), MAPKs, Akt/NF-?B, and intracellular Ca2+ mobilization but with no effect on Fyn and Lyn. On the other hand, spinacetin suppressed IgE/Ag-induced activation of RBL-2H3 cells with inhibition against phosphorylation of extracellular signal regulated-protein kinase (ERK), c-Jun-NH2-terminal kinase (JNK), p38 MAPKs, PLC?, translocation of cPLA2, and Akt/I?B?/NF-?B signal. However, spinacetin had no effect on PMA and A23187-induced activation of HMC-1. Furthermore, oral administration of spinacetin dose-dependently attenuated IgE/Ag-mediated PCA reaction in mouse model. Taken together, spinacetin showed the activities in preventing inflammatory processes, which might be at least partially attributed to the abolishment of Syk-dependent activation of IgE/Ag-mediated mast cells.
Project description:Allergic diseases including allergic rhinitis, asthma, and atopic dermatitis are increasing worldwide. Common medications used to treat these inflammatory disorders are anti-histamines and corticosteroids, but they have their own limitations such as short duration and severe side effects. Thus, interest in complementary and alternative medicine is continually growing. Here, we investigate the anti-inflammatory mechanisms of Tonggyu-tang (TGT), a traditional Korean medicine that has been used to treat patients with allergic nasal disorders.We measured mRNA expressions and production of pro-inflammatory cytokines such as interleukin (IL)-4, IL-6, IL-8 and tumor necrosis factor alpha (TNF-?) by RT-PCR and ELISA assays in HMC-1 (human mast cell line-1) and HaCaT cells, immortalized human keratinocytes. Moreover, we evaluated the effect of TGT on two major inflammation-related pathways, mitogen activated protein kinase (MAPK) and NF-?B signaling pathway in these two cells.Our results revealed that that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-? in the agonist-treated HMC-1 and HaCaT cells. We also found that TGT suppressed MAPK signaling pathway including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) as well as NF-?B pathway, which are known to regulate inflammatory cytokine expression.Taken together, our results demonstrate that TGT inhibits expression of pro-inflammatory cytokines by suppressing MAPK and NF-kB pathway in both mast cells and keratinocytes, suggesting the potential use of TGT in treating allergic inflammatory diseases.
Project description:AIM:To detect and evaluate the antibodies against Helicobacter pylori (H pylori) neutrophil-activating protein (HP-NAP) in patients with gastric cancer and other gastroduodenal diseases. METHODS:Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coli. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 (IL-8) and growth-related oncogene (GRO(alpha)) cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected. RESULTS:The serum positivity and mean absorbance value of HP-NAP-specific antibodies in the gastric cancer group (97.7% and 1.01 +/- 0.24) were significantly higher than those in the chronic gastritis group (85.7% and 0.89 +/- 0.14, P < 0.005) and healthy control group (27.7% and 0.65 +/- 0.18, P < 0.001). The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly up-regulate IL-8 production in gastric epithelial cell lines but had no effect on GRO(alpha) production. CONCLUSION:Infection with virulent H pylori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma. rHP-NAP-based ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.
Project description:There are several clinical trials worldwide using bone marrow stromal cells (BMSCs) as a cellular therapy to modulate immune responses in patients suffering from various inflammatory conditions. A deeper understanding of the molecular mechanisms involved in this modulatory effect could help us design better, more effective protocols to treat immune mediated diseases. In this study, we demonstrated that human BMSCs express H1, H2, and H4 histamine receptors and they respond to histamine stimulation with an increased interleukin 6 (IL-6) production both in vitro and in vivo. Using different receptor antagonists, we pinpointed the importance of the H1 histamine receptor, while Western blot analysis and application of various mitogen-activated protein kinase inhibitors highlighted the role of p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase kinases in the observed effect. When BMSCs were pretreated with either histamine or degranulated human mast cells, they exhibited an enhanced IL-6-dependent antiapoptotic effect on neutrophil granulocytes. Based on these observations, it is likely that introduction of BMSCs into a histamine-rich environment (such as any allergic setting) or pretreatment of these cells with synthetic histamine could have a significant modulatory effect on the therapeutic potential of BMSCs.
Project description:BACKGROUND:Gastric cancer arises, mainly, on an inflammatory background. Helicobacter pylori neutrophil activating (HP-NAP) protein functions as a potent pro-inflammatory mediator. Similarly, IL-4 plays a critical role in the inflammation pathway, the levels of which are altered by C to T transition at position -590 in its promoter region. Here, we have aimed to assess the risk of gastritis and gastric cancer in the co-presence of these two inflammation modulating mediators. METHODS:Gastritis (n=58) and gastric cancer (n=31) patients were evaluated and compared with H. pylori-positive asymptomatic controls (n=46), for serum antibodies against recombinant HP-NAP and IL-4 C-590T single nucleotide polymorphism using immunoblotting and PCR-RFLP, respectively. Multivariable logistic regression, adjusting for age, gender and ethnicity, was used for data analysis. RESULTS:In terms of susceptibility to gastritis, seropositivity to HP-NAP projected a risk impact of 4.62 fold (OR=4.62, 95% CI=1.50-14.22), which when present in IL-4 -590 T carriers augmented the risk up to 9.7 fold (OR=9.70, 95% CI=2.06-45.69). A similar pattern, but of a stronger magnitude, occurred for the risk of gastric cancer, which was estimated at 9.07 fold (OR=9.07, 95% CI=1.99-42.0) for HP-NAP-seropositive subjects and was drastically amplified (OR=33.64, 95% CI=2.06-548.68), when double-positive (HP-NAP seropositive/IL-4 -590 T carrier) subjects were examined against double negatives (HP-NAP seronegative/IL-4 -590 CC). CONCLUSION:Our preliminary data indicate that serum antibodies against HP-NAP represent a state of risk, which is further exacerbated in IL-4 -590 T carriers. These biomarkers, if validated in larger prospective studies, can be used to screen for gastric cancer susceptibility.
Project description:1.We investigated the role of Annexin (ANX)-A1 and its receptor, ALX/FPR2, in the regulation of mast cell degranulation produced by compound 48/80. 2.Both human cord-blood derived mast cells (CBDMCs) and murine bone marrow derived mast cells (BMDMCs) release phosphorylated ANX-A1 during treatment with glucocorticoids or the mast cell 'stabilising' drugs ketotifen and nedocromil. 3.Compound 48/80 also stimulated ANX-A1 phosphorylation and release and this was also potentiated by nedocromil. Anti-ANX-A1 neutralising monoclonal antibodies (Mabs) enhanced the release of pro-inflammatory mediators in response to compound 48/80. 4.Nedocromil and ketotifen potently inhibited the release of histamine, PGD2, tryptase and ?-hexosaminidase from mast cells challenged with compound 48/80. Anti-ANX-A1 neutralising Mabs prevented the inhibitory effect of these drugs. 5.BMDMCs derived from Anx-A1?/? mice were insensitive to the inhibitory effects of nedocromil or ketotifen but cells retained their sensitivity to the inhibitory action of hu-r-ANX-A1. 6.The fpr2/3 antagonist WRW4 blocked the action of nedocromil on PGD2, but not histamine, release. BMDMCs derived from fpr2/3?/? mice were insensitive to the inhibitory effects of nedocromil on PGD2, but not histamine release. 7.Compound 48/80 stimulated both p38 and JNK phosphorylation in CBDMCs and this was inhibited by nedocromil. Inhibition of p38 phosphorylation was ANX-A1 dependent. 8.We conclude that ANX-A1 is an important regulator of mast cell reactivity to compound 48/80 exerting a negative feedback effect through a mechanism that depends at least partly on the FPR receptor.