| Background:DCs are major antigen-presenting cells that play important roles in atopic diseases such as asthma. They present allergens to Th lymphocytes, prime Tho lymphocytes toward Th2differentiation to secrete Th2type cytokines such as IL-4, IL-5and IL-13to constitute the salient features of asthma, such as goblet-cell hyperplasia, airway-wall remodeling and bronchial hyper-reactivity. Therefore, they play a critical role in initiating and maintaining the airway inflammation of asthma. However, the precise mechanisms underlie these effects of DCs are still not completely understood. Store-operated calcium entry (SOCE) is the main Ca2+influx pathway of dendritic cells (DCs). DCs primed with histamine facilitate Th2immune response via different types of histamine receptors. Histamine induces DCs to release Ca2+from internal store. Therefore, we wonder that whether histamine could activate SOCE in DCs through its receptors, and what's the functional relevance of the Ca2+influx through SOCE induced by histamine in Th2response.Methods:Mononuclear cells were separated from human venous blood. Monocytes were positively selected by CD14+magnetic microbeads. Isolated monocytes were cultured and induced into dendritic cells (DCs). Cell toxicity of the reagents was evaluated by AlamarBlue reduction assay and cell counting after trypan blue exclusion. Cells were loaded with Fluo-3/AM and then the intracellular Ca2+concentration were measured by confocal Ca2+fluorescence imaging system. Western blotting was used to determine the protein expression levels of STIM1and Orail. Real-time PCR were used to half-quantify the mRNA expression of STIM1and Orai1in DCs. Flow cytometry was used to estimate the expression of specific surface markers of DCs. The levels of IL-10and IL-12p70in culture supernatants of DCs were measured by Elisa kits. DCs were co-incubated with allogenic lymphocytes for mixed lymphocyte reaction. The expression of IL-4and IFN-y secreting on CD3+CD8-lymphocytes (Th cells) were analyzed by Flow cytometry. Results:1). The major reagents at the chosen concentrations in our experiments were safe to the cells;2). Histamine induced significant Ca2+release response, re-addition of external Ca2+(2mM) evoked a Ca2+influx, which was inhibited by pretreatment with SOCE blockers SKF96365, BTP-2and2-APB;3). SOCE blocker BTP-2and H4receptor (H4R) antagonist JNJ7777120(JNJ) inhibited the increased expression of CD86induced by histamine. SOCE channel blocker SKF96365(SKF) and H1receptor (H1R) antagonist pyridylethylamine (Pyr) inhibited the increased expression of CD1a induced by histamine;4). When pretreated with JNJ or Pyr, the percentage of DCs that has an obvious Ca2+release response to histamine was decresed;5). After treatment with histamine, the protein expression of STIM1was up-regulated significantly, whereas the protein expression of Orail just has a mild up-regulation, but with no statistical significance. Real-time PCR indicated a similar result of genes expression of STIM1and Orail with western blotting;6). SOCE blockers SKF96365and BTP-2inhibited the increased IL-10secretion of mature DCs which was induced by histamine. Histamine inhibited the secretion of IL-12p70of DCs. SOCE blockers SKF95365and BTP-2also decreased the level of IL12p70in mature DCs induced by either LPS or histamine;7). In MLR, pretreatment with SOCE blockers and H1/4R antagonists of DCs could inhibited the Th2polarization of Th cells which was induced by histamine treated DCs.Conclusions:In conclusion, we found that histamine induces Ca2+influx through SOCE; histamine facilitates DCs maturation and Th2polarization by activating SOCE via its H1R and H4R and increasing the expression of STIM1; In addition, SOCE blockers and H1R/H4R antagonists alone or cooperate could reverse the effect of histamine-treated DCs on Th2polarization. Our results present new evidences for the mechanisms on Th2bias observed in allergic asthma. |
PDF Full Text Request