高迁移率族蛋白B1对小鼠调节性T细胞与CD4+CD25-T细胞相互作用的影响

目的 观察高迁移率族蛋白B1(HMGB1)对调节性T细胞(Treg)与CD4+CD25-T细胞相互作用的影响,并初步探讨其影响Treg抑制功能的机制.方法 免疫磁珠法分离正常BALB/c小鼠脾脏CD4+CD25+Treg及CD4+CD25-T细胞.采用固相包被抗CD3/可溶性抗CD28进行辅助活化,以不同时间及浓度HMGB1刺激Treg,ELISA法分析HMGB1刺激对Treg分泌IL-10的影响.将HMGB1(1000μg/L)刺激后的Treg与CD4+CD25-T细胞共培养,MTT法观察其对Treg抑制CD4+CD25-T细胞反应的作用,并分析HMGB1刺激后的Treg对CD4+CD25-T细胞IL-2生成及细胞功能极化的影响.结果 经抗CD3/CD28辅助活化的CD4+CD25+Treg在HMGB1作用下IL-2生成无显著差异(P＞0.05),但随时间延长及剂量增加,IL-10生成明显减少(P＜0.05).经HMGB1刺激的Treg对CD4+CD25-T细胞增殖的抑制反应减弱,同时诱导CD4+CD25-T细胞IL-2产生及细胞功能极化的能力均下降(P＜0.05).结论 HMGB1可通过诱导Treg抑制功能的下调,从而影响CD4+CD25-T细胞功能,进而调节炎症反应过程.

Influence of high mobility group box-1 protein on the correlation between regulatory T cells and CD4+ CD25- T cells of spleen in mice

OBJECTIVE: To investigate the influence of high mobility group box-1 protein (HMGB1) on the immunosuppression function of splenic regulatory T cells (Tregs) and its potential regulatory mechanism underlying the effect on CD4+ CD25- T cells in mice. METHODS: CD4+ CD25+ Tregs isolated from the spleens of male BALB/c mice by magnetic beads were seeded on 96-well (1 x 10(5) cells/well) cell culture plates coated with 1 microg/ml anti-CD3 and soluble CD28. After being stimulated with HMGB1 for different time and concentrations, the secretions of IL-2 and IL-10 were analyzed by ELISA. Tregs stimulated for 72 hours were cultured with CD4+ CD25- T cells together. The suppressive activity of CD4+ CD25+ Treg to CD4+ CD25- T cells was analyzed by MTT test. IL-2, IL-10, IL-4, and interferon (IFN)-gamma in the cell suspensions were determined by ELISA. RESULTS: After stimulation with HMGB1, the suppressive activity of splenic Tregs in mice were significantly down-regulated at 72 hours, when the proportion of Tregs to CD4+ CD25- T cells was 1 : 1. The secretion of IL-2 of Tregs stimulated by HMGB1 was not markedly changed (P > 0.05), while a dose-dependent decrease between IL-10 induction and HMGB1 concentration was obviously (P < 0.05). When CD4+ CD25- T cells were cultured with stimulated Tregs, comparing with unstimulated-Treg group, levels of IL-2 and IFN-gamma were elevated following the increased concentration of HMGB1 (P < 0.05 or P < 0.01). Meanwhile the secretion of IL-4 and IL-10 significantly decreased when cultured with stimulated Tregs (P < 0.05). CONCLUSIONS: These data suggested that HMGB1 stimulation can result in significant down-regulation of immunosuppression of splenic Tregs in mice. HMGB1 might be a potential immunoregulatory signal that influences the proliferation of effector T cells, secretion of IL-2 and cells-polarization by inhibiting CD4+ CD25+ Tregs activity.