高迁移率族蛋白B1对小鼠调节性T细胞表达T淋巴细胞毒性相关抗原-4水平的影响及受体机制

Potential receptor mechanism underlying the effect of high mobility group box-1 protein on expression of cytotoxic T lymphocyte-associated antigen 4 in regulatory T ceils in mice

目的观察高迁移率族蛋白B1（HMGB1）对小鼠调节性T细胞（Treg）表达细胞毒性T淋巴细胞相关抗原（CTLA）-4水平的影响及其受体机制。方法免疫磁珠法分离正常C3H／HeN[（Toll样受体4（TLR4）野生型）]小鼠脾脏CD4＋CD25＋Treg，接种于细胞培养板，各细胞培养孔均加入可溶性抗CD3和可溶性抗CD28作为辅助活化剂。应用／不应用抗TLR4抗体预封闭CD4＋CD25＋Treg表面TLR4，观察HMGB1刺激CD4＋CD25＋Treg表达CTLA-4的时间．效应关系和剂量-效应关系。结果HMGB1（0．1mg／L）刺激CIM＋CD25＋Treg，cTLA-4在24、48、72h表达水平（47．56±5．49、35．83±4．96和31．94±4．65）较正常对照组水平（79．42±2．79）均显著下降（P〈0．01），以48、72h组下降尤为明显；不同剂量HMGB1刺激CD4＋CD25＋Treg48h其CTLA-4表达水平显著下调（P〈0．01），其中以0．1mg／L和1mg／L组表达水平（分别为33．34±4．00和29．90±4．30）降低幅度尤为明显。经封闭TLR4预处理后，给予HMGB1（0．1mg／L）刺激24、48和72hCD4＋CD25＋Treg表达CTLA-4的水平（90．39±8．80、93．13±4．80和80．29±4．31）均较对正常照组（71．03±4．34）显著增强（P〈0．05或P〈0．01）；而不同剂量HMGB1刺激CD4＋CD25＋Treg48h，仅以0．1mg／L组能显著上调CTLA-4的表达水平（P〈0．01）。结论HMGB1通过TLR4负向调节CD4＋CD25＋Treg表达CTLA-4水平，从而影响Treg的免疫活性。

Objective To investigate the effect of high mobility group box-1 protein （HMGB1） on the expression of cytotoxic T lymphocyte-associated antigen 4 （CTLA-4） in regulatory T cells （Treg） and its potential receptor mechanism in mice. Methods CD4＋ CD25＋ Tregs isolated from the spleens of C3H/HeN [ Toll-like receptor 4 （TLR4） wild type l mice by magnetic beads were seeded on 96-well cell culture plates coated with 1 mg/L anti-CD3 and soluble CD28. By treatment with or without anti-mouse TLR4 antibody ,the time-and dose-dependent responses between HMGB1 stimulation and CTLA-4 expression on CD4 ＋ CD25 ＋ Tregs were studied. Results Compared to normal controls, the expression of CTLA-4 on CIM ＋ CD25＋ Tregs stimulated by 0.1 mg/L HMGB1 was significantly down-regulated at 24,48 ,and 72 h （47.56 ±5.49,35.83 ±4.96,and 31.94 ±4.65 ,respectively） （ all P 〈0.01 ） ,especially at 48 and 72 h. Meanwhile, markedly decreased expression of CTLA-4 was found CD4＋CD25 ＋ Tregs treated with HMGB1 at various concentrations （0.01,0.1 and 1 rag/L） for 48 h （58.87 ± 6.70,33.34 ±4.00 and 29.90 ±4.30, respectively, all P 〈 0.01 ）. After pretreatment with TLR4 antibody, however, the CTLA-4 expression on CD4＋ CD25 ＋ Tregs was significantly enhanced by HMGB1 （0.1 mg/L） stimulation at 24,48 and 72 h （90.39 ± 8.80,93.13±4.80 and 80.29 ± 4.31, respectively, P 〈 0.05 or P 〈 0.01 ）. After treatment with different doses of HMGB1 for 48 h ,the CTLA-4 expression on CD4 ＋ CD25 ＋ Tregs was much higher in 0.1 mg/L HMGB1 group （94.98 ± 6.35 ） than in normal controls （ P 〈 0.01 ）. Conclusion With HMGB1 stimulation,TLR4 can markedly down-regulate CTLA-4 expression levels on CD4＋ CD25 ＋ Tregs, thereby influencing the immunological activity of Tregs.