Toll样受体4在高迁移率族蛋白B1影响小鼠调节性T细胞免疫功能中的作用

目的 探讨腹腔注射高迁移率族蛋白B1(HMGB1)后不同基因型小鼠天然调节性T细胞(CD4~+CD25~+Treg)免疫功能的改变及其受体作用机制.方法 分别给C3H/HeN和C3H/HeJ分别为Toll样受体4(TLR4)野生型(TLR4~(+/+))和天然突变型(TLR4~(-/-)]小鼠腹腔注射不同剂量HMGB1(0、10、20μg/只),饲养48 h后采用免疫磁珠法分离小鼠脾脏CD4~+CD25~+Treg.体外培养12 h后采用流式细胞仪检测CD4~+CD25~+Treg表达细胞毒性T淋巴细胞相关抗原-4(CTLA-4)表达强度,并应用酶联免疫吸附试验(ELISA)检测CD4~+CD25~+Treg生成白细胞介素(IL)-10量.结果 与对照组比较,20 μg HMGB1攻击后C3H/HeN小鼠CD4~+CD25~+Treg表达CTLA-4水平显著下降(78.70±11.42与60.76±7.64,P<0.01),同时细胞牛成IL-10量也明显降低(96.89±6.25)ng/L与(47.11±4.25)ng/L,P<0.01];但不同剂量HMGB1攻击可引起C3H/HeJ小鼠CD4~+CD25~+Treg表达CTLA-4明显上调和生成IL-10量不同程度地增加(P<0.01).结论 HMGB1攻击可显著影响CD4~+CD25~+Treg介导的免疫状态,TLR4在HMGB1诱导CD4~+CD25~+Treg免疫活性过程中发挥了重要负向调控作用.

The potential role of Toll-like receptor-4 in high mobility group box-1 protein-mediated immune function of regulatory T cells in mice

Objective To investigate the effect of high mobility group box-1 protein(HMGB1) on immune function of regulatory T cells(Tregs) in two gene-type mice and its potential receptor mechanism in vivo.Methods 10 μg or 20 μg HMGB1 was administered i.P.in C3H/HeN(TLR4 wild type,TLR4~(+/+))and C3H/HeJ (TLR4 mutant type,TLR4~(-/-))mice.Mice were sacrificed at the 48th h after HMGB1 challenge,and splenic CD4~+CD25~+ Tregs were harvested by magnetic beads aseptically following another 12 h culture.Cytotoxic T lymphocyte-associated antigen 4(CTLA-4)expressed on CD4~+ CD25~+ Tregs was detected by means of flow cytometry,and the production of interleukin(IL)-10 in supematants was examined by ELISA.Results The expression levels of CTLA-4 in two gene-type mice were similar (P>0.05).Compared to the controls,the expression of CTLA-4 on CD4~+ CD25~+ Tregs was markedly down-regulated in C3H/HeN mice with a maximal response at a dose of 20μg HMGB1(78.70±1 1.42 vs 60.76±7.64,P<0.01),and IL-10 release was also significantly decreased(96.89±6.25)vs(47.11±4.25)ng/L,P<0.01].However,after treatment with various doses of HMGB1 in C3H/HeJ mice.the expression of CTLA-4 on CD4~+ CD25~+ Tregs and IL-10 release were markedly enhanced compared to the controls(P<0.01).Conclusion HMGB1 challenge can markedly influence the inununological activity of CD4~+ CD25~+ Tregs in mice,and TLR4 appears to be critically involved in down-regulation of HMGB1-mediated CTLA-4 expression and IL-10 production of CD4~+ CD25~+ Tregs in vivo.