外源性髓鞘抗原的清除在实验性自身免疫性脑脊髓炎中的作用

Role of clearance of exogenous myelin antigen in experimental autoimmune encephalomyelitis

目的探讨外源性髓鞘抗原的清除对其诱导实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的作用。方法用髓鞘少突胶质细胞糖蛋白35~55肽(myelin oligodendrocyte glycoprotein 35-55,MOG_(35-55))或FITC标记的MOG_(35-55)免疫C57BL/6J小鼠诱导EAE,通过分析过继转移的CFSE标记的mT/mG-2D2 CD4^(+)T细胞在脾脏内的增殖情况评估免疫系统内外源性髓鞘抗原的含量;免疫组织化学法和流式细胞术分析接种部位外源性髓鞘抗原的释放;组织切片HE染色探讨外源性髓鞘抗原快速清除的原因;通过对比分析背部和足垫免疫诱导的EAE,以及可溶性MOG_(35-55)的治疗效果验证外源性髓鞘抗原的清除在EAE中的作用。结果免疫第2天小鼠脾脏内mT/mG-2D2 CD4^(+)T细胞增殖占比显著高于免疫第7天[(52.6±6.8)%vs(18.5±4.9)%,P<0.01];在发病小鼠(第13天)脾脏内mT/mG-2D2 CD4^(+)T细胞增殖与在初始小鼠脾脏内的增殖差异无统计学意义[(4.4±1.5)%vs(2.5±1.4)%,P=0.11];免疫组织化学法和流式细胞术结果显示,MOG_(35-55)在接种部位释放并被CD11b细胞吞噬,EAE发病时接种部位已无MOG_(35-55)释放;组织病理切片显示,EAE发病时乳化剂周围形成的肉芽肿阻止乳化剂释放抗原,使抗原与外周免疫系统完全隔离;经足垫免疫诱导的小鼠不易发病与MOG_(35-55)持续刺激免疫系统有关,与CD4^(+)调节性T细胞没有直接关系;持续腹腔注射可溶性MOG_(35-55)可预防和治疗EAE。结论EAE小鼠免疫系统内外源性髓鞘抗原已完全清除,EAE的发生依赖外源性髓鞘抗原的完全清除。

Objective To investigate the role of the clearance of exogenous myelin antigen in experimental autoimmune encephalomyelitis(EAE).Methods EAE was induced in C57BL/6J mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35-55(MOG_(35-55))or FITC-MOG_(35-55).The concentration of exogenous myelin antigen was assessed by analyzing the proliferation of the transferred CFSE-labeled mT/mG-2D2 CD4^(+)T cells in spleen tissues.The release of exogenous myelin antigen from the inoculation sites was analyzed by immunohistochemistry and flow cytometry.HE staining was used to investigate the mechanism underlying the rapid clearance of exogenous myelin antigen.The role of the clearance of exogenous myelin antigen in EAE was investigated by comparative analysis of EAE induced by subcutaneous immunization in the back and footpads,and analyzing the therapeutic effect of soluble MOG_(35-55).Results The proliferation of mT/mG-2D2 CD4^(+)T cells in mice was enhanced on day 2 than on day 7 after immunization[(52.6±6.8)%vs(18.5±4.9)%,P<0.01].There was no significant difference in the proliferation of mT/mG-2D2 CD4^(+)T cells between EAE mice(day 13)and naive mice[(4.4±1.5)%vs(2.5±1.4)%,P=0.11].Immunohistochemistry and flow cytometry showed that MOG_(35-55) was released and engulfed by CD11b+cells at the inoculation sites,and no more MOG_(35-55) was released at the onset of EAE.HE staining showed that granuloma that formed surrounding the antigen emulsion during EAE development prevented antigen release from the emulsion,completely isolating the antigen from the peripheral immune system.The incidence of EAE was relatively low in mice immunized via footpads,which was related to the sustained release of MOG_(35-55),but had no direct relation to CD4^(+)regulatory T cells.Continuous intraperitoneal injection of soluble MOG_(35-55) could prevent and treat EAE.Conclusions Exogenous myelin antigen has been completely cleared in EAE mice,and the occurrence of EAE depends on the clearance of the myelin antigen.