Th1、Th17细胞对小鼠视网膜星形细胞的杀伤作用

摘要

背景 C57BL/6及B10RⅢ小鼠是实验性自身免疫性葡萄膜炎(EAU)动物模型常用的小鼠种系,其中C57B L/6小鼠免疫后眼部炎症较轻,而B10RⅢ小鼠免疫后眼部表现为典型的后葡萄膜炎病理改变.目的 观察培养的光感受器间维生素A类结合蛋白(IRBP)抗原特异性Th1、Th17细胞对小鼠视网膜星形细胞的杀伤作用,研究EAU中Th1及Th17细胞的致病机制. 方法 选取C57BL/6小鼠、B10RⅢ小鼠各10只,尾根部及躯干皮下注射200 μl含有200 μg IRBP1-20或IRBP161-180抗原及完全弗氏佐剂(CFA)的乳化液,共均匀注射6个点以免疫动物.流式细胞仪分析B10RⅢ小鼠模型的眼内浸润T细胞类别.分离培养C57BL/6小鼠淋巴结及脾脏IRBP特异性T细胞,分别加入白细胞介素2(IL-2)或IL-23以适于Th1、Th17细胞生长.将培养至第5天的Th1、Th17细胞分别加入到经干扰素-γ(IFN-γ)预处理的单层视网膜星形细胞中,观察细胞间的相互作用,检测肿瘤坏死因子-α( TNF-α)的质量浓度. 结果 B10RⅢ小鼠EAU模型眼球中有大量炎性细胞浸润,流式细胞仪检测证实含有IFN-γ+、IL-17+细胞和CD45+细胞,分别占9.5％、5.1％和41.4％.IRBP1-20刺激后6d,含IL-2和IL-23培养基中IFN-γ+细胞分别为44.0％、8.0％,IL17+细胞分别为1.0％、26.0％.Th1、Th17与视网膜星形细胞相互作用24 h后,可见视网膜星形细胞死亡脱落,Th17较Th1具有更强的杀伤作用.Th1、Th17细胞分别与星形细胞共培养48 h,两种培养基中TNF-α的质量浓度分别为(500±10)、(801±24) μg/L,差异有统计学意义(t=-20.36,P=0.00).结论 Th1、Th17对视网膜星形细胞均有杀伤作用,Th17细胞杀伤作用更强,在葡萄膜炎致病过程中发挥更重要的作用.杀伤过程中既有细胞直接接触作用,又有通过细胞因子介导的间接作用.%Background C57BL/6 and B10R Ⅲ are routine murine species used in experimental autoimmune uveitis (EAU).The inflammation is light for mouse after immunization whereas it is prominent for B10R Ⅲ. Objective This study was to observe the killing effect of interphotoreceptor retinoid binding protein (IRBP) 1-20-specific T cells on mouse retinal astrocyte.Th1 and Th17 cells effect in the EAU mechanism was discussed.Methods B10RllⅢ mice and C57BL/6 mice were immunized with IRBP 161-180 and IRBP 1-20 in complete Freund adjuvant (CFA).The infiltrating cells of diseased B10R Ⅲ eyes were analyzed by flow cytometry.IRBP 1-20-specific T cells were isolated from the drainage lymph node and spleen and cultured in IL-2 or IL-23 for Th1 and Th17 cells polarization,respectively.Th1 and Th17 cells cultured for 5 days were seeded on the mouse retinal astrocyte monolayer pretreated with gamma interferon.Cell interaction was observed and the quantity of TNF-α was tested by ELISA.Every test was repeated 6 times and the mean was calculated.The maintenance of experimental animals complied with the Statement of ARVO. Results There were lots of infiltrating cells in the eyes of B10Rm mice after immunization,including 9.5％ IFNγ+ cells,5.1％ IL-17+cells and 41.4％ CD45+ cells.Six days after IRBP1-20 stimulation and cultured by IL-2 and IL-23,44.0％ and 8.0％ cells were IFNγ+,and 1.0％ and 26.0％ cells were IL17+.Twentyfour hours after the interaction between Th1 or Th17 and retinal astrocyte,retinal astrocyte died and detached.The killing effect of Th17 was stronger than Th1.48 hours after co-culture of Th1 or Th17T cells with astrocytes,the concentrations of TNF-α were ( 500± 10 ) and ( 801 ±24 μg/L) μg/L,respectively,with a significant statistical difference (t =-20.36,P =0.00). Conclusions Both Th1 and Th17 can kill retinal astrocyte,but Th17 plays a key role in the EAU pathogenesis process.The killing effect is caused by intercellular contact and interaction under the induction of cytokines.