γδ T细胞在实验性自身免疫性葡萄膜炎小鼠脾脏中的动态表达及作用机制

摘要

背景 以往研究证明葡萄膜炎的发病机制与γδ T细胞相关,γδ T细胞在实验性自身免疫性葡萄膜炎(EAU)中的作用尚不完全清楚. 目的 研究γδ T细胞在EAU小鼠脾脏中的动态变化,探讨γδ T细胞在EAU病程进展中的作用机制.方法 应用随机数字表法将45只C57 BL/6(B6)小鼠随机分为正常对照组6只和模型组39只.用抗原肽光感受器间维生素A类结合蛋白1-20(IRBP1-20)及完全弗氏佐剂(CFA)的乳化液在B6小鼠的足垫、尾根部及躯干部均匀注射6个点,用Genesis-D动物眼部照相机观察并记录正常小鼠及免疫后不同时间点(4、8、12、16、20、24、28、32和36 d)EAU小鼠的发病情况,参照Thurau的评分标准进行炎症评分.颈椎脱臼法处死小鼠后摘取右侧眼球,切片后行组织病理学评分.同时在相同时间点分离模型小鼠脾脏淋巴细胞,流式细胞仪检测γδ T细胞数量和激活状态.免疫磁珠分选γδ T细胞,并用流式细胞仪检测其细胞内表达白细胞介素-17A(IL-17A)的变化.向EAU小鼠回输激活的γδ T细胞,观察炎症变化. 结果 经IRBP1-20及CFA乳化液免疫后小鼠于第12天可见轻度葡萄膜炎症,炎症反应在免疫后第16 ～20 d达峰,至第28天炎症明显减轻.组织病理学观察发现,免疫小鼠视网膜外核层皱褶,玻璃体、视网膜全层炎性细胞浸润及内界膜层增厚.造模后不同时间点的炎症症状评分和病理学炎症评分的总体比较,差异均有统计学意义(F=51.399,P=0.000;F=47.342,P=0.000).流式细胞仪检测发现,EAU发病高峰期小鼠的脾脏中γδ T细胞数量增加,造模后16 d和20 d分别为(5.67±0.49)％和(5.78±0.55)％,与造模前的(1.53±0.14)％比较,差异均有统计学意义(均P＜0.05),且呈活化状态.EAU发病高峰期小鼠的脾脏中γδ T细胞分泌的IL-17A明显增多,造模后16d和20 d分别为(13.40±0.50)％和(17.80±2.37)％,与正常对照小鼠的(1.53±0.19)％比较,差异均有统计学意义(P=0.000、0.001).体内回输激活的γδ T细胞后EAU炎症加重,炎症症状评分为1.00(1.00,2.00),明显高于未回输激活的γδ T细胞的0.75(0.05,1.00),二者比较差异有统计学意义(Z=27.00,P=0.03).结论 EAU中γδ T细胞比例在炎症的高峰期明显升高,且呈激活状态;活化性Yδ T细胞在EAU模型中发挥的免疫调节作用可能是通过分泌IL-17A进行的.%Background Previous studies showed that the pathogenesis of uveitis is related to γδ T cells.However,it remains unclear that how these cells are involved in experimental autoimmune uveitis (EAU).Objective This study aimed to observe the dynamic changes of γδ T cells in EAU and explore the role of γδ T cells in the pathological process of EAU.Methods Forty-five C57BL/6(B6) mice were assigned to the normal control group (six mice) and EAU model group (thirty-nine mice).The mice were immunized subcutaneously at 6 spots on the footpads,tail base,and flank with emulsion containing human interphotoreceptor retinoid binding protein1-20 (IRBP1-20) emulsified in complete Freund's adjuvant.After immunization,the mice were examined for clinical signs of EAU by using a Genesis-D camera.The changes of histopathology were compared by hematoxylin and eosin staining.Mouse lymphocytes were isolated and purified from the spleens of IRBP1-20-immunized or normal B6 mice by using a γδ T-cell isolation kit.Flow cytometry was used to detect the changes of intracellular expression of interleukin-17A (IL-17A),and then transferred the activated γδ T cells into EAU models to analyze the changes of clinical signs and histopathology of EAU.Experimental study program as well as the use and feeding of the animals were authorized by the Animal Management and Use Committee of Shandong Traditional Chinese Medicine University.Results The inflammatory symptoms in mouse eyes appeared on day 12 after modeling.The initial changes were fundal blood vessel thickening and minimal inflammatory cell infiltration.Then,multifocal chorioretinal lesions,serious vasculitis and linear lesions were observed on days 16-20,along with abundant lymphocyte infiltration in the vitreous and retinal disorganization.The inflammation symptom scores and the pathological inflammation scores at different time points after modeling had statistically significant differences (F =51.399,P =0.000;F =47.342,P =0.000).The inflammatory symptoms in the eyes began to abate from day 28 onwards.The number of γδ T cells was obviously increased during the inflammation phase of EAU at day 16-20 after modeling,with the number of γδ T cells was (5.67 ±-0.49) ％ and (5.78 ±±0.55) ％,respectively,which was significantly higher than (1.53 ± 0.14) ％ before modeling,with significant differences between them (both at P＜0.05),meanwhile CD69 levels and the integrin lymphocyte function-associated antigen-1 (LFA-1) and secreted IL-17A were elavated.The secretion level of IL-17A was (13.40±0.50)％ and (17.80±2.37)％ on day 16 and day 20 after modeling,respectively,which was significantly higher than (1.53 ± 0.19) ％ before modeling,with significant differences between them (P =0.000,0.001).The activated γδ T cells were transferred into EAU model,the inflammation symptom scores were 1.00 (1.00,2.00) after activated γδ T cells were transferred into EAU model,which was significantly higher than 0.75 (0.05,1.00) of the untransferred group (Z =27.00,P =0.03),and the symptoms of EAU were aggravated.Conclusions The proportion of γδ T cells reaches peak in inflammation of EAU,and the cells are activated.The activated γδ T cells in the EAU model play a immune regulation role by secreting IL-17A.