白细胞介素-2与白细胞介素-23对小鼠类结合蛋白特异性T细胞向Thl、Thl7分化的诱导及致病性研究

Effects of exogenous interleukin-2, interleukin-23 on differentiation of IRBP 1-20-specific T cellstoward Thl and Thl7 and comparison of the pathogenicity of IRBP 1-20-specific T cells

目的探讨外源性白细胞介素（IL）-2和IL-23对小鼠类结合蛋白（IRBP）特异性T细胞向辅助性T细胞（Th）1、Thl7分化的诱导及致病性的研究。方法实验研究。向30只雌性C57BL／6小鼠（6-8周）皮下注射200IXl含200tag人IRBP1-20和0．8mg结核分枝杆菌的乳糜液，建立实验性自身免疫性葡萄膜炎（EAU）动物模型。在免疫后第13天取出小鼠脾脏和引流淋巴结，预留部分脾脏通过逆转录（RT）-PCR法检测其中干扰素（IFN）-γ mRNA与IL-17mRNA的表达水平，作为正常对照组。其余脾脏在纱网研磨后用尼龙毛富集的方法提取小鼠T细胞，将其分为IL-2组和IL-23组，均加入抗原递呈细胞和IRBP1-20，分别加入外源性小鼠重组IL-2与IL-23。在培养后第2天用Ficoll密度梯度离心方法分离T细胞，RT-PCR法检测T细胞中IFN-γ mRNA与IL-17mRNA的表达情况，同时取T细胞上清液用酶联免疫吸附试验检测上清中IFN-γ与IL-17的含量。继续培养到第4天，利用流式细胞仪检测不同干预条件下IL-17＋ γδ＋T细胞的比例。取30只小鼠建立EAU模型后，取出T细胞，按照以上方法建立IL-2组和IL-23组IRBP特异性T细胞。另取6只小鼠随机分为两组，分别腹腔内注射IL-2组与IL-23组的T细胞，剂量为3．5×10^6个／只。在注射后第3、7、14天通过间接眼底镜观察小鼠的眼部发病情况，在第21天处死小鼠取出小鼠眼球做病理切片，观察IL-2组和IL-23组小鼠眼部病理变化。分别采用秩和检验、单因素方差分析和配对t检验对实验数据进行统计分析。结果在抗原和外源性IL刺激后第2天，T细胞出现明显的“玫瑰花环”现象。RT-PCR检测结果显示，IL-2组中IFN-1mRNA的表达水平（0．26±0．02）明显高于正常对照组（0．12±0．05）和IL-23组（0．10±0．00）（F=80．51，P=0．003）。IL-2组T细胞上清中IFN-γ的含量[（13124．67±107．73）μg/L]高于IL-23组[（3953．67±117．34）μg／L]（t=169．61，P=0．000），而IL-17的含量[（588．67±77．43）μg/L]低于IL-23组[（5038．33±88．00）μg／L]（t=-361．71，P=0．000）。流式细胞仪检测结果显示，IL-23组中分泌IL-17的78T细胞的比例为（9．93±0．55）％，高于IL-2组的（3．23±0．28）％（t=-33．18，P=0．001）。IL-2组和IL-23组小鼠在过继性转移免疫后第3天均未出现炎症反应，在第14天时两组小鼠炎症反应最重，IL-23组可见大量炎症细胞浸润、视网膜出血和视网膜脱离，明显重于IL-2组。结论IL-23对抗原特异性T细胞向Thl7细胞分化具有促进作用，其中Thl7细胞又以78T细胞为主，而IL-2对其却具有抑制作用。进一步的研究证实，IRBP特异性Thl7细胞的致病陡明显高于Thl细胞。

Objectives To explore the effects of exogenous interleukin （IL）-2 and IL-23 on differentiation of IRBP 1-20-specific T cells originated from C57BL/6 mouse with experimental autoimmuneuveitis （EAU） and to investigate the difference in pathogenicity. Methods Experimental study. Thirty C57BL/6 mice were immunized subcutaneously with 200μl emulsion containing 200 μg interphotoreceptor retinoid-binding protein （IRBP） 1-20 and 0. 8 mg mycobacterium tuberculosis in complete Freund＇s adjuvant ,distributed over six spots at the tail base and on the flank. On postimmunization Day 13 ,spleens and draining lymph nodes were removed from mice, and a part of spleens, as the control group, was reserved for examining the expression of IFN-3, mRNA and IL-17 mRNA by RT-PCR. T cells were isolated from the rest of spleens and lymph nodes by passage through a nylon wool column, and then T cells were stimulated for 48 hours with IRBP 1-20 in the presence of antigen-presenting cell and mouse recombinant cytokine IL-2 or IL-23. The IRBP 1-20-specific T cells were separated by Ficoll gradient centrifugation, the expressions of IFN-γ mRNA and IL-17 mRNA were assessed by RT-PCR,and IFN-γ and IL-17 in T cells supernatant were detected using a commercial ELISA kit. The T cells were cultured for another 48 hours, and then the proportions of IL-17＋ γδ＋T cells were analyzed by flow cytometry. EAU models were built in 30 C57BL/6 mice, T cells from which were randomly divided into two groups according to the methods mentioned above： IL-2 group and IL-23 group. Transfer EAU models were built in other 6 mice and divided into two groups, IL-2 group and IL-23 group, by intraperitoneal injection of 3.5 ×10^6IRBP-special-T cells from IL-2 group or IL-23 group respectively . Clinical changes were observed by indirect ophthalmoscopy on postimmunization day 3,7,14. For histopathological evaluation, whole eyes were collected on postimmunization day 21. Rank sum test, one-way ANOVA and paired t test were used to analyze the results. A comparison of pathogenicity was made between Thl and Thl7 through clinical observation and histopathological evaluation of B6 mouse. Results Rosette formation was found among T cells on poststimulation day 2. There was a statistical difference in the expression of IFN-γ mRNA between the IL-2 group and normal group or IL-23 group （0. 26±0. 02 vs. 0. 12 ±0.05 or 0. 10 ± 0.00） （F = 80. 51, P = 0. 003 ） ; however, the experssion of IFN-γ in the IL-2 group [ （ 13 124. 67±107. 73 ） μg/L] was higher than that of the IL-23 group [ （ 3953.67±117. 34） μg/L] （t = 169. 61 ,P =0. 000） ;and the expression of IL-17 mRNA in the IL-2 group[ （588.67±77.43）μg/L] was lower than that of the IL-23 group [ （5038.33±88.00） μg/L ] （ t = - 361.71, P = 0. 000）. Flow cytometer showed that the concentration of IL-17 in the IL-2 group[ （3.23 ±0. 28 ）% ] was significantly lower than that in the IL-23 group[ （9. 93±0. 55）% ] （t = -33.18,P =0. 001 ）. There was a significant difference in the proportion of IL-17＋ γδ＋T cells between the IL-23 group and IL-2 group （9. 93±0. 55 vs. 3.23± 0. 28） （t = - 33. 18, P = 0. 001 ）. Inflammatory response could not be detected in either group three days after injection of IRBP 1-20-specific T cells. Both groups of mice had the most severe inflammatory response on the 14 th day, of which that of the IL-23 group was significantly more severe. Conclusions IL-23 facilitates IRBP 1-20-specific T cells to differentiate into Thl7, whereas IL-2 inhibits this process and induces these cells to differentiate towards Thl. Further studies showed that the pathogenicity of Thl7 cells was significantly higher than that of Thl cells.