实验性自身免疫性神经炎中NKR-P1+细胞对IL-12和IL-18协同作用的影响

Effect of NKR-P1+ cells on synergism of interleukin-12 and interleukin-18 in pathogenesis of experimental autoimmune neuritis

目的探讨实验性自身免疫性神经炎(Experimental autoimmune neuritis,EAN)中IL-12和IL-18的协同作用以及NKR-P1+细胞对该作用的影响。方法用200μl免疫乳剂(含230μg特异性肽段P2 53-78、2 mg结核菌素、100μl PBS和100μl弗氏不完全佐剂)免疫Lewis大鼠,建立EAN动物模型,在发病高峰期取出大鼠淋巴结,制备单核细胞悬液,在NKR-P1+细胞存在或不存在的条件下,分别或同时与IL-12(20 ng/ml)、IL-18(25 ng/ml)共培养,同时加入20μg/ml P2 53-78刺激,孵育24 h后,以1×107个活细胞/只鼠回输至正常Lewis大鼠体内,每天观察发病情况,并进行临床评分以及组织病理学检测,采用ELISA法检测细胞培养上清中IFNγ、TNF-α和IL-4的分泌水平,淋巴细胞增殖试验检测P2 53-78特异性淋巴细胞的增殖反应。结果 P2 53-78特异性淋巴细胞在IL-12或IL-18单独刺激下,可引发中等发病程度的EAN;而在IL-12和IL-18共同刺激下,引发EAN的发病程度明显加重;当删除淋巴细胞中的NKR-P1+细胞后,可引发严重的EAN;而NKR-P1+细胞删除后的P2 53-78特异性淋巴细胞与IL-12和IL-18共培养,其自身反应活性得到了抑制,并低表达IFNγ,从而抑制Th1细胞的分化。结论IL-12和IL-18的共同刺激能够增强P2 53-78特异性淋巴细胞的自身反应性;IL-12和IL-18能够诱导出高水平的IFNγ,从而促进Th1细胞的分化,其作用部分是通过NKR-P1+细胞实现的。

Objective To investigate the role of synergism of interleukin （IL）-12 and IL-18 in pathogenesis of experimental autoimmune neuritis （EAN） as well as the effect of NKR-PI＋ cells on the synergism. Methods Rat model of EAN was established by immunization of Lewis rats with 200 μl of immune emulsion containing 230 Ixg of specific peptide P2 53-78, 2 mg of tuberculin, 100 μl of PBS and 100 μl of incomplete Freund adjuvant. The lymph nodes of rats at peak incidence were isolated, from which monocyte suspension was prepared and, in presence or absence of NKR-P1 ＋ cells, co-cuhured with IL-12 （20 ng/ ml）, IL-18 （25 ng/ml） and IL-12 （20 ng/ml） ＋ IL-18 （25 ng/ml） respectively. The monoeyte suspension in each group was stimulated with 20 p^g/ml P2 53-78 for 24 h, and transferred to normal Lewis rats at a dosage of 1 x 107 live monoeytes per rat. The rats were observed for signs of disease daily and subjected to clinical score, of which the sciatic nerves were subjected to histopathological examination. The secretion levels of IFN＂/, TNF-o~ and IL-4 in culture supematant of monocytes were determined by ELISA, while the proliferative reaction of P2 53-78 specific lymphocytes by lymphocyte proliferation test. Results P2 53-78 specific lymphocytes stimulated with IL-12 or IL-18 alone caused moderate EAN, while those co-stimulated with IL-12 and IL-18 caused aggressive EAN. However, the lymphocytes with NKR-PI＋ cells deleted caused severe EAN and, after co-culture with IL-12 ＋ IL-18, induced low IFN＇y level and inhibited Thl differentiation, of which the auto-reactivity was inhibited. Conclusion The co-stimulation with IL-12 and IL-18 enhanced the anto-reactivity of P2 53-78 specific lymphocytes, induced high levels of IFNy and promoted the differentiation of Thl cells partially through NKR-P1 ＋ cells.