来氟米特对狼疮患者树突状细胞作用机制的初探

An exploratory study on in vitro effects and mechanism of leflunomide on dendritic cells of systemic lupus erythematosus

目的 探讨来氟米特(LEF)处理前后系统性红斑狼疮(SLE)患者树突状细胞(DC)表面标志及功能的改变,揭示LEF治疗SLE的作用机制,为开展“抑制性DCs”治疗SLE奠定实验基础。方法 (1)分离SLE患者外周血单核细胞,用细胞因子诱导DC成熟, LEF组再加入A7717262(来氟米特的活性代谢产物)培养。第9天收集DC细胞,流式细胞仪检测CD80、CD83、CD86和HLA DR的表达。(2)分别将A771726处理或不处理的第9天DC和T细胞进行培养, 72h后用MTT法检测DC刺激淋巴细胞增殖的能力,FACS检测T细胞亚群和ELISA检测培养上清中IL 10和IFNγ水平。结果A771726处理后虽DC形态无改变,但DC表达CD83、CD86和HLA DR百分数较对照组均明显降低(72 70±1 77vs 79 36±4 80, 63 50±14 06vs. 83 91±9 81, 80 44±12 56vs. 90 51±8 63,P值均<0 01)。A771726处理后的DC,其刺激T细胞增殖相应的吸光度值明显降低,混合培养的上清液中IL 10水平较无A771726处理的DC与T细胞的混合培养上清液明显降低,而IFNγ两者间无显著差异;但见CD+4 CD+25CTLA+4 T细胞百分比增高。结论 LEF在体外可抑制SLE患者外周血DC的成熟;未成熟DC能抑制T细胞增殖及T细胞向Th2 细胞转化,诱导CD+4 CD+25CTLA+4 T细胞产生,从而纠正SLE患者的部分免疫紊乱。

Objective To detect the effects of leflunomide to phenotype and function of dendritic cells(DCs) in systemic lupus erythematosus (SLE) patients, reveal the effective mechanism inducing remission of SLE and lay a research foundation for using ‘inhibit’ DCs to treat SLE in future Methods The monocytes were isolated from peripheral blood of SLE patients and cultivated into DCs with cytokines such as GM-CSF and IL-4 A771726(active metabolite) was added in with cytokines in leflunomide group, but not in control DCs were harvested after 9 days culture CD_ 80 , CD_ 83 , CD_ 86 and HLA-DR surface markers on DCs were detected by flow cytometry(FACS) The ability of DCs stimulating lymphocytes proliferation was detected by MTT assay IL-10 and IFNγ level in the supernatant of MLR were detected by ELISA and T cell subtype after MLR was detected by FACS Results The DCs treated with A771726 showed a lower percentage expression of CD_ 83 、CD_ 86 and HLA-DR phenotype(CD_ 83 :72 70±1 77 vs 79 36±4 80, CD_ 86 : 63 50±14 06 vs. 83 91±9 81, HLA-DR: 80 44±12 56 vs. 90 51±8 63,all P <0 01), a weaker ability to stimulating T lymphocytes proliferation(at DC∶TC= 1∶10, 0 285±0 079 vs. 0 458±0 100;at DC∶TC=1∶50, 0 194±0 054 vs. 0 382± 0 023,all P <0 01) and a lower secretive level of IL-10 in the MLR supernatant [(195 0±36 9)μg/L vs. (423 6±93 2)μg/L, P <0 01], exclude those it could still increase amount of a new T cell subtype——CD +_4CD +_ 25 CTLA +_4 T cell(12 00% & 6 23%) Conclusions A771726 can inhibit DCs maturation, the immature DCs can inhibit T cells proliferation and refrain T cells from dividing into Th_2 subtype, and also the immature DCs can induce a sort of regulate T cell(CD +_4CD +_ 25 CTLA +_4 T cell) production Through that LEF may correct part over humor immune dysfunction and get a new immune balance in SLE