IL-10诱导小鼠树突状细胞耐受的分子机制

The molecular mechanism of tolegenic dendritic cells induced by IL-10 in mouse

目的:研究白细胞介素-10(interleukin-10,IL-10)诱导小鼠来源的树突状细胞(DC)耐受及其与配对免疫球蛋白样受体(PIR-A/B)的关系。方法:以IL-10(20μg/L)诱导小鼠来源的树突状细胞系(DC2.4)6 d,即IL-10-DC组,脂多糖(LPS)刺激其48 h为成熟DC2.4细胞(LPS-DC),体外化学合成特异性针对PIR-B的小干扰RNA片段,以脂质体2 000转染IL-10组(Si-DC组)。分别应用半定量RT-PCR和流式细胞仪(FCM)检测DC2.4、IL-10组、LPS组及Si-DC组细胞PIR-A/B的表达。以[3H]-TdR标记法检测上述各组细胞刺激同种异体淋巴细胞的增殖反应(MLR),ELISA方法测混合培养上清中IFN-γ的水平变化。结果:RT-PCR结果表明,IL-10诱导PIR-B表达升高、PIR-A表达下降,LPS则下调PIR-B、上调PIR-A的表达。FCM检测IL-10组和LPS组的PIR-A/B胞外区PIR表达均升高,且前者明显高于后者。同正常DC2.4和LPS组相比,IL-10可抑制MLR,小干扰RNA沉默PIR-B表达可增强MLR,伴随MLR反应上清中IFN-γ的水平升高。结论:IL-10诱导DC高度表达免疫抑制性受体PIR-B,使其获得耐受,上调PIR-B的表达是IL-10诱导DC耐受的分子机制之一。

AIM： To investigate the relation of tolerogenic dendritic cells （DC） induced by interleukin - 10 （IL- 10） and the paired immunoglobin- like receptor （PIR） A and B （PIR- A and PIR- B） in mouse. METHODS： The mouse dendritic cell line, DC2.4 cells were cultured with the IL - 10 to develope the IL - 10 - DC and were stimulated by lipopolysaccharide （LPS） for 48 h to induce the mature dendritic cells （ LPS- DC ）. Special small inference RNA （siRNA） molecule of PIR - B was chemically synthesized and was transfected into IL - 10 - DC by Lipofectamine 2000 （ Si - DC）. The expression of PIR A and PIR B on DC2.4 cells were measured by semi - quantitative RT - PCR and flow cytometry （FCM）. The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction （MLR） using [3^H] -thymidine incorporation test. The concentration of IFN -γ in supernatants of MLR from distinct groups was analyzed by ELISA. RESULTS： IL- 10 up -regulated the PIR -B and down- regulated the PIR- A by semi- quantitative RT - PCR. On the contrary, LPS down - regulated the PIR - B expression and up - regulated the PIR - A expression. The expression of PIR, which is the common extra - membrane of PIR - A and PIR - B, was increased in both the IL - 10 - DC and the LPS - DC groups by FCM detection, but the higher expression was found in IL - 10 - DC group than that in LPS group. The IL - 10 induced the higher PIR - B expression, inhibited allogenefic T cell proliferation and down - regulated the IFN - γ secretion. Special siRNA molecules of PIR - B in IL - 10 group promoted the T cell proliferation and enhanced the IFN-γ secretion in MLR. CONCLUSION： IL- 10 up- regulates the PIR -B expression and makes DC tolerance. Up - regulated PIR - B expression may be the molecular mechanism of tolerogenic dendritic cells induced by IL - 10 in mouse.