IL-4调控DC-SIGN表达对树突状细胞免疫学功能的影响及与结核病发生的关系

Effect of IL-4-regulated DC-SIGN on immune function of dentritic cells and relationship of its expression with tuberculosis

目的研究IL-4调控树突状细胞特异性非整合素(dendritic-cell specific ICAM-3 grabbing non-integrin,DC-SIGN)的表达对树突状细胞免疫学功能的影响,以探讨DC-SIGN表达与结核病发生的关系。方法用不同剂量的IL-4(5、10、50、100、200ng/ml)调控DCs表面DC-SIGN的表达水平;根据加入IL-4剂量将DCs分为5ng/ml组和50ng/ml组,分别加入100ng/ml的脂多糖(lipopolysaccharide,LPS)、结核杆菌H37Rv菌悬液共培养。应用流式细胞仪检测各组DCs表型(CD11c、DC-SIGN、CD83、CD86、HLA-DR),ELISA法检测细胞培养液中IL-12、IL-10含量,混合淋巴细胞培养法检测各组DCs刺激T淋巴细胞增殖的能力。结果①IL-4能调控DC-SIGN的表达水平,并呈剂量依赖性,当加入IL-4的剂量由5ng/ml增加到50ng/ml时,DC-SIGN的表达水平显著增加(P<0.05)。当IL-4的剂量由50ng/ml增至200ng/ml时,DC-SIGN的表达水平虽仍随IL-4剂量增加而增加,但差异无统计学意义(P>0.05)。②表达不同水平DC-SIGN的DCs成熟度无显著差异(P>0.05)。③表达不同水平DC-SIGN的DCs加入LPS、H37Rv诱导后,培养液中IL-12含量无显著差异(P>0.05),加入LPS诱导后培养液中IL-10含量也无显著差异(P>0.05),但DC-SIGNhigh-DCs加入H37Rv诱导后培养液中IL-10含量明显高于DC-SIGNlow-DCs(P<0.05)。④表达不同水平DC-SIGN的DCs加入LPS诱导后刺激同种异体T淋巴细胞增殖能力差异无统计学意义(P>0.05)。DC-SIGNhigh-DCs加入H37Rv诱导成熟后与DCs加入LPS诱导成熟后刺激同种异体T淋巴细胞增殖的能力相当(P>0.05),但DC-SIGNlow-DCs加入H37Rv诱导后刺激同种异体T淋巴细胞增殖能力明显低于其他3组(P<0.05)。结论DC-SIGNhigh-DCs加入H37Rv诱导后分泌IL-10水平明显高于DC-SIGNlow-DCs,但DC-SIGNlow-DCs加入H37Rv诱导后刺激T淋巴细胞增殖的能力明显降低。DC-SIGN表达过高或过低均不利于机体抗结核免疫应答。

Objective To investigate the effect of the expression level of dendritic-cell specific ICAM-3 grabbing non-integrin （DC-SIGN） which was regulated by IL-4 on the immune function of dentritic cells （DCs） and analyze the relationship between the expression level and tuberculosis. Methods IL-4 at different doses （5, 10, 50, 100 and 200 ng/ml） was used to regulated the expression level of DC-SIGN in DCs. The DCs were divided into 2 groups according to the different doses of IL-4, which were 5 ng/ml group and 50 ng/ml group, and then were co-cultured with 100 ng/ml LPS or the suspension of Mycobacterium Tuberculosis H37Rv. The phenotypes （CDllc, DC-SIGN, CD83, CD86, and HLA-DR）of DCs were analyzed by flow cytometry. The concentrations of IL-12 and IL-10 in the DCs supernatant were measured by ELISA. The ability of DCs to stimulate T cell proliferation was detected by mixed leukocytes reaction （MLR）. Results IL-4 regulated the expression level of DC-SIGN in a dose-dependent manner. When the dose of IL-4 was increased from 5 ng/ml to 50 ng/ml, the expression level of DC-SIGN was elevated obviously （P 〈 0.05）. But when the dose was in- creased from 50 ng/ml to 200 ng/ml, the expression level also was risen, though with no statistical difference （P 〉 0.05 ）. There was no significant difference in the maturity degree of DCs which had different expression levels of DC-SIGN （P 〉 0.05 ）. No significant difference was observed in the concentrations of IL-12 in the supernatant of DCs with high （50 ng/ml IL-4） or low levels （5 ng/ml IL-4） DC-SIGN expression in present of LPS or H37 Rv （P 〉 0.05）. When co-cultured with LPS, the DCs with different expression of DC-SIGN secreted similar amount of IL-10 （P 〉 0.05）. But when co-cultured with H37Rv, the DCs with high expression level of DC-SIGN produced significantly more IL-10 than the cells with low expression level （ P 〈 0.05 ）. After the treatment of LPS, the DCs with different expression level of DC-SIGN had the similar ability to stimulate allogenic T cell proliferation （P 〉0.05 ）. Compared with LPS, H37Rv treatment showed similar ability of DCs with high DC-SIGN expression to stimulate allogenic T cell proliferation （ P 〉 0.05 ）, but for the DCs with low DC-SIGN expression, the ability was much lower than those of the other group did （P 〈0.05）. Conclusion DCs with high expression level of DC-SIGN secreted more IL-10 when co-cultured with H37 Rv than the cells with low expression, but the later cells have lower ability to stimulate allogenic T cell proliferation. Expression of DC- SIGN no matter upregulated or downregulated has disadvantage to the outcome of the tuberculosis immunity.