Objective: To investigate mesenchymal stem cells (MSCs) immunosuppressive activity in the presence of interferon-gamma (IFN-γ) to reveal synergistic immunomodulatory effects of IFN-γ and MSCs.
Methods: ① MSCs were cultured in the presence or absence of IFN-γ（100 ng/ml）, the supernatants were collected for measurements of PGE2、HGF and TGF-β1 by ELISA kits. ② MSCs were cultured in the presence or absence of IFN-γ （100 ng/ml）for 48 h. The cDNA was analysed for the expression of human indoleamine 2, 3-dioxygenase（IDO）mRNA by semiquantitative RT-PCR. ③ Mononuclear cells (MNCs) were extracted from peripheral blood of healthy donors. The T cell proliferation was tested in the co-culture system added with MSCs, recombinant human IFN-γ (100 ng/ml) and anti-IFN-γ mAb (5 μg/ml) by BrdU ELISA kit.
Results: ①The immunosuppressive cytokines PGE2、HGF and TGF-β1 were detectable within 24-48 h in the supernatants. Their expressions were significantly up-regulated in the presence of IFN-γ. Concentrations of these cytokines were as of (1715.5±628.6) pg/ml vs (1344.5±709.4) pg/ml (P=0.001);(4031.8±1496.8) pg/ml vs (2452.4±1375.3) pg/ml（P=0.011）;(1753.5±413.8) pg/ml vs (1026.6±450.5) pg/ml（P<0.001）,respectively. ②The expression of IDO mRNA was undetectable when MSCs were cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced in the presence of IFN-γ. ③Bone marrow-derived MSCs remarkably suppressed allogeneic T cell proliferation in vitro. Addition of exogenous IFN-γ had no significant effect on the inhibitory capacity of MSCs, the inhibitory ratios of T cell proliferation were （40.4±10.9）% vs（36.7±7.4）% (P=0.272). By contrast, the inhibitory ratio of T cell proliferation was significantly decreased in the presence of anti-IFN-γ mAb[(40.4±10.9)% vs (23.9±7.6)%,P=0.002].
Conclusion: ①Human MSCs constitutively expressed immunosuppressive concentrations of PGE2, HGF and TGF-β1, and their expressions were significantly up-regulated by IFN-γ. ②IFN-γ-induced expression of IDO on MSCs involved in tryptophan catabolism. ③MSCs notably suppressed allogeneic T cell proliferation in vitro. IFN-γ promoted the immunosuppressive capacity of human MSCs, indicating the synergistic immunomodulatory effect of IFN-γ and MSCs.
目的 探讨干扰素-γ（IFN-γ）对正常人骨髓间充质干细胞（MSC）免疫活性的影响,阐明IFN-γ与MSC在免疫抑制中的协同作用。方法 ① ELISA法检测IFN-γ（终浓度100 ng/ml）对正常人骨髓来源MSC前列腺素E2（PGE2）、肝细胞生长因子（HGF）和转化生长因子β1（TGF-β1）表达水平的影响。②RT-PCR法检测100 ng/ml IFN-γ作用下MSC内吲哚胺2,3-双加氧酶（IDO）基因表达水平变化。③ 体外刺激正常人外周血T细胞增殖,并与正常人骨髓来源MSC共培养,检测T细胞增殖水平;在共培养体系中分别加入重组人IFN-γ（100 ng/ml）及鼠抗人IFN-γ单克隆抗体（单抗,5 μg/ml）,检测T细胞增殖水平变化。结果 ①MSC单独培养24~48 h可检测到PGE2、HGF、TGF-β1三种细胞因子表达;IFN-γ可促进MSC上述细胞因子的表达[实验组及对照组PGE2、HGF、TGF-β1表达水平分别为(1715.5±628.6) pg/ml对(1344.5±709.4) pg/ml;(4031.8±1496.8) pg/ml对(2452.4±1375.3) pg/ml;(1753.5±413.8) pg/ml对(1026.6±450.5) pg/ml,P值分别为0.001、0.011及<0.001]。②MSC单独培养48 h后,RT-PCR法未检测到IDO基因表达。与IFN-γ共培养后,IDO基因呈高水平表达。 ③在体外MSC可以显著抑制T细胞增殖,外源性IFN-γ不影响MSC对T细胞增殖的抑制作用[T细胞增殖抑制率分别为（40.4±10.9）%和（36.7±7.4）%,P=0.272];在反应体系中加入抗IFN-γ单抗后,MSC抑制T细胞增殖作用显著减弱[T细胞增殖抑制率分别为（40.4±10.9）%和（23.9±7.6）%,P=0.002]。结论 MSC组成性表达PGE2、HGF及TGF-β1等免疫抑制性细胞因子,在一定水平IFN-γ作用下,MSC分泌上述3种细胞因子水平明显上调,IDO mRNA表达水平显著增高,MSC免疫活性增强。骨髓MSC在体外可以显著抑制T淋巴细胞增殖,IFN-γ与MSC在免疫抑制中具有协同效应。