Steinbrenner Holger, Nguyen Thi-Bang-Tam, Wohlrab Ulrike, Scherbaum Werner A, Seissler Jochen
German Diabetes Research Institute at the University of Duesseldorf, Germany.
Endocrinology. 2002 Oct;143(10):3839-45. doi: 10.1210/en.2002-220583.
Cytokines released from activated antigen-presenting cells and T-lymphocytes are crucially involved in the pathogenesis of type 1 diabetes. Previous studies have shown that proinflammatory cytokines play an important role in the induction of autoimmunity and beta-cell damage. Inhibition of insulin expression has been described, but their effects on other major target autoantigens, such as the tyrosine phosphatase-like protein IA-2, is not known. In the present study, we established sensitive real-time RT-PCR to measure IA-2, insulin, and inducible nitric oxide (NO) synthase (iNOS) mRNA expression. Rat insulinoma INS-1 cells were stimulated with IL-1beta, TNF-alpha, interferon (IFN)-gamma, and IL-2 as well as with two combinations of these cytokines (C1: IL-1beta + TNF-alpha + IFN-gamma; C2: TNF-alpha + IFN-gamma). Treatment with IL-1beta, TNF-alpha, or IFN-gamma alone caused a significant down-regulation of IA-2 and insulin mRNA levels in a time and dose-dependent manner, whereas IL-2 had no effect. Exposure to cytokine combinations strongly potentiates the inhibitory effects. Incubation of cells with C1 and C2 for 24 h induces a significant inhibition of IA-2 mRNA levels by 78% and 58%, respectively. Under these conditions, an up to 5 x 10(4)-fold increase of iNOS gene expression was observed. The hypothesis that the formation of NO is involved in IA-2 regulation was confirmed by the finding that the coincubation of C1 with 4 mM L-N(G)-monomethyL-L-arginine, an inhibitor of the iNOS, partly reversed the down-regulation of IA-2. Further, incubation with the synthetic NO-donor S-nitroso-N-acetyl-D-L-penicillamine significantly decreased IA-2 mRNA level to 51% of basal levels. In conclusion, we have demonstrated for the first time that IL-1beta, TNF-alpha, and IFN-gamma exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1beta may be partly mediated by the activation of the NO pathway.
活化的抗原呈递细胞和T淋巴细胞释放的细胞因子在1型糖尿病的发病机制中起关键作用。先前的研究表明，促炎细胞因子在自身免疫诱导和β细胞损伤中起重要作用。胰岛素表达受到抑制已有报道，但它们对其他主要靶自身抗原，如酪氨酸磷酸酶样蛋白IA-2的影响尚不清楚。在本研究中，我们建立了灵敏的实时逆转录聚合酶链反应（RT-PCR）来检测IA-2、胰岛素和诱导型一氧化氮合酶（iNOS）的mRNA表达。用白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、干扰素（IFN）-γ、IL-2以及这些细胞因子的两种组合（C1：IL-1β+TNF-α+IFN-γ；C2：TNF-α+IFN-γ）刺激大鼠胰岛素瘤INS-1细胞。单独用IL-1β、TNF-α或IFN-γ处理以时间和剂量依赖性方式显著下调IA-2和胰岛素mRNA水平，而IL-2无作用。暴露于细胞因子组合可强烈增强抑制作用。用C1和C2孵育细胞24小时分别诱导IA-2 mRNA水平显著抑制78%和58%。在这些条件下，观察到iNOS基因表达增加高达5×10⁴倍。iNOS抑制剂4 mM L-N(G)-单甲基-L-精氨酸与C1共同孵育可部分逆转IA-2的下调，这一发现证实了NO的形成参与IA-2调节的假说。此外，用合成的NO供体S-亚硝基-N-乙酰-D-L-青霉胺孵育可使IA-2 mRNA水平显著降低至基础水平的51%。总之，我们首次证明IL-1β、TNF-α和IFN-γ对糖尿病自身抗原IA-2的表达有强烈抑制作用。IL-1β的作用可能部分由NO途径的激活介导。
