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Effect of Proinflammatory Cytokines on Gene Expression of the Diabetes-Associated Autoantigen IA-2 in INS-1 Cells

German Diabetes Research Institute at the University of Duesseldorf, Duesseldorf D-40225, Germany

Address all correspondence and requests for reprints to: J. Seissler, M.D., German Diabetes Research Institute, University of Duesseldorf, Auf'm Hennekamp 65, D-40225 Duesseldorf, Germany. E-mail: .

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 ß-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-1ß, TNF-, interferon (IFN)-, and IL-2 as well as with two combinations of these cytokines (C1: IL-1ß + TNF- + IFN-; C2: TNF- + IFN-). Treatment with IL-1ß, TNF-, or IFN- 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 10⁴-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-1ß, TNF-, and IFN- exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1ß may be partly mediated by the activation of the NO pathway.

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