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

• Published in: Endocrinology (Philadelphia) 2002-10, Vol.143 (10), p.3839-3845
• Main Authors: Steinbrenner, Holger, Nguyen, Thi-Bang-Tam, Wohlrab, Ulrike, Scherbaum, Werner A, Seissler, Jochen
• Format: Article
• Language: English
• Subjects: Animals; Antigen-presenting cells; Arginine; Autoantigens; Autoantigens - genetics; Autoantigens - metabolism; Autoimmunity; Beta cells; Cytokines; Cytokines - pharmacology; Cytokines - physiology; Diabetes; Diabetes mellitus (insulin dependent); Diabetes Mellitus - immunology; Down-regulation; Enzyme Inhibitors - pharmacology; Gene expression; Gene Expression - physiology; IL-1β; Inflammation; Inflammation Mediators - pharmacology; Inflammation Mediators - physiology; Insulin; Insulin - genetics; Insulinoma; Insulinoma - metabolism; Interleukin 2; Lymphocytes; Lymphocytes T; Membrane Proteins - genetics; Membrane Proteins - metabolism; Monomethyl-L-arginine; Nitric oxide; Nitric Oxide - physiology; Nitric Oxide Synthase - antagonists & inhibitors; Nitric Oxide Synthase Type II; Nitric-oxide synthase; omega-N-Methylarginine - pharmacology; Pathogenesis; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases - genetics; Protein Tyrosine Phosphatases - metabolism; Protein-tyrosine-phosphatase; Rats; Real time; Receptor-Like Protein Tyrosine Phosphatases, Class 8; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Time dependence; Time measurement; Tumor Cells, Cultured; Tumor necrosis factor-α; Tyrosine; γ-Interferon
• ISSN: 0013-7227; 1945-7170
• 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 β-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-1β, TNF-α, interferon (IFN)-γ, and IL-2 as well as with two combinations of these cytokines (C1: IL-1β + TNF-α + IFN-γ; C2: TNF-α + IFN-γ). Treatment with IL-1β, TNF-α, or IFN-γ 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 × 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-NG-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-1β, TNF-α, and IFN-γ exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1β may be partly mediated by the activation of the NO pathway.