title: Effect of proinflammatory cytokines on gene expression of the diabetes-associated autoantigen IA-2 in INS-1 cells.
abstract: 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.
authors.affiliation: German Diabetes Research Institute at the University of Duesseldorf, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Steinbrenner
authors.forename: Thi-Bang-Tam
authors.initials: TB
authors.lastname: Nguyen
authors.forename: Ulrike
authors.initials: U
authors.lastname: Wohlrab
authors.forename: Werner A
authors.initials: WA
authors.lastname: Scherbaum
authors.forename: Jochen
authors.initials: J
authors.lastname: Seissler
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology / Q000502:physiology*; D003920:Diabetes Mellitus / Q000276:immunology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology*; D018836:Inflammation Mediators / Q000494:pharmacology / Q000502:physiology*; D007328:Insulin / Q000235:genetics; D007340:Insulinoma / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D001324:Autoantigens; D016207:Cytokines; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D007328:Insulin; D008565:Membrane Proteins; D012333:RNA, Messenger; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516864:Ptprn protein, rat; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8