Expression of an insulin/interleukin-1 receptor antagonist hybrid gene in insulin-producing cell lines (HIT-T15 and NIT-1) confers resistance against interleukin-1-induced nitric oxide production.
A hybrid gene consisting of the insulin gene enhancer/promoter region, the signal sequence, the insulin B- and C-chains, and the human interleukin-1 receptor antagonist (IL-1ra) gene was constructed. This hybrid gene was transfected together with the pSV2-neo construct into the insulin-producing cell lines HIT-T15 and NIT-1. One of the geneticin-selected clones, HITra2, expressed a 1.4-kb mRNA, which hybridized both to insulin and IL-1ra-cDNA in Northern blot analysis. Three proteins, with the mol wt 23, 17, and 14 kD, were immunoprecipitated with anti-IL-1ra antibodies from [35S]methionine-labeled HITra2 cells. Both at a low and at a high glucose concentration, 4-5 ng of IL-1ra/10(6) cells (ELISA) was released from these cells. On the other hand, a high glucose concentration evoked a three-fold increase in the release of insulin, suggesting that IL-1ra was released constitutively. Measured by nitrite production, transfected HIT, and NIT-1 cells exhibited a more than 10-fold decrease in IL-1 beta sensitivity. Since the conditioned culture media from the HITra2 cells exhibited an anti-IL-1 beta activity of only 0.5 U/ml, and mixed culture of HITra2 cells and isolated rat islets prevented IL-1 beta induced inhibition of insulin release, it is likely that IL-1ra acts locally at the cell surface. It is concluded that expression of a hybrid insulin/IL-1ra gene confers resistance to IL-1 and that this technique may be used to elucidate the role of IL-1 in autoimmune disorders such as insulin-dependent diabetes mellitus.
Welsh
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Department of Medical Cell Biology, Uppsala University, Sweden.
Bendtzen
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Welsh
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D000818:Animals; D015152:Blotting, Northern; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics; D007340:Insulinoma; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D010190:Pancreatic Neoplasms; D011233:Precipitin Tests; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured
C507763:IL1RN protein, human; C507764:Il1rn protein, mouse; D007328:Insulin; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D009569:Nitric Oxide; D005947:Glucose