INTERLEUKIN-1 STIMULATES THE EXPRESSION OF TYPE-I AND TYPE-II INTERLEUKIN-1 RECEPTORS IN THE RAT INSULINOMA CELL-LINE RINM5F - SEQUENCING ARAT TYPE-II INTERLEUKIN-1 RECEPTOR CDNA

The insulin secreting rat Rinm5F cells are often used to study the cyt otoxic actions of interleukin-1 (IL-1) on pancreatic beta-cells. We de monstrate here that Rinm5F insulinoma cells express both type I and ty pe II interleukin-1 receptor (IL-1R) mRNAs and gene products. IL-IR ag onists, recombinant murine IL-1 alpha (rmIL-1 alpha, 10 ng/ml) and rec ombinant rat IL-1 beta (rrIL-1 beta, 100 pg/ml or 10 ng/ml) induce the upregulation of mRNA expression for both types of IL-1 receptors (IL- 1Rs). This effect of rrIL-1 beta is antagonised by preincubation with recombinant human interleukin 1 receptor antagonist protein (rhIL-1ra, 5 mu g/ml). Furthermore, this rrIL-1 beta induced upregulation of IL- 1R mRNAs is blocked by actinomycin D (7.5 mu g/ml), whereas cyclohexim ide (20 mu g/ml) has no effect. The phorbol ester PMA (20 nM) upregula tes the expression of mRNAs both IL-1 receptors, whereas glucose (50 m M) upregulates the expression of the type I IL-1R mRNA only. Pretreatm ent of cells with pertussis toxin (100 ng/ml) partially blocks the rrI L-1 beta induced expression of mRNA for the type I and, to a lesser ex tent, the type II IL-1R. Incubation of the cells with rrIL-1 beta also induces a time-dependent expressionof c-fos, interleukin-6 (IL-6) and tumour necrosis factor alpha. (TNF-alpha) mRNAs. Binding studies with I-125-recombinant human IL-1 beta (I-125-rhIL-1 beta) indicate that I L-1R gene products, with the ligand binding characteristics of the typ e I IL-1R, are constitutively present on Rinm5F cells. Treatment with rrIL-1 beta (6h) increases the number of I-125-rhIL-1 beta binding sit es on Rinm5F cells. We have also demonstrated that the number of type II IL-1R binding sites increases after induction with rrIL-1 beta (6h) , by indirect immunofluorescence using a monoclonal antibody (ALVA 42) raised against the human type II IL-1R. Furthermore, we have sequence d the type II IL-1R cDNA in the rat insulinoma Rinm5F cells. The compa rison of the amino acid sequence of the rat type II IL-1R with that of the mouse and human type II IL-1Rs shows 90% and 62% amino acid ident ity, respectively. The most important difference between the human and murine type II IL-1Rs, and this rat type II IL-1R cDNA, is an open re ading frame coding for a six amino acid longer, strongly charged (QIKE MK), cytosolic domain. Another detected difference is a putative longe r signal sequence in the rat type II IL-1R, as compared to human and m urine cDNA.