Interleukin-1 stimulates the expression of type I and type II interleukin-1 receptors in the rat insulinoma cell line Rinm5F; sequencing a rat type II interleukin-1 receptor cDNA
Bristulf, J.; Gatti, S.; Malinowsky, D.; Bjork, L.; Sundgren, A.K.; Bartfai, T.
European Cytokine Network 5(3): 319-330
ISSN/ISBN: 1148-5493 PMID: 7524717 Accession: 008898234
The insulin secreting rat Rinm5F cells are often used to study the cytotoxic actions of interleukin-I (IL-1) on pancreatic beta-cells. We demonstrate here that Rinm5F insulinoma cells express both type I and type II interleukin-1 receptor (IL-1R) mRNAs an gene products. IL-1R agonists, recombinant murine IL-1-alpha (rmIL-1-alpha, 10 ng/ml) and recombinant 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 cycloheximide (20 mu-g/ml) has no effect. The phorbol ester PMA (20 nM) upregulates the expression of mRNAs both IL-1 receptors, whereas glucose (50 mM) upregulates the expression of the type I IL-1R mRNA only. Pretreatment of cells with pertussis toxin (100 ng/ml) partially blocks the rrIL-1-beta induced expression of mRNA for the type I and, to a lesser extent. the type II IL-1R. Incubation of the cells with rrIL-1-beta also induces a time-dependent expression of c-fos, interleukin-6 (IL-6) and tumour necrosis factor cc (TNF-alpha) mRNAs. Binding studies with 125I-recombinant human IL-1-beta (125I-rhIL-1-beta) indicate that IL-1R gene products, with the ligand binding characteristics of the type I IL-IR, are constitutively present on Rinm5F cells. Treatment with rrIL-1-beta (6h) increases the number of 125I-rhIL-1-beta binding sites on Rinm5F cells. We have also demonstrated that the number of type II II-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 sequenced the type II IL-1R cDNA in the rat insulinoma Rinm5F cells. The comparison 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 identity, respectively. The most important difference between the human and murine type II IL-1Rs, and this rat type II II-1R cDNA, is an open reading frame coding for a six amino acid longer, strongly charged (QIKEMK), cytosolic domain. Another detected difference is a putative longer signal sequence in the rat type II IL-1R, as compared to human and murine cDNA.