Mouse IL-1 receptor antagonist isoforms: complementary DNA cloning and protein expression of intracellular isoform and tissue distribution of secreted and intracellular IL-1 receptor antagonist in vivo
Gabay, C.; Porter, B.; Fantuzzi, G.; Arend, W.P.
Journal of Immunology 159(12): 5905-5913
1998
ISSN/ISBN: 0022-1767 PMID: 9550387 Document Number: 488669
Previous studies showed that interleukin-1 R antagonist (IL-1 Ra) was a competitive inhibitor of the binding of IL-1 to IL-1R. IL-1Ra showed 2 forms of the protein derived from the same gene by alternate splicing of 2 different first exons. One protein contained a leader sequence and was secreted (sIL-1 Ra), whereas the other remained intracellular (icIL-1 Ra). The present paper described the cloning of mouse icIL-1 Ra cDNA, the expression of the recombinant mouse icIL-1Ra protein, and the tissue distribution of sIL-1 Ra and icIL-1 Ra mRNA and of icIL-1 Ra protein in control and lipopolysaccharide (LPS)-injected mice. As described in rabbits, mouse icIL-1 Ra protein differed from mature mouse sIL-1 Ra protein by 7 amino acids at the amino terminus. Regulation of IL-1 Ra isoforms was examined in normal mice and after LPS injection. Circulating levels were undetectable in control mice, but were strongly increased 4 h after LPS injection. A ribonuclease protection assay (RPA) showed that icIL-1 Ra mRNA was expressed constitutively in skin and in LPS-stimulated RAW 264.7 mouse macrophages. Consistent with the RNA studies, Western blot analysis showed that mouse icIL-1 Ra protein was constitutively expressed in skin and in LPS-stimulated RAW 264.7 cells. In contrast, sIL-1 Ra mRNA was not detected by RPA in control mice, but was strongly up-regulated in the lung, spleen and liver after LPS injection. Using RPA, primer extension assay and 5' rapid amplification of cDNA ends, different transcription start sites for murine sIL-1 Ra mRNA were demonstrated.