MOUSE IL-1 RECEPTOR ANTAGONIST ISOFORMS - COMPLEMENTARY-DNA CLONING AND PROTEIN EXPRESSION OF INTRACELLULAR ISOFORM AND TISSUE DISTRIBUTIONOF SECRETED AND INTRACELLULAR IL-1 RECEPTOR ANTAGONIST IN-VIVO

IL-1R antagonist (IL-1Ra) is a competitive inhibitor of the binding of IL-l to IL-1R. IL-l Ra refers to two different proteins derived from the same gene by alternate splicing of two different first exons. One protein contains a leader sequence and is secreted (sIL-1Ra), whereas the other remains intracellular (icIL-1Ra). We describe the cloning of mouse icIL-1Ra cDNA, the expression of the recombinant mouse icIL-1Ra protein, and the tissue distribution of sIL-1Ra and icIL-1Ra mRNA and of icIL-1Ra protein in control and LPS-injected mice. As described in the human and the rabbit, mouse icIL-1Ra protein differs from mature mouse sIL-1Ra protein by seven amino acids at the amino terminus. In a ddition, human and mouse icIL-1Ra are 77% identical. Regulation of IL- 1Ra isoforms was examined in normal mice and after LPS injection. Circ ulating levels were undetectable in control mice, but were strongly in creased 4 h after LPS injection. Using a ribonuclease protection assay (RPA), we found that icIL-1Ra mRNA was expressed constitutively in sk in and in LPS-stimulated RAW 264.7 murine macrophages. Consistent with the RNA studies, Western blot analysis showed that murine icIL-1Ra pr otein was constitutively expressed in skin and in LPS-stimulated RAW 2 64.7 cells. In contrast, sIL-1Ra mRNA was not detected by RPA in tissu es of 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, we were able to demonstrate the p resence of different transcription start sites for murine sIL-1Ra mRNA .