Intracellular IL-1Ra exhibits unique antiinflammatory properties

IL-1Ra exists in four isoforms, three of which are created by alternate transcriptional splice mechanisms and remain in the cytoplasm. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra, the 18 kDa type (icIL-1Ra1), bound specifically to cytoplasmic proteins and exerted antiinflammatory effects inside cells. A yeast two-hybrid screen with HeLa cell lysates indicated binding of icIL-1Ra1 to the third component of the COP9 signalosome complex (CSN3). The CSN complex contains eight subunits, is found in the cytoplasm and nucleus of all mammalian cells, and appears to function as an interface between signal transduction pathways and ubiquitin-dependent proteolysis [1]. The binding of icIL-1Ra1 to CSN3 was confirmed by far western blot analysis and sedimentation in a glycerol gradient; icIL-1Ra1 bound only to CSN3 and not to the other seven components of the CSN. Glutathione pull-down experiments showed that only icIL-1Ra1, and not the other isoforms of IL-1Ra, bound to CSN. Co-immunoprecipitation experiments also indicated binding only of icIL-1Ra1 to the CSN. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of IκB, p53 and c-Jun by CSN-associated kinases. Furthermore, icIL-1Ra1 blocked p53 phosphorylation by recombinant protein kinase 2 and protein kinase D, two kinases associated with the CSN.

To examine the biological function of icIL-1Ra1, this cDNA was transfected into the intestinal epithelial cell line Caco-2, which expresses no endogenous IL-1Ra isoforms. The transfected icIL-1Ra1 inhibited IL-1-induced IL-6 and IL-8 production; this effect was not due to release of icIL-1Ra1 by the cells and blockade of IL-1 receptors [2]. Furthermore, the levels of IL-1-induced IL-6 and IL-8 production in various keratinocyte cell lines were inversely related to the baseline levels of icIL-1Ra1 in the cells. The keratinocyte line A431 contained large amounts of icIL-1Ra1 and exhibited weak production of IL-6 and IL-8 after culture with IL-1. In contrast, KB cells contained no icIL-1Ra1 and exhibited a robust production of IL-6 and IL-8 after stimulation with IL-1. Transfection of icIL-1Ra1 into KB cells led to inhibition of IL-1-induced IL-6 and IL-8 production. To explore whether these antiinflammatory effects of icL-1Ra1 are mediated through interaction with CSN3, experiments are in progress to examine IL-1-induced cytokine production in keratinocyte cell lines after blockade of CSN3 or icIL-1Ra1 production using specific siRNAs. Similar experiments on the effects of icIL-1Ra1/CSN3 interactions on cytokine and matrix metalloproteinase production in human synovial fibroblasts are also in progress.