Interleukin-1 (IL-1) has been implicated in the pathogenesis of infection and other inflammatory diseases. IL-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of IL-1 activity that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity. IL-1Ra can be found as a secreted protein (sIL- 1Ra) which is primarily produced by monocytes, whereas keratinocytes produce an intracellular form of IL-1Ra (icIL-1Ra). Both IL-1 and IL- 1Ra production are increased during inflammation. There is increasing evidence that exogenously administered IL-1Ra reduces the severity of disease in a variety of models, but there is a paucity of studies examining the role of endogenous IL-1Ra in inflammation. The experiments proposed in this grant are designed to examine what happens if the synthesis of endogenous IL-1Ra is inhibited by antisense or its actions are neutralized by anti-IL-1Ra antibodies. Preliminary studies indicate that anti-human IL-1Ra antibody prevents recombinant IL-1Ra from inhibiting IL-1-induced cytokine synthesis in human blood mononuclear cells. Studies will examine the effect of neutralizing endogenous sIL-1Ra on the cascade of cytokines synthesized in human monocytes stimulated with agents such as endotoxin. Similar experiments will be performed using antisense oligodeoxynucleotides complementary to human sIL-1Ra. In keratinocytes, antisense specific for icIL-1Ra will be used to determine the role of icIL-1Ra in IL-1-induced proliferation and stimulus-induced cytokine synthesis. Since studies suggest that IL- 1 can affect the transcription of some genes by binding directly to DNA, it will be ascertained whether a reduction of icIL-1Ra levels with antisense icIL-1Ra increases translocation of IL-1 from the cell-surface to the nucleus. The role of IL-1Ra in vivo will also be assessed by examining the effect of antisense icIL-1Ra on survival in mice injected with endotoxin. To identify stimuli which trigger the icIL-1Ra promoter, epithelial cells will be transfected with the icIL-1Ra promoter linked to the chloramphenicol acetyltransferase gene. These studies will link regulation of icIL-1Ra production to cellular events induced by inflammatory stimuli.
