Inflammation and fibrosis in the human lung are regulated by a complex cytokine network(s). The ability of interleukin-1 (IL-1) and tumor necrosis factor (TNF) to synergistically regulate lung fibroblast function is a very important aspect of this network. However the mechanisms of IL-1-TNF synergy (or any form of cytokine-cytokine synergy) have not been adequately investigated. We demonstrated that interleukin-1 (IL-1) and tumor necrosis factor (TNF) interact in a synergistic fashion in regulating lung fibroblast interleukin-6 (IL-6) production and that this synergistic interaction is the result of a selective stabilization of IL-6 mRNA. More recently we demonstrated that IL-1 and transforming growth factor-beta1 (TGF-beta) synergistically stimulate lung fibroblast IL-6 production and that this interaction is mediated at the level of gene transcription. To further understand the processes mediating cytokine-cytokine synergy, we propose to further investigate and compare the mechanisms by which IL-1 plus TNF and IL-1 plus TGF-beta synergistically regulate IL-6 production. We will: I.Determine whether the synergistic effects of IL-1 plus TNF or IL-1 plus TGFbeta are associated with one cytokine to altering the expression of the receptor for the other. II.Characterize the second messenger pathways involved in the synergistic effects of IL-1 plus TNF and IL-1 plus TGF-beta. The importance of cyclic AMP, calcium-calmodulin dependent processes, protein kinase C, phospholipid turnover, intracellular calcium, and G proteins will be investigated. III.Characterize the processes regulating IL-6 transcription in cells stimulated with IL-1 and/or TGF-beta. The cis-elements and trans-acting proteins that mediate the effects of the cytokines will be characterized. IV.Characterize the processes regulating IL-6 mRNA degradation in unstimulated fibroblasts and define the alterations in these processes induced by IL-1 and/or TNF. This will entail: A.Characterization of the elements that determine the stability of IL-6 mRNA. beta-globin-IL-6 constructs will be used to determine whether specific sequences in the IL-6 molecule determine its instability and whether modification of these sequences alters IL-6 mRNA stability. B.Characterization of the cellular events degrading IL-6 MRNA. We will determine whether exoribonucleases and/or endoribonucleases degrade IL-6 mRNA. C. Characterization of the alterations of IL-6 mRNA degradation induced by IL-1 and/or TNF. We will determine whether cytokine induced alterations In IL-6 mRNA degradation are the result of increased ribonuclease activity, increased ribonuclease inhibitors, or alterations in the susceptibility of IL-6 mRNA to degradation. D. Determination of the cytokine response element(s) of IL-6. We will determine if specific segments in the IL-6 transcript mediate the stabilizing effects of the cytokines.
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