Mononuclear phagocytes are frequent participants in the host response to tumor and may contribute to the recruitment and activation of other immune effector cells via the secretion of cytokines or chemokines. These functions are dependent upon new gene expression which reflects the macrophage response to the array of stimuli encountered in the tissue micro-environment. Prototypic stimuli such as INFgamma and lipolysaccharide (LPS) induce or enhance inflammatory function while anti-inflammatory cytokines such as IL-10 suppress such responses. Work supported by this award has focused upon mechanisms of enhanced transcription of chemokine genes. The emphasis of this proposal is the anti-inflammatory action of IL-10. The mechanisms through which IL-10 suppresses gene expression, although incompletely characterized, appear to be diverse and include reductions in transcription, stability of mRNA translation. This proposal will test the following related hypotheses: (A) that a principal site of IL-10 action is the control of mRNA function: specifically the de-stabilization of mRNA and (B) that a secondary mode IL-10 action is the reduction of transcription of genes which are regulated indirectly by the products of LPS (and IL-10) sensitive genes. This hypothesis will be tested in the following specific experimental aims. 1. Determine the role of mRNA primary sequence in IL-10 mediated post-transcriptional control by assessing whether the mRNA sequence is sufficient to determine IL-10 sensitivity, and identifying the specific sequence region(s) of the mRNA which confer sensitivity to IL-10. 2. Evaluate the relationship between mRNA translation and stability in IL-10 mediated suppression. IL-10 may act to reduce mRNA stability requiring coincident target mRNA translation or may inhibit translation with subsequent increased decay of mRNA. 3. Determine if IL-10 indirectly inhibits selective transcription through modulation of required intermediate gene expression. Is LPS-induced IFN alpha/beta a direct target of IL-10 whose suppression leads indirectly to transcriptional inhibition of LPS-induced IP-10? 4. Evaluate the role of sequence specific mRNA binding activity and identify proteins/factors which recognize/bind the IL-10 sensitivity sequence(s). In these experiments the investigator will seek RNA binding proteins which recognize IL-10 sensitivity sequence motif(s), assess their stimulus-dependence and attempt to determine their structural identity.
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