Interferons (IFNs) have been implicated in the restriction of a variety of nonviral pathogens. One mechanism by which IFN inhibits pathogens is the induction of indoleamine 2,3-dioxygenase (IDO), which depletes tryptophan required for pathogen growth. Specific cytokines and immunomodulating agents can enhance the antimicrobial effect of IFNs in macrophages by potentiating IDO induction. Interleukin-1 (IL-1) increases the amount of IDO activity induced in IFNs treated macrophages by increasing expression of IDO mRNA, suggesting that potentiation occurs at the nucleic acid level. The objective of this research project is to extend observations made with IL-1 to other cytokines known to potentiate IDO induction, to characterize molecular mechanisms by which IDO mRNA concentration is increased , and to assess the role of arachidonic acid metabolites as second messengers in IDO potentiation. The long-term goal of this research is to understand better the ways cytokine regulate the immune response to intracellular infection. To identify intracellular molecular mechanisms of IDO potentiation, reverse transcription of mRNA followed by polymerase chain reaction amplification of IDO cDNA will be used to quantify increases in IDO mRNA following treatment with IFNs alone, IFNs combined with potentiators, and potentiators alone. Initial experiments will use a monocyte cell line; later experiments will use primary macrophage cultures. In each case, expression of IDO mRNA will be compared with expression of IDO enzymatic activity. If mRNA expression correlates with enzymatic activity, nuclear run-off transcription will be used to determine if increases in IDO mRNA are due to increased rate of transcription of mRNA stabilization. If no correlation is observed, alternative mechanisms will be explored. To confirm the hypothesis that arachidonic acid or its metabolites are integral to signal transduction pathway that mediate IDO potentiation, three goals must be achieved. It must be shown that potentiation trigger the release and metabolism of arachidonic acid, that addition of arachidonic acid or its metabolites mimics the effect of potentiation, and that specific inhibition of the metabolic pathway inhibits the potentiating effect of positive regulators. The association of arachidonic acid release and metabolism with IDO potentiation will be established by scintillation spectrometry and RIA. Arachidonic acid and metabolites will be tested for their capacity to potentiate IDO. Release of arachidonic acid will be induced by exogenous phospholipase A2 to determine if its release substitutes for the signal delivered by cytokine binding. Specific inhibitors of phospholipase A2, 5-lipoxygenase and cyclooxygenase will be used to modulate IDO potentiation. The identification of effects of cytokines on IDO mRNA expression, the establishment of molecular mechanisms leading to enhanced IDO induction, and the determination of the role of arachidonic acid metabolism in IDO regulation are all areas requiring further research to facilitate unraveling the complexity of cytokine- mediated immunity to intracellular infection. This proposal addresses these areas.
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