The ability of IFN-gamma to activate macrophages has been exploited as a model to study the potent immunomodulatory activities of this lymphokine. Two classes of genes have been identified which preferentially respond to IFN-gamma; those which appear to represent cell-specific effects of the lymphokine, such as the IgG Fc receptors and those of more general cellular response, exemplified by IP-10 and IP-30. The goals of this proposal remain to define the mechanisms of gene activation by IFN-gamma and the uncover the functions of these induced molecules in the array of IFN-gamma activities. The isolation and structural characterization of several examples of both classes of genes over the past four years have provided the substrates for the continued characterization of the mechanism of IFN- gamma induction. Transcriptional and post-transcriptional events regulate the accumulation of mRNA for these proteins. An interferon stimulated response element (ISRE), defined by it ability to regulate the expression of IFN-gamma induced genes. The role of this and other elements will be assessed by deletional and mutational analysis of the isolated regulatory regions coupled to reporter genes and their re-introduction into cells which are capable of IFN-gamma induction of these genes. Protein interactions with these regulatory sequences will follow by footprinting and gel-retardation analysis of appropriate IFN-gamma induced cellular extracts. Clues to the potential functions of these gene products in the IFN-gamma response have been obtained from studies on the primary structure, localization, correlation with specific inflammatory events and relationship to other inflammatory proteins. To define specific biochemical activities associated with these induced molecules, recombinant protein is being overexpressed and purified from a variety of sources for use in vitro studies. In addition, overexpression of these molecules in a variety of target cells will address in vivo activities. Finally, re- introduction of these transfected cells into mice will determine the role of these molecules in the more complex interactions observed in IFN-gamma action.
