The means by which interferons (IFNs) activate gene expression through interaction with cellular receptors is being actively investigated. It has been shown that intracellular human IFN-gamma can activate characteristic responses in murine cells despite the normal species-specificity barrier presumed to be due to the receptor. This effect is not due to induced synthesis of murine IFNs or TNF. Experiments to be performed will determine whether other secondary mediators could be responsible. If they are not, alternative mechanisms by which intracellular IFN-gamma may activate gene expression and the relationship between this response and the normal physiological pathway for IFN-gamma action will be investigated. To examine the role of secondary mediators, the induction of MHC class II mRNA expression in cells will be assayed in cells expressing intracellular IFN-gamma transiently or under a regulatable promoter, or after delivery of IFN-gamma protein by liposome fusion. Neutralizing antibodies and inhibitors of protein synthesis will be added to such cells to block the action or production of secondary mediators. To examine mechanisms by which intracellular IFN-gamma can activate responses, a search will be made to identify cytoplasmic proteins which interact with free IFN- gamma. Mutations of the IFN-gamma molecule will be used to explore the role of C-terminal domains in both normal extracellular and intracellular activity. The "comon wisdom" is that inteferons exert their effects, such as induction of specific gene expression, on target cells via receptor-mediated signal transduction. The proposed work embraces an unconventional concept, i.e., that an extracellular ligand can have an intracellular function. The experiments proposed will explore the hypothesis that IFN acts directly, possibly within the nucleus, to turn on specific genes. The information obtained from this study could turn out to be applicable to signaling mechanisms used by other cytokines and growth factors.
