Macrophages play a key role in the generation and regulation of cellular and humoral immunity. T-cell responses are triggered by macrophages through antigen processing and presentation in the context of class II MHC molecules. In addition, macrophages regulate immune responses by the production and release of cytokines and are important immune effectors; carrying out phagocytosis, clearance of immune complexes, ADCC, and the killing of intracellular pathogens and tumor cells. Most of these cellular functions are not preformed constitutively but are acquired as the result of macrophage activation. Macrophage activation occurs in response to cytokines and bacterial cell wall products such as LPS. Macrophage activation primarily occurs through gene activation. Although several genes have been shown to be turned on by macrophage activation, many of the genes activated in macrophages remains to be identified. The goal of this research will be to identify and characterize the expression of genes activated in murine peritoneal macrophages in response to interferon-gamma. To accomplish this goal, cDNA clones will be isolated from cDNA subtraction libraries constructed using mRNA isolated from macrophages stimulated with interferon-gamma. cDNA clones will be characterized by DNA sequence analysis; gene mapping; analysis of expression in various tissues, macrophage subpopulations, and macrophage cell lines; and analysis of the regulation of expression by cytokines and LPS. cDNA clones will also be subcloned into expression vectors and antiserum and monoclonal antibodies raised against the fusion proteins. The antibodies will be used in biochemical analysis of the interferon-gamma induced protein and in immunofluorescence and imunnoelectron microscopy studies to determine the intracellular location of interferon-gamma induced proteins.
