Macrophages are vital cells in host defense against facultative and obligate intracellular infectious agents because one of their capabilities is preventing microbial replication. Consequently it is important to understand how macrophages inhibit microbial replication. This function of murine macrophages is studied in vitro by measuring inhibition of replication of the pathogenic fungus, Cryptococcus neoformans. Fungistasis correlates with consumption of L-arginine from the medium. The l-arginine consumed is metabolized by at least 3 routes: 1) encorporation onto cell protein, 2) conversion to ornithine and urea by macrophage arginase, 3) oxidation to form nitrite and nitrate anions. Fungistatic capability correlates with pathway 3) Metabolism of L-arginine by macrophages in relation to fungistatic activity will be investigates.
The specific aims are: i. Determine what role, if any, pathways 1) and 2) play in contributing to macrophage fungistasis (measure protein synthesis and arginase activity). ii. Identify the intermediates and products of pathway 3) and measure its stoichiometry (separation by HPLC and identification by chemical assays). iii. Determine whether membrane transport is required for macrophage fungistasis (measure transport using radiolabelled arginine). iv. Investigate the relationship of the macrophage activation sequence as induced by lymphokines, monokines, and triggering signals, to the acquisition of pathway 3). v. Isolate the L-arginine consuming enzyme of pathway 3) by affinity chromatography and characterize this activity in a subcellular assay system. The results of each set of experiments will be interpreted in relation to the biological function of intact macrophages, namely their capability for inhibiting fungal replication. These studies are important because they will provide knowledge toward understanding what is required for the macrophage to inhibit microbial replication. This in turn may give insight in explaining certain failures in host defense against intracellular pathogens.
