The polymorphonuclear leukocyte (PMN) is a critical component of host defense against microbial pathogens as evidenced by the frequent and severe infections seen in patients with an inadequate number of normally functioning PMNs. Optimal microbicidal action occurs in the presence of oxygen and numerous investigators have described various features of this oxygen-dependent system. However, the molecular basis for activation of PMN oxidative metabolism is still incompletely understood. Work proposed herein is designed to use murine monoclonal antibodies (MAbs) as probes to dissect the stimulus-coupled responses in PMN oxidative metabolism. MAbs which alter activation of PMN oxidative metabolism, such as the prototype MAb PMN 7C3 described herein, will be selected. The principles of protein biochemistry, immunochemical analysis, and membrane structure will be used to isolate integral membrane proteins identified by such MAbs in order to define the biochemical nature of important PMN membrane constituents. Radiolabelled MAbs, or fragments thereof, will be used in binding studies to determine the membrane sites available under various conditions, including PMN activation and exposure to secretogogues. Studies will be performed to define the interaction of functionally important membrane proteins with the cytoskeleton and to determine the fate of critical membrane proteins in the activated PMN. Endocytosis of the MAb-membrane protein complex will be studied both with radiolabelled and fluorescented MAb fragments to determine if such proteins are recycled back to the PMN surface or digested intralysosomally with the MAb. These same methods will be applied to establish if intracellular reserves of critical membrane proteins exist and to determine their role in PMN activation. Lastly, the genesis of critical proteins during myeloid development will be examined in a human cell line in culture. These studies will further understanding of the biochemical basis of PMN microbicidal action and aid in unraveling the biochemical abnormalities underlying important clinical disorders such as chronic granulomatous disease.
Showing the most recent 10 out of 17 publications
