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 PMN activation work proposed herein is designed to use murine monoclonal antibodies (MAbs) as probes to dissect the stimulus-coupled responses in PMN activation. Both previously derived MAbs as well as new MAbs will be employed for this study. MAbs which inhibit oxidative metabolism (PMN 7C3), as well as MAbs directed against erythrocyte band 3 and the leukocyte adhesion glycoprotein Mo1 will be used. MAbs directed against integral membrane proteins (IMPs) in lysosomal membranes will be generated. 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. Labelled MAbs, or fragments thereof, will be used in binding studies to determine the membrane sites available under various conditions as well as intracellular translocation of lysosomal IMPs during PMN activation and exposure to secretogogues. Studies will be performed to define the interaction of functionally important membrane proteins with the submembranous and transcellular 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. Biosynthesis and intracellular processing and transport of lysosomal IMPs will be examined in a cultured cell line (HL-60) and the role of such IMPs in regulating trafficking of nascent enzymes into the correct lysosomal compartment. 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.
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