HUMAN BLOOD MONOCYTE RECEPTOR EXPRESSION AND MODULATION

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Monocyte/macrophage stimulation by their Fc-gamma receptors induces signal transduction leading to phagocytosis. Among the Fc-gamma receptors, Fc-gamma-RI is unique in that it has an extracellular domain (EC) which binds ligand with high affinity and a distinct 61 amino acid cytoplasmic domain (CY) which lacks tyrosines. We have established that members of all 3 human Fc-gamma receptor classes can induce phagocytosis in the absence of any other human Fc-gamma receptor in epithelial cell and murine macrophage transfectants, but only Fc-gamma- RI transmits a signal through both its own alpha chain CY and the CY of an associated gamma subunit. We have also determined that they chain CY is necessary for phagocytosis by Fc-gamma-RI and that Syk kinase, associated with the gamma chain in monocytes/macrophages, enhances phagocytosis by Fc-gamma-RI several fold. In addition to establishing that transfection of specific Fc-gamma receptors and chimeric Fc-gamma receptors into such epithelial cells as CHO cells and hepatocytes renders these cells capable of phagocytosis, we have observed that hepatocytes transfected with Fc-gamma receptors in vivo have the potential to phagocytose IgG coated cells. However, despite the importance of phagocytosis in physiologic and pathophysiologic processes, the intracellular fate of Fc-gamma receptors and their associated signaling molecules during phagocytosis is not well defined. Specifically, we will explore the following: 1) The kinetics of the interactions between Fc-gamma-RIalpha, the gamma chain and Syk kinase during phagocytosis and their role in intracellular trafficking. The association of Fc-gamma-RIalpha, gamma and Syk will be probed using fluorescent protein tags, fluorescence imaging and energy transfer techniques. Our hypothesis is that the gamma chain plays a role in determining the intracellular routing of Fc-gamma-RIalpha, and that gamma, but not Syk kinase, is required for phagosomal maturation. We will also examine the trafficking of Fc-gamma-RIIA, a single chain Fc- gamma receptor which also interacts with Syk kinase, but induces phagocytosis in the absence of the gamma chain; 2) The role of clathrin, other components of the clathrin coated pit and ubiquitination in the routing of Fc-gamma receptors; and 3) Approaches for producing hepatocytes highly efficient in the clearance of IgG coated cells. A major theme of our proposal is to further study Fc-gamma receptor phagocytosis in epithelial cells in vitro (CHO and hepatocytes) and in vivo (hepatocytes) in order to impart to hepatocytes the ability to clear IgG coated cells. Our long term goal is to systematically insert Fc-gamma receptors into hepatocytes thus establishing the potential to enhance host defense and to enhance the clearance of immune complexes.