Human Blood Monocyte Receptor Expession and Modulation

Schreiber, Alan

Abstract

Monocyte/macrophage stimulation by their Fc-gamma receptors induces signal transduction leading to phagocytosis and cytokine release. 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 (CY) domain which lacks tyrosines. The PI has established that members of all 3 human Fc-gamma receptor classes can induce phagocytosis in the absence of any other 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 domain and the CY domain of an associated gamma subunit. The PI has also determined that the gamma chain CY domain is necessary for phagocytosis by Fc-gamma-RI and that the Syk kinase, associated with the gamma chain in monocytes/macrophages, enhances phagocytosis by Fc-gamma-RI several fold. However, during phagocytosis the intracellular fate of Fc-gamma-RI-alpha and its associated signaling molecules is not well defined. In addition, the PI has recently observed that the Fc-gamma-RI-alpha CY domain (which lacks tyrosines) is required for the release of the important cytokine IL-6 from macrophages, suggesting a unique mechanism by which the Fc-gamma-RI-alpha/gamma receptor complex mediates important biologic functions. Specifically, The PI will explore the following questions: 1) The initial interaction in the trafficking of Fc-gamma-RI-alpha, the gamma chain and Syk kinase during phagocytosis. The association of Fc-gamma-RI-alpha, the gamma chain and Syk will be probed using fluorescent protein tags, fluorescence imaging and energy transfer techniques. The PI's hypothesis is that the CY domain of Fc-gamma-RI-alpha plays a role in determining the intracellular routing of Fc-gamma-RI-alpha and that the gamma chain, but not Syk kinase, is required for phagosomal maturation. The PI will also define the role of clathrin and other components of the clathrin coated pit in the routing of the Fc-gamma-RI-alpha/gamma complex. 2) The role(s) of the Fc-gamma-RI-alpha and gamma chains in IL-6 release and structure/function relationships of the Fc-gamma-RI-alpha CY domain in IL-6 release and Ca2+ signaling. The PI's hypothesis is that serine residues in the Fc-gamma-RI-alpha CY domain are necessary for Fc-gamma-RI mediated IL-6 release by macrophages. 3) Finally, the EC of Fc-gamma-RI-alpha binds monomeric (serum) IgG with high affinity and its ability to bind immune complexes and IgG coated cells in vivo is unknown. Therefore, the ability of Fc-gamma-RI-alpha to bind IgG coated cells in vivo will be addressed by directly testing the ability of the EC domain of Fc-gamma-RI-alpha to bind and phagocytose IgG coated RBCs in vivo. The PI's hypothesis is that the extracellular domain of Fc-gamma-RI-alpha will enhance the clearance of IgG coated RBCs in vivo.