HOMEOSTATIC CONTROL OF Fc gamma RECEPTOR TRIGGERED MACROPHAGE FUNCTION

Ohio State University, Columbus OH

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Abstract

Our aim is to understand how the biologic functions mediated by macrophage Ef receptors for IgG (FcgammaR) are homeostatically controlled. FcgammaR clustering triggers a variety of macrophage functions such as endocytosis of opsonized particles, the production of inflammatory cytokines, and the generation of oxygen radicals. The signal cascade begins with phosphorylation of tyrosine based motifs (ITAM) in the cytoplasmic tails of the clustered receptors. These phosphorylated ITAM in turn assemble a variety of positive-acting kinases such as Syk and PI2K. The cascade must be tightly regulated to contain the inflammatory response. At least three levels of control are apparent. First, ITIM-bearing FcgammaRIIb, when co-clustered with ITAM-receptors, recruits the inositol phosphatase SHIP which tempers the forward response. Second, FcgammaRIIb expression, and thus SHIP recruitment, is tightly regulated by cytokines. Third, ITAM-bearing receptors such as FcgammaR1 and FcgammaRIIa, which are know agonists, activate SHIP as well, integrating positive and negative signals from a single ITAM. Building on several years of work in this area, we bring to this project three innovations. First, we apply a new antibody recognizing human FcgammaRllb that, for the first time, will allow us to study the function of this receptor in human cells. Second, we have found that FcTRIIb expression is greatly enhanced by IL-4 and culture density. Third, we show that not only ITIM-but ITAM-FcTR as well activate SHIP, providing a new paradigm for FcTR-mediated regulation of downstream functions. Our two major hypotheses, then, are that SHIP works through both ITAM- and ITIM-FcTR to regulate FcTR function and that the regulated expression of FcgammaRIIb in turn modulates SHIP inhibition of FcgammaR-mediated responses. Testing several predictions of these hypotheses we propose three aims. First, we explore implications of the notion that ITAM activates SHIP. Second, we define the molecular detail surrounding IL-4 enhancement of FcTRllb expression focusing on its ultimate regulation of SHIP activity and differential regulation of the two FcgammaRIIb isoforms. Third, we dissect the molecular events downstream of SHIP asking whether there are differences between ITAM- and ITIM-mediated activation, whether the downstream SHIP effects are enzymatic or due to adaptor functions, and whether the multiple FcgammaR-mediated functions are differentially regulated by SHIP. Our work focuses on human cells of the mononuclear phagocyte lineage although we take advantage ofmurine models where relevant. Our technical approaches to the various aims are broad based and multidisciplinary and include the tools of molecular and cellular biology, biochemistry and immunology. The significance of this work is that understanding these regulatory mechanisms at the molecular level holds the promise of novel therapeutic approaches for modulating antibody-triggered inflammation and killing.