Studies on the regulatory mechanisms of Fcγ receptor function
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Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leukocytes, thereby linking the innate with the adaptive branches of immunity. This thesis undertook a series of studies to further investigate the role of Fc receptors in innate immune responses, using in vitro approaches to examine functional activity and underlying regulatory mechanisms.FcγRIla (CD32a) is a member of the Fcγ receptor family and mediates binding of multivalent IgG. Although CD32a is expressed by a number of myeloid cell types, including neutrophils, macrophages, eosinophils and monocytes, we observed that ligand binding to this receptor was suppressed, as evidenced by the low levels of CD32a-mediated IgG binding to these cells. We therefore aimed to determine the mechanisms that account for the myeloid-specific suppression of CD32a ligand binding. A series of experiments were performed to examine the effects of proteases and sialylation of CD32a in the regulation of IgG binding. In addition, the role of association of CD32a with plasma membrane microdomains rich in cholesterol and sphingolipids in the regulation of IgG binding to CD32a was examined. These membrane microdomains, also termed lipid rafts, have previously been reported to be essential for efficient receptor signalling. Chemical disruption of lipid raft structure by depletion or sequestration of membrane cholesterol greatly inhibited CD32a-mediated IgG binding, strongly implicating lipid rafts in the control of CD32a function. To further investigate this suggestion, specific CD32a mutants were generated, which would be predicted to have altered association with lipid rafts. Both these mutants showed reduced association with lipid rafts (A224S and C241A) and displayed decreased levels of IgG binding compared with wild type CD32a. Additionally, we generated a chimaeric CD32a receptor containing a glycophosphatidylinositol (GPI] lipid anchor consensus sequence, which would constitutively associate with lipid rafts. GPI-anchored CD32a exhibited increased capacity for IgG binding compared with the full-length transmembrane CD32a. Our findings clearly suggest a major role for lipid rafts in the regulation of IgG binding and more specifically, that suppression of CD32a-mediated IgG binding in myeloid cells is achieved by receptor exclusion from lipid raft membrane microdomainsThis thesis also describes an investigation of the association of Fcγ receptor genetic variants with idiopathic pulmonary fibrosis (IPF) susceptibility and progression. In particular, two allelic variants of CD32a (H131/R131) and CD16 (NA1/NA2) were examined that confer altered IgG binding and may therefore contribute to disease pathogenesis. Copy number variation of the FCGR3B gene was also determined using a quantitative PCR-based approach. Susceptibility to IPF was found to be associated with the NA1 allele of FcγRIIIb and increased FCGR3B copy number. In addition, IPF disease severity at disease presentation and progression over a 1 2-month period following diagnosis was found to be linked to the FcγRIIa H131 variant. These results support the involvement of Fcγ-mediated interactions in IPF and reveal a novel role of Fcγ receptors in IPF disease pathogenesis and progression.