Regulation of CD32A in neutrophils

点击翻译按钮获取中文摘要

EXCEED THE SPACE PROVIDED. In many autoimmune diseases such as arthritis, and lupus the tissue injury is caused by the interaction of inflammatory cells such as neutrophils with the immune complexes (IC) deposited on tissues. Such a tissue injury also results in glomerulonephritis leading to kidney failure and death in these disease states. Recent studies with gone knock out mice have clearly demonstrated that Fc gamma receptors (Fc,/Rs) play a major role in IC mediated autoimmune diseases. Therefore understanding the regulation of function Fcq,Rs has important therapeutic implications. In humans, two types of low affinity FcyRs for IgG, CD32A and CDI6B, are coexpressed on neutrophils. Both bind ligands with overlapping specificity, however, only CD32A is capable delivering signal for phagocytosis. Recent studies from our laboratory have demonstrated that CD32A is functionally inactive in resting neutrophils. However, once neutrophils are activated by fMLP, a bacterial chemoattractant peptide, CD32A is converted to a functionally active state and can bind ligand efficiently. On the contrary, activation of neutrophils with PMA, a neutrophil activating phorbol ester, completely abolished CD32A binding to antibody coated erythrocytes. Interestingly, the neutrophils expressed on cultured cell lines are constitutively active. These results suggest that the avidity modulation is cell type and activation signal specific and the regulation of ligand binding may be one of the mechanisms by which human neutrophil regulates CD32A function. We hypothesize that the molecular changes that occur during neutrophil activation alter the functional state of CD32A. In this grant we propose to determine the molecular basis for this signal specific and cell type dependent regulation of CD32A functional state. Specifically, we will: 1. determine whether the neutrophil activators alter the CD32A-dependent EA binding by influencing the 2D and 3D affinity of CD32A; 3, determine whether phosphorylation of ITAM motif of CD32A is altered by cell activation and correlates with change in ligand binding using immunoblotting, 2D amino acid analysis, and CD32A cytoplasmic domain mutation studies; 3. analyze whether neutrophil activation alters the cytoskeleton interaction of CD32A, receptor clustering, and change in the lateral mobility of CD32A. Since neutrophil activation occurs in rive during infectious and autoimmune diseases it can be hypothesized that dysregulation of CD32A functional state occurs in vivo leading to the expression of high avidity CD32A which enables the ncutrophils bind IC efficiently resulting in tissue injury in these diseases. The data obtained from the proposed research will be useful in understanding and designing therapies to inhibit IC mediated tissue injury in autoimmune and infectious diseases. PERFORMANCE SITE ========================================Section End===========================================