The objectives center on two closely related membrane receptors referred to as CR3 and CR4, CD11b and CD11c, or Mac-1 and p150,95. These Beta2- integrin molecules mediated a variety of phagocytic functions, and their absence in leukocyte adhesion deficiency (LAD) is associated with chronic, life-threatening, bacterial infections. Despite their recognized importance in the functions of phagocytic cells, little is known of their functions or importance on NK cells. An interesting feature is their variable ability to mediate intercellular adhesion events with a broad spectrum of ligands. Binding site affinity is apparently regulated by cytoskeletal proteins that attach to receptor cytoplasmic domains. In addition, the cytoskeleton also permits CR3 and CR4 to mediate phagocytosis. In the current award period, acquisition of the ability to mediate phagocytosis or homotypic aggregation was shown to be associated with phosphorylation of the beta-subunit of CR3, CD18. Inhibition of protein kinase C blocked these CR3-dependent functions. The proposed specific aims are based on the central hypothesis that CR3 and CR4 are important receptors for recognition of diverse ligands expressed by pathogenic microorganisms (e.g. Beta-glucan and LPS), tumor cells (e.g., fixed iC3b generated by the alternative pathway), or normal tissue counter receptors (e.g. Beta-glucan and LPS). With NK cells it is further hypothesized that activation with beta-glucan allows CR3 and CR4 (CR3/4) to recognize tumors and initiate cytotoxicity.
For aim 1 cytoskeletal proteins associated with resting CR3/4 on unactivated neutrophils and monocyte/macrophages will be compared to those proteins associated with activated CR3/4 to determine which cytoskeletal proteins are responsible for triggering the activated state. A synthetic peptide representing the cytoplasmic domain of CD18 will be used for both affinity chromatography of solubilized phagocytes and for direct binding studies with purified cytoskeletal proteins are responsible for triggering the activated state. A synthetic peptide representing the cytoplasmic domain of CD18 will be used for both affinity chromatography of solubilized phagocytes and for direct binding studies with purified cytoskeletal proteins known to interact with Beta1 or Beta3 integrins. Cytoskeletal protein associations demonstrated with purified proteins will be confirmed by immunofluorescence microscopy with intact cells.
For aim 2 it is hypothesized that Beta-glucan activation of neutrophils, monocytes, or NK cells make CR3.4 capable of triggering phagocytosis and/or cytotoxicity of targets that express either an endogenous ligand or exogenously fixed iC3b/C3dg. Investigations will be determine whether CR3/4 activation is mediated by direct binding of beta-glucan to CR3/4, or whether there is a distinct beta-glucan receptor. Also, attempts will be made to identify the CR3 ligand recognized by beta-glucan-activated NK cells on K562 cells.
For aim 3 it is hypothesized that fixed iC3b and C3dg are present on many types of human tumor cells in vivo because of the presence of natural and induced antibodies to breast cancer mucin that promote activation of the classical pathway of C. It is further proposed that activation of NK cell or monocyte CR3/4 with soluble beta- glucan will promote the cytotoxicity of such iC3b/C3dg-targeted tumor cells. Focusing on breast cancer, parallel studies will examine breast tumors for the presence of fixed C3 fragments and breast tumor cell lines for their ability to activate C and be killed by beta-glucan-activated NK cells or monocytes. If this aim is successful, it may lead to a new form of therapy for breast cancer in which soluble Beta-glucan is combined with IL-2.
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