Gangliosides are ubiquitous components of eukaryotic plasma membranes. They may function as receptors for bacterial toxins, peptide hormones, viruses, and immunomodulatory agents (interferon, MIF). A complex and unique pattern of gangliosides is associated with lymphoid tissue. More than twenty new gangliosides appear when murine macrophages are activated by endotoxin or other activating agents. The physiologic role of these new gangliosides is totally unknown. Endotoxins interact with macrophages and trigger a variety of biochemical responses many of which clearly play a role in host defenses. The molecular mechanisms by which endotoxins act on macrophages are totally unknown. The long term goal of this application is to elucidate the mechanisms by which endotoxins interact with macrophages. Specific attention will be paid to macrophage gangliosides as potentially critical components of the plasma membrane which may be involved in the transduction of activating signals. This application proposes to determine whether activating agents, both endogenous and exogenous, interact with gangliosides from mononuclear phagocytes; to assess the expression of specific gangliosides in different macrophage populations using monoclonal antibodies directed against specific gangliosides; and to transfer gangliosides between cell populations and assess whether new functions may be acquired by the altered macrophages. A two-dimensional TLC system has been developed in our laboratory to display gangliosides purified from lymphoid cells. Monoclonal antibodies will be prepared using endotoxin-ganglioside complexes as immunogens and a solid phase immunoassay to detect positive clones. These antibodies will be used in direct binding assays on TLC plates to identify specific compounds. In similar fashion, the binding of bacterial endotoxins and gamma interferon to macrophage gangliosides will be assessed. The physiologic significance of a particular ganglioside composition will be assessed using fusogenic liposomes to transfer the gangliosides between populations of macrophages. Phagocytosis and assays of oxidative metabolism will be used to assess macrophage function. These experiments will further define the molecular basis of endotoxin-induced cellular responses. Furthermore they will help elucidate the significance of macrophage gangliosides as potential receptors for immunomodulatory agents.
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