Macrophage cells in the immune system destroy foreign targets by recognizing them and ingesting them. Receptors for the constant region of IgG antibodies that are on the surfaces of macrophages recognize IgG bound to a foreign target and signal the macrophage to begin phagocytosis. Proposed is a set of experiments designed to investigate the molecular events that occur in the region of initial contact between a macrophage and a potential target, leading both to target recognition and the onset of phagocytosis. The proposed research involves the investigation of two complementary model systems. In the first, physically and chemically well-defined phospholipid target membranes containing hapten-conjugated phospholipids will be deposited on solid planar substrates. The behavior of hapten-specific IgG at the membranes (binding curves, binding kinetic rates, lateral mobility, oligomerization) will be carefully characterized under different conditions. Next, the molecular requirements for and effects of the metabolic activation and/or binding of macrophage-related cell lines at the planar target membranes in the presence of hapten-specific IgG will be examined. In the second model system, purified mouse macrophage cell-surface receptors for IgG will be reconstituted into phospholipid bilayers deposited on planar substrates. The behavior of hapten-specific IgG, followed by soluble monovalent haptyens or multivalent hapten-bearing targets at these planar models of the macrophage cell-surface will be investigated. Several very new techniques in fluorescence microscopy will be refined and employed to probe molecular motion, interaction, and distribution at or near planar membranes, including fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy. The proposed research stands an excellent chance of providing new and important information about the cell-surface molecular mechanisms of macrophage receptor-mediated phagocytosis. It is likely that similar mechanisms operate in other immunological cell types that participate in antibody-mediated target recognition. Development and refinement of techniques in protein reconstitution and in surface fluorescence microscopy will have wide applicability in cell-surface biology, in membrane biophysics, and in biotechnology.
