Pulmonary macrophages (PM) aid lung defense by removing a variety of deposited pathogens through the process of phagocytosis. This process begins with the specific interaction of particle surface ligands with ligand receptors in the macrophage cell membrane. Unlike immunologically opsonized pathogens, PM receptors for pathogens which do not generate an immune response remain largely undefined. We have developed new techniques to investigate phagocytosis. These include the ability to distinguish and quantitate bound versus ingested particles as well as the ability to evaluate the heterogeneity of these and other characteristics within the PM population. Using a series of in vitro experiments, we propose to characterize the mechanisms by which particles with different surface charges and particles opsonized with immunoglobulin are phagocytized. Particles will be prepared by coating fluorescent latex beads with appropriate proteins. The quantitation of attached versus ingested particles will be made through the combined use of flow cytometry and fluorescence microscopy. Particles will also be used to isolate the cell surface receptors to which they bind. Antibodies raised against these receptors will allow us to determine if changes in particle binding reflect changes in receptor number or particle- receptor affinity. Another tool we have developed is a monoclonal antibody against an age-related cell surface antigen specific to hamster PM. This antigen may be the receptor for negatively-charged particles. Interestingly, our antibody and calcium ionophore inhibit ingestion, presumably by raising intracellular calcium levels. This contention must be reconciled with the fact that calcium ionophore also stimulates leukotriene synthesis. Particles, our monoclonal antibody, calcium ionophore and metals will be used to test hypothesis concerning the role of calcium and leukotrienes in the mechanism which couples particle-receptor binding to particle internalization. The integration of this information will enhance our understanding of phagocytosis and its cellular rregulation in vivo.
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