Our laboratory is interested in the consequences of particular ligand-receptor interactions which mediate cell entry on the ultimate intracellular compartmentalization and fate of obligate intracellular microorganisms. Methods were developed to purify and characterize phagosomes from human neutrophils ingesting Salmonella typhimurium. Taking advantage of the fact that neutrophils will endogenously iodinate particles which are phagocytosed in the presence of 1251 Na, we characterized iodinated constituents, humoral ligands, and cell surface receptors for C3 fragments and for IgG were iodinated and incorporated into the phagosomes regardless of whether the ligand on Salmonella was C3 or IgG. Of particular interest, lactoferrin, a marker for neutrophil secondary granules, was iodinated and incorporated within phagosomes containing IgG-coated S. typhimurium but not C3 coated S. typhimurium. Efforts have also been initiated to purify and characterize phagosomes from murine macrophages ingesting yeast or various life cycle stages of Trypanosoma cruzi. Murine macrophages provide a distinct advantage for many of these studies since a number of variants and alternative cell lines exist which have well characterized properties. For example, phagolysosomes are rapidly formed in the J774 cell line, as assessed by incorporation of lysosomal enzyme markers, whereas these markers are missing in compartments purified from P388D1 cells, lacking the mannose-6- phosphate receptor responsible for targeting lysosomal enzymes. Using yeast coated with eosinophil peroxidase, we have shown that plasma membrane receptors, and in particular CR3, form part of the phagosome membrane. Initial experiments with T. cruzi internalization by J774 cells indicate that CR3 is prominently iodinated when eosinophil peroxidase-coated epimastigotes are internalized.
