We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D, jl-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins. (J. Clin. Invest. 1992. 90:1978

A method of determining the stoichiometry of metastable phases from nucleation characteristics is described and is applied to calcium phosphate precipitation. The stoichiometric ratio [Ca]/[P] is shown to be 1.49 ± 0.02 in the pH range 7.0–9.1, indicating that it is not hydroxyapatite which normally forms first in the physiological pH region. Characterization of the product by radio isotopes and by infrared spectroscopy and electron diffraction confirms the stoichiometry and indicates that the calcium phosphate nucleated from solution at high pH is an amorphous or soft metastable material. Some biological implications of these findings are pointed out.

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Several studies have suggested that prostaglandin E2 (PGE2) might influence the phagocytic activity of macrophage cells. The present study was designed to examine the in vivo effects of PGE2, the prostaglandin synthesis inhibitor meclofenamate, the prostaglandin precursor arachidonic acid, and the biologically inactive fatty acid 11,14,17-eicosatrienoic acid on phagocytosis by peritoneal macrophage cells in the rat. Following 3 days of treatment with either agent, fluorescent methacrylate microbeads were injected intraperitoneally into all rats. Peritoneal exudates were harvested after administration of the microbeads and the percent phagocytosis determined in macrophage cells using a fluorescence-activated cell sorter (FACS II). The administration of PGE2 was associated with a significant decrease in the percentage of peritoneal macrophages ingesting the fluorescent methacrylate microbeads. In contrast, treatment with arachidonic acid or 11,14,17-eicosatrienoic acid significantly enhanced the percentage of phagocytic macrophage cells. A significant increase in the number of macrophages undergoing phagocytosis of the methacrylate microbeads was also observed in rats treated with meclofenamate. This later observation, taken together with the inhibitory effect induced by PGE2 on macrophage phagocytosis, points to a potential modulator role of PGE2 on the phagocytic activity of macrophages. These data also suggest that arachidonic acid might influence macrophage phagocytosis by a mechanism independent of PGE2.