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Receptors for the Fc portion of immunoglobulin (Ig)G (Fc gamma R) mediate phagocytosis of IgG-opsonized particles by a process that can be divided into four major steps: receptor-ligand binding, pseudopod extension, internalization, and lysosomal fusion. We have expressed single classes of Fc gamma R in COS fibroblasts to examine the structural determinants necessary to complete the four steps of phagocytosis. Using phase contrast, fluorescence, confocal, and electron microscopy we have demonstrated that Fc gamma R-expressing COS cells can phagocytose in a manner similar to that of professional phagocytes. We have further analyzed the capacity of the three classes of Fc gamma R to phagocytose, placing special emphasis on the Fc gamma RIA-gamma chain complex, which allowed us to examine independently the roles of the ligand-binding unit (Fc gamma RIA) and the signaling unit (gamma chain). We found that receptor complexes containing a conserved tyrosine activation motif (ITAM), as found in the cytoplasmic domain of Fc gamma RIIA and in the gamma chain associated with Fc gamma RIA and Fc gamma RIIIA, readily internalized target particles. In contrast, Fc gamma RIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of gamma chain with Fc gamma RIA restored the ability of the receptor to internalize target particles. A mutant Fc gamma RIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the gamma chain nor the cytoplasmic domain of Fc gamma RIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all Fc gamma R, but did not block pseudopod extension. Staining the Fc gamma RIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during Fc gamma RIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.

The microtubule cytoskeleton of human leukocytes has been difficult to study, in part, due to the lack of a reliable protocol for the indirect immunofluorescence staining of microtubules in these cells. We report here the development of a simple and reliable immunocytochemical labeling protocol for the examination of microtubules in leukocytes including monocytes, neutrophils, and eosinophils. The dynamic properties of microtubules in both monocytes and neutrophils were examined by indirect immunofluorescence staining of cells following exposure to nocodazole. Nocodazole-induced depolymerization is extremely rapid in both cell types, as is the regrowth of microtubules following removal of the nocodazole. Rapid reorganization of the microtubule cytoskeleton was also observed in neutrophils undergoing chemotactic stimulation. Bundling of microtubules was observed in both monocytes and neutrophils isolated from patients undergoing taxol infusion chemotherapy. The taxol-induced bundles were transient in nature as they were absent from samples collected 48 h following the completion of the taxol infusion. These results demonstrate the unique dynamic properties of leukocyte microtubules and indicate that they can be altered in vivo. The development of this staining protocol should allow for the further analysis of leukocyte microtubules as related to the normal functional response of these cells and form the basis for correlating alterations in microtubule dynamics with the effects of taxol on leukocyte function.

The NADPH oxidase complex catalyzes the formation of superoxide (O2.-) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed.