Paracrine downregulation of FcγRIII in human monocyte-derived macrophages induced by phagocytosis of nonopsonized particles

Abstract

Monocytes and macrophages can take up nonopsonized particles through a direct phagocytic process and IgG-coated particles through combined mechanisms of nonspecific phagocytosis and internalization mediated by specific Fc receptors for IgG (FcγR) on the plasma membrane. In this study, we report the effect of phagocytosis of nonopsonized latex beads on the levels of expression of Fcγ receptors in human monocyte-derived macrophages (MDM) as measured by flow cytometry (fluorescence-activated cell sorter [FACS]). Macrophages were exposed to green fluorescent 1-μm polystyrene beads before labeling for FcγRI, FcγR-II, and FcγR-III, respectively, with 32.2, 2E1, and 3G8 monoclonal antibodies (MoAbs) and a phycoerythrin (PE)- conjugated secondary Ab to permit dual-channel analysis of fluorescence intensity by FACS. Macrophages that had phagocytosed at least one bead showed reduced levels of surface FcγR-III but not FcγR-I or FcγR-II when compared with cells that had never been exposed to beads. Moreover, cells that were not in direct contact with beads, but that shared medium with cells that had phagocytosed beads also had reduced levels of FcγR-III but not FcγR-I or FcγR-II, suggesting a cytokine-mediated mechanism of FcγR-III downregulation. Phagocytosis of 1-μm beads alone stimulated macrophages to release tumor necrosis factor-α (TNF-α). The medium from macrophages phagocytosing beads could stimulate other macrophages not in direct contact with beads to release TNF-α as well, but such paracrine-triggered release could be reduced by more than 50% if the medium from the phagocytosing cells was first treated with a neutralizing anti-TNF-α-antibody. Moreover, the paracrine downregulation of FcγR-III described above could also be blocked if the neutralizing anti-TNF-α antibody was added to the medium of phagocytosing cells. Treatment of macrophages with recombinant human TNF-α in the absence of beads induced decreased levels of FcγR-III but not of FcγR-I or FcγR-II. These results show that paracrine downregulation of FcγR-III is mediated by a TNF-α-dependent mechanism. In parallel with cell surface FcγR-III levels, phagocytosis of IgG-opsonized fluorescent beads by MDM decreased after stimulation of MDM with nonopsonized beads, whereas the nonopsonic phagocytic activity was unchanged, indicating that the downregulation of cell surface FcγR-III expression induced by nonopsonic phagocytosis can result in changes in FcγR-mediated macrophage functions.