Functional Separation of Pseudopod Extension and Particle Internalization during Fcγ Receptor–mediated Phagocytosis

Abstract

Receptors for the Fc portion of immunoglobulin (Ig)G (FcγR) 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 FcγR 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 FcγR-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 FcγR to phagocytose, placing special emphasis on the FcγRIA–γ chain complex, which allowed us to examine independently the roles of the ligand-binding unit (FcγRIA) and the signaling unit (γ chain). We found that receptor complexes containing a conserved tyrosine activation motif (ITAM), as found in the cytoplasmic domain of FcγRIIA and in the γ chain associated with FcγRIA and FcγRIIIA, readily internalized target particles. In contrast, FcγRIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of γ chain with FcγRIA restored the ability of the receptor to internalize target particles. A mutant FcγRIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the γ chain nor the cytoplasmic domain of FcγRIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all FcγR, but did not block pseudopod extension. Staining the FcγRIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during FcγRIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.

Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

“…Based on this divergence and assuming an inhibitory role for human CD47, human macrophages might be expected to effi ciently phagocytose opsonized sheep red blood cells (RBCs), which is certainly consistent with years of data showing sheep RBCs are readily phagocytosed by human macrophages ( Lowry et al, 1998 ;Botelho et al, 2000 ;Cooney et al, 2001 ). We show below that human-CD47 can inhibit P hagocytosis of foreign cells or particles by macrophages is a rapid process that is ineffi cient when faced with " self " cells that display CD47 -although signaling mechanisms in self-recognition have remained largely unknown.…”

Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

“…When the vacuole contains active Fc␥RIIB1 or B2 receptors, fusion with lysosomes and acidification develop (24). Importantly, unlike Fc␥RIIA, the B1 and B2 isoforms are unable to trigger phagocytosis of IgG-coated inert particles, such as RBC (10,40,41). This implies that signals generated by interaction of Toxoplasma with other cellular components are required for Fc␥RIIB1 or B2-induced phagosomal formation and maturation.…”

A Comparison of Mediators Released or Generated by IFN-γ-Treated Human Mast Cells Following Aggregation of FcγRI or FcεRI

“…The ␥-chain cytoplasmic domain contains immunoreceptor tyrosine-based activation motifs (ITAMs) and the ␥-chain cytoplasmic domain is both necessary and sufficient for Fc␥RI␣-induced functions (33,34). Biochemical studies have shown that both cross-linking of Fc⑀RI␣-␥-chain complex and the Fc␥RI␣-␥-chain complex result in activation of Src family kinases and the tyrosine kinase p72 Syk (4,(35)(36)(37)(38).…”