Inhibition of Fcγ Receptor-Mediated Phagocytosis by a Nonphagocytic Fcγ Receptor

Abstract

There are three major classes of human Fcγ receptors (FcγRI, FcγRII, and FcγRIII) and various isoforms of each class are capable of mediating phagocytosis. FcγRIIA is an unusual Fcγ receptor in that it transmits a phagocytic signal in the absence of an additional receptor subunit. The cytoplasmic domain of FcγRIIA contains a conserved motif containing two copies of the sequence YXXL. The tyrosines (Y) within the motif are phosphorylated after receptor crosslinking and the integrity of these conserved sequences is required for efficient phagocytosis. The FcγRIIB receptors, FcγRIIB1 and FcγRIIB2, contain one copy of the cytoplasmic YXXL sequence and do not transmit a phagocytic signal. In B cells, FcγRIIB negatively regulates B-cell activation by the B-cell antigen receptor. Human macrophages express both FcγRIIA and FcγRIIB and while FcγRIIA mediates phagocytosis, the function of FcγRIIB in these cells is unknown. Using the epithelial/fibroblast-like cell line COS-1 as a model to examine the molecular events that regulate the phagocytosis of IgG-coated cells (EA), we investigated the effect of FcγRIIB on FcγRIIA signaling. FcγRIIB inhibited phagocytosis mediated both by FcγRIIA and by a chimeric FcγRIIA receptor containing the extracellular domain of FcγRI and the transmembrane and cytoplasmic domains of FcγRIIA. This inhibition occurred at an early signaling stage because tyrosine phosphorylation of the FcγRIIA cytoplasmic domain was inhibited after concurrent stimulation of these receptors with EA. FcγRIIB mutations showed the importance of the FcγRIIB YXXL for inhibition of FcγRIIA-mediated phagocytosis. Deletion of the FcγRIIB YXXL or conservative replacement of the YXXL tyrosine substantially reduced the inhibitory signal. FcγRIIB had a lesser inhibitory effect on phagocytosis by the Fcγ receptor FcγRIIIA, which requires a γ subunit to mediate a phagocytic signal. These results show that FcγRIIB negatively regulates phagocytic signaling by FcγRIIA and suggests that FcγRIIB plays a role in modulating FcγRIIA function in vivo.

PHAGOCYTOSIS PLAYS an important role in host defense against microbial infection and is a major function of monocytes and macrophages. Monocytes and macrophages induce a phagocytic signal through their cell-surface Fcγ receptors (FcγR) which detect and bind IgG coated cells through the constant (Fc) region of IgG.1-3 Human macrophages express receptors from all three FcγR classes: FcγRI, FcγRII, and FcγRIII. Although Fcγ receptors share similar structures, including homologous extracellular IgG binding domains, the individual Fcγ receptors differ significantly in their cytoplasmic regions. The divergence in the structures of the Fcγ receptor cytoplasmic domains largely accounts for their distinct functions.

The FcγRII class of receptors is encoded by three genes: FcγRIIA, FcγRIIB, and FcγRIIC.4 FcγRIIA is expressed on phagocytic cells such as monocytes and macrophages and is the only FcγR present on platelets. FcγRIIB receptors are expressed on monocytes and macrophages as well as on lymphoid cells (B cells and some subpopulations of T cells) and mast cells.5 The two major isoforms of FcγRIIB, FcγRIIB1, and FcγRIIB2 are identical except for a 19-amino acid insertion in the FcγRIIB1 cytoplasmic domain resulting from alternative splicing of the FcγRIIB gene.6 FcγRIIB isoforms are homologous to FcγRIIA in their extracellular and transmembrane regions, but the cytoplasmic domains of the FcγRIIB receptors share little homology with FcγRIIA. Because of the difficulty associated with examining individual Fcγ receptors in hematopoietic cells where several different FcγR classes may be expressed, we have used the fibroblast/epithelial-like cell line COS-1 as a model system to evaluate individual FcγR function. FcγRIIA, when transfected into these cells, can efficiently phagocytose antibody coated erythrocytes (EA).7

Much attention has focused on the presence of the pair of tyrosine containing sequences (YXXL) found within the cytoplasmic domains of most receptors of the Ig gene superfamily or their associated subunits.8-10 This immunoreceptor tyrosine-based activation motif (ITAM) is required for signal transduction through these receptors. FcγRIIA contains an ITAM-like consensus motif consisting of two YXXL sequences separated by 12 amino acids. Phosphorylation of the tyrosines (Y) within this motif is essential for FcγRIIA-mediated phagocytosis and the importance of the ITAM-like region in FcγRIIA-mediated signaling has been established by extensive mutation and deletion studies.10 Although FcγRIIB receptors do not contain a consensus ITAM in their cytoplasmic domain, they do contain one YXXL sequence (YSLL). This YSLL is contained within an ITIM sequence (for immunoreceptor tyrosine-based inhibitory motif). The ITIM sequence found in FcγRIIB was first studied in B lymphocytes where it inhibits B-cell receptor–mediated Ig production.11 12 When transfected into COS-1 cells, FcγRIIB isoforms do not mediate phagocytosis of opsonized cells although they bind EA avidly.13 These studies suggest that although FcγRIIB is expressed in cells of myeloid origin, it does not play a direct role in mediating phagocytosis in vivo.

Since FcγRIIB contains an ITIM sequence, we used our COS-1 cell model to examine the hypothesis that FcγRIIB may regulate FcγRIIA-mediated phagocytosis. Both FcγRIIB1 and FcγRIIB2 inhibited FcγRIIA-mediated phagocytosis whereas phagocytosis by the Fcγ receptor, FcγRIIIA, was only minimally reduced. Using a chimeric FcγRIIA receptor containing the cytoplasmic domain of FcγRIIA, we further showed that the FcγRIIB YSLL tyrosine is important for the inhibitory effect. Signaling through the FcγRIIA cytoplasmic domain and tyrosine phosphorylation of the FcγRIIA chimeric receptor are decreased after costimulation of the FcγRIIA chimera and FcγRIIB. These data indicate that one Fcγ receptor isoform, FcγRIIB, can regulate phagocytosis mediated by another monocyte/macrophage Fcγ receptor isoform, FcγRIIA.