Divergent Intracellular Sorting of FcγRIIA and FcγRIIB2*

Abstract

The human low affinity Fc␥RII family includes both the activating receptor Fc␥RIIA and the inhibitory receptor Fc␥RIIB2. These receptors have opposing signaling functions but are both capable of internalizing IgG-containing immune complexes through clathrin-mediated endocytosis. We demonstrate that upon engagement by multivalent aggregated human IgG, Fc␥RIIA expressed in ts20 Chinese hamster fibroblasts is delivered along with its ligand to lysosomal compartments for degradation, while Fc␥RIIB2 dissociates from the ligand and is routed separately into the recycling pathway. Fc␥RIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase activity or ubiquitylation of receptor lysine residues. The sorting of Fc␥RIIB2 away from a degradative fate is not due to its lower affinity for IgG and occurs even upon persistent receptor aggregation. Upon co-engagement of Fc␥RIIA and Fc␥RIIB2, the receptors are sorted independently to distinct final fates after dissociation of co-clustering ligand. These results reveal fundamental differences in the trafficking behavior of different Fc␥ receptors.Fc␥ receptors (Fc␥R) 2 are key players in immune responses. Widely expressed on cells of the hematopoietic system, these receptors mediate a multitude of biological responses that are triggered upon receptor engagement by multivalent IgG-containing immune complexes (1). These responses include production of inflammatory cytokines, antibody-dependent cellular cytotoxicity, and induction of dendritic cell maturation. Fc␥R also mediate the internalization of immune complexes. Soluble immune complexes are internalized by clathrin-mediated endocytosis whereas large (Ͼ0.5 m) antibody-coated particles are internalized via phagocytosis. These uptake processes are important in host defense against infection; moreover, defects in immune complex clearance are associated with the development of autoimmunity (2).Fc␥Rs can be categorized into two functional groups. The activating Fc␥R have an immunoreceptor tyrosine-based activation motif (ITAM) within the cytoplasmic domain of the receptor itself or an associated FcR␥ signaling subunit. Phosphorylation of tyrosine residues in the ITAM by Src family kinases after receptor aggregation initiates signaling cascades that trigger downstream effector responses (3). In contrast, inhibitory Fc␥R contain an immunoreceptor tyrosine-based inhibitory motif (ITIM), which recruits phosphatases that antagonize ITAM-mediated signaling. Therefore, the responsiveness of effector cells to immune complexes is determined by the balance between activating and inhibitory receptors (1).A feature of the human immune system that distinguishes it from that of mouse is the presence of both activating and inhibitory members of the low affinity Fc␥RII subfamily. Fc␥RIIA is an activating Fc␥R unique to humans and other primates, and is the most widely expressed Fc␥R on human leukocytes (4). In addition, Fc␥RIIA is unusual among activating Fc␥R in having both ligand binding and ITA...

Inhibitory FcγRIIb-Mediated Soluble Antigen Clearance from Plasma by a pH-Dependent Antigen-Binding Antibody and Its Enhancement by Fc Engineering

“…It has been said that multivalent immune complexes were eliminated by FcgR in vivo and the fact that multivalent immune complexes are constitutively eliminated through FcgRII expressed on liver sinusoidal endothelial cells in mice (11)(12)(13)(14)(15) indicates that multivalent immune complexes bound to mFcgRII would be internalized and transferred to lysosome in vivo. Alternatively, it was also shown by an in vitro study that hFcgRIIb has a recycling capability and that an immune complex internalized by hFcgRIIb is constitutively recycled to the cell surface after internalization (25,26). Considering these conflicting observations, the in vivo behavior of FcgRII needs further evaluation.…”

Human FcγRII Cytoplasmic Domains Differentially Influence Antibody-Mediated Dengue Virus Infection

“…3). It is possible that upon engagement of dengue immune complexes, FcgRIIa is delivered along with its ligand to lysosomal compartments for degradation, whereas FcgRIIb is dissociated from the ligand and routed separately into the recycling pathway, as suggested in a study using transfected cells and nonpathogenic particles (24). These postreceptor signaling and sorting differences in activatory versus inhibitory FcgRII isoforms could favor differential cellular activation, differential viral replication (when immune complexes engage one receptor type versus the other), and differential production in FcgRIIa-expressing cells versus FcgRIIb-expressing cells.…”

“…These postreceptor signaling and sorting differences in activatory versus inhibitory FcgRII isoforms could favor differential cellular activation, differential viral replication (when immune complexes engage one receptor type versus the other), and differential production in FcgRIIa-expressing cells versus FcgRIIb-expressing cells. As a counterbalance, the FcgRIIb pathway inhibits phagocytosis and signaling and could provide a means to control inflammatory responses after exposure to immune complexes (24). This activation and inhibition by FcgRIIa and FcgRIIb, respectively, in cells could be operating in regulating net ADE during DENV disease.…”

“…However, internalization via FcgRIIa was more efficient with a 2-log increase in viral copy number per million cells (Fig.3B, black bars; log10 6.8 versus 10 4.5 viral RNA copies; p = 0.01). It was shown previously that Ag endocytosed by the inhibitory receptor FcgRIIb accesses a nondegradative intracellular compartment that recycles to the cell surface (23,24). Therefore, the efficiency of entry and postentry routing differences could contribute to overall retention of virus derived from immune complexes engaged with respective FcgRII isoforms.…”

Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain

“…Although normal human B cells only express the inhibitory Fc␥R, Gamberale et al (21) observed heterogeneous expression of Fc␥RIIa in malignant B cells from patients with chronic lymphocytic leukemia. Fc␥RIIa shares ϳ93% homology with Fc␥RIIb in the extracellular and transmembrane domains but differs substantially in the intracellular domain (22) because it contains an immunoreceptor tyrosinebased activatory motif (ITAM) rather than an ITIM (23). From the high degree of homology between the extracellular and transmembrane domains of Fc␥RIIa and Fc␥RIIb, we anticipated that Fc␥RIIa would also augment internalization of RTXligated CD20.…”