P. Mark Hogarth

Molecular Basis of Fc Receptor Function

Publisher Summary This chapter focuses on studies of the murine and human leukocyte FcγR and FCɛR I, with particular reference to the structural characterization of these receptors, the molecular nature of their interaction with immunoglobulin (Ig), and their mechanisms of signal transduction. In addition, the chapter also reviews different aspects of FcμR, FcαR, the poly Ig receptor, the receptor for the transport of Ig in neonatal gut, and receptors for IgD. The characterization of the proteins, transcripts, and genes of the different classes of both these FcR families— FcγR and FCɛR I—has largely been completed and those for IgM, IgD, and IgA receptors still to be completed. Attention is now turning to understanding the nature of the mechanisms by which these receptors and their subunits are able to mediate their functions. The structural analysis of the functional basis of FcγR and FCɛR signaling is beginning to shed some light on the way these receptors trigger biological responses, and the molecular dissection of the pathways involved in signaling is also underway. The understanding of these interactions may provide the means to devise strategies to inhibit pathophysiological effects of FcR function that would have far reaching implications in the treatment of antibody induced hypersensitivity.

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG.

The low affinity receptor for IgG, FcγRII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. FcγRII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human FcγRII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by FcγRII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between FcγRII and the structurally related FcεRI α chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by FcγRII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of FcγRII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.

The second and third extracellular domains of FcγRI (CD64) confer the unique high affinity binding of IgG2a

FcgammaRI (CD64) is functionally unique as it is the only FcgammaR able to bind monomeric IgG with high affinity. FcgammaRI is also structurally distinct, containing an extracellular Ig-interactive region of three Ig-like domains in contrast to the two domains of the low affinity receptors FcgammaRII and FcgammaRIII. Previous studies have demonstrated that the third domain of FcgammaRI plays a crucial role in high affinity IgG binding of the receptor, with the first and second domains together forming a low affinity IgG binding motif. In this study the individual functional contributions of the first and second domains of FcgammaRI to IgG binding have been investigated. Chimeric FcgammaR were generated by exchanging extracellular domains between mouse FcgammaRI and the structurally related yet distinct low affinity receptor for IgG, mouse FcgammaRII. The replacement of both domains 1 and 2 of FcgammaRI with domains 1 and 2 of FcgammaRII results in a dramatic change in IgG binding characteristics, as this receptor loses the capacity to bind monomeric IgG with high affinity and also demonstrates a broader specificity (binding not only IgG2a but also IgG1 and 2b. IgG3 was not tested). However, the substitution of FcgammaRII domain 2 of this chimeric receptor with domain 2 of FcgammaRI (generating a chimeric receptor with domain 1 of FcgammaRII linked to domains 2 and 3 of FcgammaRI) was found to reconstitute the specific high affinity monomeric IgG2a binding of wild-type FcgammaRI, albeit with a slightly reduced affinity (1.8-fold lower than wild-type FcgammaRI). These findings suggest that it is the specific interaction between domains 2 and 3 of FcgammaRI, with domain 1 playing a supporting role in maintaining the conformational stability of the receptor, that is the major structural requirement to confer the unique Ig binding characteristics of FcgammaRI.

The inhibitory co-receptor, PECAM-1 provides a protective effect in suppression of collagen-induced arthritis.

Studies of PECAM-1−/− mice have identified that PECAM-1 functions as an inhibitory co-receptor to modulate immunological responsiveness. In this study, we describe the in vivo consequences of PECAM-1 deficiency in mouse models of collagen-induced arthritis (CIA) and K/BxN passive transfer model that resembles many of the features of human rheumatoid arthritis. Immunization of PECAM-1−/− C57BL/6 (H-2b) mice with chicken collagen type II induced CIA with an incidence of 82% by day 49, while 33%; of wild-type and 100% of DBA/1 mice developed arthritis in a similar time frame. The mean onset of disease for PECAM-1−/− C57BL/6 mice was day 32 compared to day 51 for wild-type C57BL/6 mice and day 18 for DBA/1 mice (H-2q susceptible). In terms of disease severity, the mean maximal arthritic index for PECAM-1−/− C57BL/6 mice was comparable to DBA/1 mice (8.91 ± 0.91 vs 11.67 ± 0.82). This mean maximal index in PECAM-1−/− C57BL/6 mice was significantly higher than wild-type C57BL/6 mice (5.00 ± 0.73). IgG1 and IgG2b antibody responses against CII were elevated in arthritic PECAM-1−/− C57BL/6 mice compared to wild-type C57BL/6 mice. Histological examination of arthritic paws of PECAM-1−/− C57BL/6 mice revealed inflammatory infiltrates of lymphocytic/monocytic cells and cartilage/bone destruction similar to CIA-induced DBA/1 arthritic paws. In the K/BxN model, the arthritis was not augmented in PECAM-1−/− mice compared to wild-type mice. In contrast, in active CIA, PECAM-1−/− mice developed severe disease comparable to susceptible DBA/1 mice and profoundly more severe than C57BL/6 mice, where only one third developed a mild/moderate disease. Together these observations suggest that PECAM-1 plays a crucial role in the suppression of development of autoimmune arthritis.

Identification of the Immunoglobulin Binding Regions (IBR) of FcγRII and FcɛRI

Fc receptors (FcR) are cell surface glycoproteins that bind the Fc region of immunoglobulin (Ig) and are found on the cell surface of the vast majority of leukocytes, FcR have been defined for all classes of Ig and play important roles in the protection of an organism against infection by providing the host with a link between immunological function (antibody production) and paraimmunological functions such as immune complex removal by macrophages and neutrophils or the sensitization of mast cells with IgE. The biological roles of FcR, their diversity, function and early biochemical characterization as well as their recently understood molecular genetics have been extensively reviewed elsewhere and will not be covered here (Spiegelberg 1984, Unkeless et al. 1988, Van de Winkel & Anderson 1991, Ravetch & Kinet 1991). This manuscript will review our attempts to define the regions of IgG and IgE FcR that are involved in Ig binding and develop a model of the structure of Fc receptors. Three distinct classes of human and murine FcR for IgG (FcyR) have been defined on the basis of their distinct structures, specificities and affinities for IgG. Thus FcyRI has a high affinity for monomeric IgG (10^M) and shows preferential binding of certain IgG subclasses, whereas FcyRII and FcyRIII have a very low affinity for IgG monomer (< 10*M) but avidly bind IgG complexes (Unkeless et al. 1988, Van de Winkel & Anderson 1991, Ravetch & Kinet 1991). A single high-affinity receptor for IgE (FceRI) is present on mast cells and binds IgE with affinity of 10M and is distinct from an unrelated low-affinity IgE receptor FccRII or CD23 (Metzger et al. 1986, Ravetch & Kinet 1991).

Fcγ receptors: Gene structure and receptor function

Molecular studies of murine FcγR have revealed much exciting new information about the structure and regulation of FcγRI and FcγRII genes and of the FcγRI protein. The FcγRI gene is composed of six exons, whereas the FcγRII gene is composed of ten. The extracellular domains are encoded by individual exons in both genes (three in FcγRI and two in FcγRII); however, the FcγRII gene shows greatest complexity in the region encoding the cytoplasmic tail and membrane spanning region, which is encoded by four exons compared to only one in the FcγRI gene. Expression of FcγRII is controlled by elements within the first 641 bases upstream of the transcription initiation site. The function of the domains of FcγRI has been defined with the surprising finding that in the absence of the third domain the first two extracellular domains function as a broadly specific low affinity FcγRII-like receptor.

The Structural Basis of the Interaction of IgE and FcεRI

FceRI is capable of inducing one of the most powerful and violent pharmacological responses known. Indeed the association of IgE with FceRI, and subsequent aggregation is a most important interaction in the induction of human disease, and causes more chronic misery (in the West at least) than the engagement of any other immunological receptor. As approximately one in five people are afflicted with IgE dependent allergies—most notably allergic rhinitis or ‘hay fever’ and asthma—there has been a large effort made by many groups in studying this receptor, its ligand and the consequences of its activation. The impetus to study this receptor probably stems from its pathological role rather than its physiological one, which is still somewhat undefined, but with evidence pointing to an anti-parasite role.1

The human FCG1 gene encoding the high-affinity FcγRI maps to chromosome 1q21

FcyRI is an interferon inducible high-affinity receptor for IgG which is important in antibody dependent killing, the binding of immune complexes and internalization of antibody-coated particles (Unkless et al. 1988; Ravetch and Kinet 1991). Biochemical characterization of FcyRI indicates that it is related to two other FcyR: the low-affinity FcyRII and FcyRIII as well as the highaffinity receptor for IgE-FceRI. Indeed, these receptors form a subfamily of the Ig superfamily (reviewed in Ravetch and Kinet 1991). Genetic characterization of these receptors indicates that in man there are at least three genes encoding FcyRI (Ernst et al. 1992), three encoding FcyRII, and two encoding FcyRIII proteins (Qiu et al. 1990). Our previous studies using somatic cell hybrids mapped the human FCG1 locus to chromosome 1 (Osman et al. 1992), but the precise location on this chromosome is unknown. By contrast, the genes encoding the lowaffinity FcyRII and III as well as the high-affinity IgE receptor Fc~RI(z have been mapped to a relatively small segment of chromosome 1 (lq23-24; Sammartino et al. 1988; Grundy et al. 1989; Hupp et al. 1989; Tepler et al. 1989; Kingsmore et al. 1990; Le Coniat et al. 1990; Webber et al. 1990), where they clearly arose by gene duplication and crossover (Qiu et al. 1990). Based on our studies of the mouse where the Fcgl gene is located on chromosome 3 (Oakey et al. 1992; Osman et al. 1992) but Fcg2, Fcg3, and Fcel are on chromosome 1, it would appear that the human FCG1 gene may not be part of the cluster of FcR-encoding genes on lq23-24. Radioactive chromosomal in situ hybridization was performed to map and determine independently the pre-

The Fc receptor family structure based strategies for the development of anti-inflammatory drugs

Summary. Leukocyte Fc receptors play key roles in the initiation of antibodyxad mediated cellular responses to pathogens. However, they are also involved in the pathogenesis of destructive immune complex inflammatory diseases. The solving of the FcR crystallographic structures together with molecular modelling has provided an opportunity to examine closely the mode of interaction between receptor and ligand. Also such work has allowed the design of novel antixad inflammatory agents that act to block receptor:ligand interactions. These are potentially effective therapeutic agents in the treatment of immune complex mediated inflammatory diseases.

Immunoglobulin Fc receptors: Diversity, structure, and function

Publisher Summary This chapter discusses the diversity, structure, and function of immunoglobulin (Ig) Fc receptors. Receptors for immunoglobulins, Fc receptors (FcR), play a fundamentally important role in immunity and resistance to infection by providing the means of linking humoral immunity to cellular effector mechanisms. Indeed, as will be seen below, these widespread and abundant receptors participate in the removal and destruction of antibody-coated particles; activate and induce cells to kill antibody-coated targets; are induced by, and, after cross linking, induce the production and release of cytokines and inflammatory mediators; transport immunoglobulins; and are also involved in the regulation of immunity. Fc receptors are cell membrane molecules that specifically bind the Fc portion of an immunoglobulin heavy chain. These receptors have been defined on leukocytes for all Ig classes—IgM, IgD, IgG, IgE, and IgA for which they are named, that is, the IgG receptors are Fc γR, IgE receptors are FcɛR, and so forth. Where more than one class of receptor is defined for an Ig isotype, the receptors are designated thus: FcγRI, FcγRII, or FcɛRI, FcɛRII, and so forth. The exception to these rules are the receptors expressed on non-leukocytes—that is, the receptor for polymeric Ig called the poly Ig receptor, or a unique receptor in the gut of neonatal rats, the FcRn. In addition to their specificity for Ig classes, Fc receptors are also classified based on reactivity with monoclonal antibodies and their affinity for Ig.