Fcy Receptor III on Human Neutrophils Allelic Variants Have Functionally Distinct Capacities

Introduction As a model system to explore the functional consequences of structural variants of human Fcy receptors (FcyR), we have investigated Fc-yR-mediated phagocytosis in relation to the NA1-NA2 polymorphism of FcyRIII (CD16) on neutrophils (Fc'yRIIIpMN). The neutrophil-specific NA antigen system is a biallelic polymorphism with codominant expression demonstrating a gene dose effect with the anti-NAl MAb CLB-gran 11 in a large donor population. To explore the impact of this allelic variation of FcyRIIIPMN on phagocytosis, we used two FcyRIII-dependent probes, IgG-sensitized erythrocytes (EA) and concanavalin A-treated erythrocytes (E-ConA). Comparison of FcyR-mediated phagocytosis by PMN from NA1 subjects and from NA2 subjects showed lower levels of phagocytosis of both probes by the NA2 individuals. The difference was most pronounced with lightly opsonized EA: at the lowest level of sensitization the phagocytic index was 72% lower for NA2 donors, whereas at the highest level of sensitization it was 21% lower (P < 0.003). Blockade of FcyRII with MAb IV.3 Fab amplified by threefold the difference between NAI and NA2 donors. NA1 and NA2 individuals had identical phagocytic capacities for the non-FctyRIII probes, serum-treated and heattreated zymosan. These individuals did not show differential quantitative cell surface expression of FcyRIIIPMN measured by a panel ofanti-CD16 MAb (3G8, CLB FcR-gran 1, VEP13, BW209/2) and by Scatchard analysis of '251-IgG dimer binding. The difference in Fc'yR-mediated phagocytosis was not explicable on the basis of differential collaboration of Fc7yRIIIPMN alleles with FcyRII, since (a) the difference in phagocytic capacity between NAI and NA2 individuals was readily apparent with the E-ConA probe (which is independent of FcyRII) and (b) the difference in phagocytosis of EA was magnified by Fc-yRII blockade. The demonstration that allelic polymorphisms in FcyR can have significant consequences for physiological functions implies that within the structural complexity of human FcyRs, including both allelic forms and cell type-specific isoforms, there will be differences in quantitative, and perhaps qualitative, function with potential importance for disease processes. (J. Clin. Invest. 1990. 85:1287-1295.) Fcy receptor * neutrophil phagocytosis Address reprint requests to Dr. Salmon, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021. Received for publication 13 July 1989 and in revisedform I November 1989. Receptors for the Fc portion ofIgG are essential participants in many immune system effector functions including phagocytosis ofopsonized and certain nonclassically opsonized particles, release of inflammatory mediators, and antibody-dependent cellular cytotoxicity (1, 2). In humans three structurally distinct families of Fc-y receptors (FcyR),' defined by molecular weights, ligand binding properties, and certain monoclonal antibodies (MAbs), represent distinct gene products and are present on characteristic populations of leukocytes (3, 4). Within these families of FcyR, several structural polymorphisms have been recognized. For example, two different forms of FcyRII, which influence the affinity of binding of various murine immunoglobulin isotypes to the human receptor, have been demonstrated by isoelectric focusing of isolated receptor from normals (5, 6). This polymorphism of FcyRII impairs the ability of the FcyRII with the "nonresponding" phenotype to provide sufficient cross-linking of murine anti-CD3 IgG 1 MAb for in vitro T-cell mitogenesis (7-9). Another FcyR polymorphism, identified on FcyRIII as the neutrophil-specific NA antigen system by serological typing with alloantisera and by certain MAbs (10-12), reflects differences in protein sequences and number of glycosylation sites (13, 14) and has no known functional significance. Systematic differences in FcyR-specific phagocytosis in certain immunogenetically defined disease-free individuals with normal numbers of surface FcyR (15-17) have raised the possibility that structural variations in FcyR may lead to functional differences. At present some differences in FcyR function between cell types have been defined (18-22), but few data about structure-function relationships for polymorphic Fc-yR within a single cell type are available (5). As a model system to explore the relationships between structural polymorphisms and functional properties of FcyR, we have investigated the NA polymorphism of FcyRIII on neutrophils (FcyRIIIPMN)Using quantitative flow cytometry in a large donor population, we have demonstrated that the NA polymorphism of FcyRIIIPMN shows a clear allelic gene dose effect and is a suitable model for study of the functional implications of allelic structural forms. We have examined the functional conse1. Abbreviations used in this paper: ANOVA, analysis of variance; Bm. , maximum binding; E, erythrocytes; EA, IgG-sensitized bovine erythrocytes; E-ConA, concanavalin A-treated rabbit erythrocytes; Fc-yR, receptors for Fc portion of IgG; Fc-yRIIIPMN, the 50-78-kD receptor on human neutrophils for the Fc portion of IgG; HTZ, heattreated zymosan; Ka, association constant; mIgG, murine IgG; %P, percentage of neutrophils with internalized particles; PE, phycoerythrin; PI, phagocytic index; STZ, serum-treated zymosan. Functional Consequences ofAlleles in Fcty Receptors 1287 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/90/04/1287/09 $2.00 Volume 85, April 1990, 1287-1295 quences of this NA 1-NA2 polymorphism for quantitative phagocytosis using IgG-sensitized erythrocytes (EA) and concanavalin A-treated erythrocytes (E-ConA). These Fc~yRIIIPMN-dependent probes are internalized by classical ligand-mediated and nonclassical lectin-carbohydrate-mediated mechanisms, respectively (2). Our results show that neutrophils from NA2 donors have a lower capacity to mediate phagocytosis compared with that ofNA 1 donors, whether the FcTR is ligated by its classical IgG ligand-binding site or by lectin-carbohydrate interactions. Since these different functional capacities cannot be explained on the basis of altered numerical expression of FcyRIIIPMN, the allelic polymorphism per se appears to have direct impact on quantitative receptor function.