Demonstration of the existence of two distinct Fc receptors for IgG isotypes on guinea-pig macrophages by the use of monoclonal antibodies

As reported in a previous paper by the authors (J. Biochem. 99, 227-235, 1986), the Fab' of a monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea-pig peritoneal macrophages with a myeloma cells line, completely inhibits the binding of ovalbumin (OA)-complexed IgG1 antibody to macrophages, but only partially the binding of OA-complexed IgG2 antibody. Based on these results, it was proposed that the cells have at least two types of Fc receptor (FcR) for homologous IgG isotypes: FcR2 for IgG2 and FcR1.2 for both IgG2 and IgG1, and also that VIA2 IgG1 is anti-FcR1.2 antibody. Thereafter, complete inhibition of the binding of OA-complexed IgG2 antibody to macrophages occurred when the Fab' of another monoclonal antibody, VIIA1 IgG1 was added to the Fab' of VIA2 IgG1, whereas the former did not affect the binding of OA-complexed IgG1 antibody. This effect of the Fab' of VIIA1 IgG1 indicates that VIIA1 IgG1 is a monoclonal antibody capable of selectively blocking the binding of OA-complexed IgG2 antibody to FcR2. When the antigen of VIIA1 IgG1 was isolated by affinity chromatography on the F(ab')2 of the antibody coupled to Sepharose, it gave a single band with a mol. wt of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It moved slightly faster than the FcR1.2 with a mol. wt of 55,000, which was isolated by the use of VIA2 IgG1, and corresponded to the fast moving portion of the broad band of FcRs isolated with OA-complexed IgG2 antibody. These results strongly suggest that VIIA1 IgG1 is a monoclonal antibody to FcR2.     

A monoclonal antibody inhibiting human placental Fc gamma-receptor activity

Fc gamma receptor (FcR) from human placenta was solubilized using EDTA and 2-mercaptoethanol and purified by affinity chromatography on human IgG-coated Sepharose 4B. BALB/C mice were immunized with FcR and monoclonal antibodies were obtained by growing hybridoma cells following fusion of spleen cells with P3 X 63Ag8 myeloma cells. Using an immunofluorescence technique, the IgG1 monoclonal antibody secreted by clone B1D6 stained the FcR-positive areas in sections of placental tissue. The endothelium of the foetal stem vessels stained more strongly than did the trophoblasts. The antibody also inhibited the haemadsorption to placental tissue of erythrocytes (E) sensitized with IgG antibodies (A), (EA), and inhibited the agglutination of EA by FcR. The data indicate that the monoclonal antibody reacts with the placental FcR at the binding site for IgG, or with an epitope close to the binding site. Apparently, the FcR in different anatomical areas in the placenta have a common antigenic determinant.     

A radioimmunoassay with monoclonal antibodies for the detection of antigenic cell-free Fc receptor

Making use of 125I-labelled monoclonal rat antibodies against mouse Fc receptor we have developed a solid-phase radioimmunoassay for cell-free antigenic mouse Fc receptor (FcR). An 80% acetone solution was used for fixation of relatively large amounts of soluble proteins on PVC microtiter plates. As a result of this treatment FcR practically lost its activity to bind the Fc portion of an IgG molecule but retained its antigenicity, the binding of specific anti-FcR (aFcR) antibody being increased following acetone fixation. Concentrations of cell-free FcR in NP-40 extracts of FcR-expressing cells were calculated from the linear part of a standard curve and expressed in units of antigenic activity, 1 unit being the amount of antigenic FcR capable of binding 1 microgram of 125I-aFcR. The method may be used for detecting cell-free FcR as a minor constituent in a mixture of proteins.     

Monoclonal opsonic mouse antibodies specific for streptococcal IgG Fc-receptor

Spleen cells from mice immunised with group-A type-M15 streptococci and boosted with purified IgG Fc-receptor (FcR) from this type were fused with Sp 2/0 mouse myeloma cells. The resulting hybridomas were screened by ELISA for antibody production. Two IgM-secreting cell lines were selected. The monoclonal antibodies and ascites fluids inhibited the binding of 125I-labelled human IgG and IgG Fc-fragments to group-A type-M15 streptococci. The monoclonal antibodies also displaced purified FcR towards the anode in electrophoresis. They opsonised group-A type M-15 streptococci for phagocytosis by human granulocytes in the presence of fresh human serum. It was concluded that FcR is important for group-A streptococcal virulence.     

Identification of an Fc receptor for IgG1 and IgG2 on guinea-pig polymorphonuclear leukocytes

Ovalbumin (OA)-complexed guinea-pig IgG1 and IgG2 antibodies were found to bind to homologous polymorphonuclear leukocytes (PMNs). As these bindings are assumed to be mediated by certain Fc receptors (FcRs) for IgG1 and IgG2, the variety and properties of the FcRs on the cells were investigated by the use of two monoclonal antibodies to guinea-pig macrophage FcRs which were prepared by Shimamura T. et al., 1987 (Molec. Immun. 24, 67-74): VI A2 IgG1 to the FcR for IgG1 and IgG2 (FcR1,2) and VII A1 IgG1 to the FcR for IgG2 (FcR2). PMNs were shown to bind the Fab' of VI A2 IgG1 (VI A2 Fab') by flow cytofluorometry, suggesting that the cells possess a certain FcR which cross-reacts antigenically with macrophage FcR1,2. In fact, VI A2 Fab' inhibited completely the binding of OA-complexed IgG1 antibody to the cells. When the FcR was isolated by affinity chromatography on the F(ab')2 of VI A2 IgG1 coupled to Sepharose, it gave a 55,000 mol. wt band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, as in the case of macrophage FcR1,2. The number of the FcR molecules per PMN cell was estimated to be 2 X 10(4) by measuring the binding of 125I-VI A2 Fab'. The binding of OA-complexed IgG2 antibody to PMNs was also inhibited with VI A2 Fab', but partially. This finding indicates that the FcR bound by VI A2 Fab' may be an FcR1,2 which is able to bind both OA-complexed IgG1 and IgG2 antibodies, and also that PMNs possess another FcR, namely FcR2 which binds IgG2 antibody alone. The Fab' of VII A1 IgG1 (VII A1 Fab'), on the other hand, did not exhibit any inhibitory activity on the bindings of OA-complexed IgG1 and IgG2 antibodies to PMNs. Since no evidence indicating the binding of VII A1 Fab' to PMN cells was obtained by flow cytofluorometry, the FcR2 of PMNs may be antigenically different from its macrophage counterpart. In conclusion, these results indicate that two distinct types of FcR for IgG isotypes exist on guinea-pig PMN cells: FcR1,2 similar to macrophage FcR1,2, and FcR2 distinct from macrophage FcR2.     

Fc receptors on mouse neutrophils and eosinophils: antigenic characteristics, isotype specificity and relative cell membrane density measured by flow cytometry.

The antigenic characteristics, isotype specificity and density of Fc receptors (FcR) on mouse neutrophils and eosinophils were studied with the aid of the rat monoclonal antibody 2.4 G2 to the mouse macrophage FcR (Unkeless, 1979). This MAb was tested for its reactivity with mouse neutrophil and eosinophil FcR, and for its ability to block the binding of sheep erythrocytes (E) coated with mouse antibodies of different isotypes to granulocytes. The use of E conjugated with fluorescein isothiocyanate (FITC) allowed an objective read-out by flow cytometry. The MAb 2.4.G2 reacted with both neutrophil and eosinophil FcR, blocking the binding of E coated with mouse IgG1, IgG2a and IgG2b in a dose-dependent manner. Blocking was specific, since it did not occur with any of several control MAb of the same rat isotype (IgG2b) as 2.4.G2. Furthermore, the binding to E through the granulocyte receptor for complement (C) was unaffected. IgG3 was unable to promote binding of E to either neutrophils or eosinophils, although it induced high levels of binding to macrophages. These results show that: (i) neutrophil, eosinophil and macrophage FcR have antigenic similarities; (ii) neutrophils and eosinophils, in contrast to macrophages, either have a common FcR for IgG1, IgG2a and IgG2b, or have different FcR for these isotypes which share the antigenic determinant recognized by 2.4.G2; (iii) in contrast to macrophages, neutrophils and eosinophils lack the FcR for IgG3. The MAb 2.4.G2 was used in an indirect immunofluorescence assay monitored by flow cytometry to measure the relative FcR density on neutrophils and eosinophils. This assay showed that neutrophils possess about 65% more FcR than eosinophils on a cell-for-cell basis, providing an explanation for the higher binding of neutrophils to IgG-coated particles at suboptimal antibody concentrations.     

Purification of a functional 40 kD human placental Fc gamma-receptor using a monoclonal antibody

F(ab')2-fragments of a mouse monoclonal antibody (B1D6) reacting with placental receptors for the Fc part of IgG (FcR) were used as affinity reagents for the purification of an antigen from placental extract (PE). The antigen agglutinated ovine erythrocytes (E) sensitized with rabbit antibodies (A) (EA), but not E or E sensitized with F(ab')2-fragments. It reduced the EA rosette-formation with mononuclear cells and the binding of soluble immune complexes to placental tissue. The antigen bound to aggregated IgG and Fc-fragments of IgG, but not to native IgG or F(ab')2-fragments of IgG. The data indicate that the purified antigen possesses FcR activity with low affinity for IgG. SDS-PAGE and Western blot showed one distinct band of approximately 40 kD. The electrophoretic mobility did not change after reduction and the band reacted with concanavalin A indicating that the FcR are single-chained glycoproteins.     

A Comparative Analysis of Cell Surface Markers on Murine NK Cells and CTL Target-Effector Conjugates

The rosetting of sheep erythrocytes (SRBC) coated with non-haemagglutinating monoclonal antibodies rather than conventional haemagglutinating antisera revealed readily detectable FcR on most splenic natural killer (NK) cells since 76% of splenic lymphocytes forming conjugates with YAC also resetted with SRBC coated with high concentrations of monoclonal anti-SRBC antibody of the IgG2b subclass and since Ficoll depletion or enrichment of splenic lymphocytes rosetting with IgG2b-coated SRBC resulted in a corresponding 4-fold decrease or increase in conjugate-forming cells and a 10-fold decrease or increase in NK cytolytic acttvity. NK cells bound much less readily to monoclonal IgG2a and not at all to monoclonal IgGI or IgM, but the degree of binding was directly proportional to the amount of antibody on the erythrocytes and was not isotype-restricted. In addition, immunofluorescent studies revealed that YAC-1-conjugated lymphocytes were Lyt-1^-, Lyt-2^-, partially Thy-1⁺ (60%), asiato-GMI ⁺ (80%), Qa-4⁺ (77%), Qa-5⁺ (79%), and Ly-5⁺ (94%). In comparison, a proportion (39%) of alloimmune peritoneal exudate cells which conjugated with P815–2 also siained by immunofluorescence with anti-asialo GM1 antisera. Most (>90%) P815- conjugated cells were Thy-1⁺, Lyt-2⁺. and a subpopulation of Lyt-l⁺2⁺ conjugates was observed (25 %). Qa-5 and Ly-5 were also expressed on most (two-thirds) cytolytic T lymphocytes (CTL) conjugates, whereas Qa-4 and FcR for IgG2b were not detected. The best phenotypic distinctions between NK cells and CTL were therefore based on the presence or absence of Lyt-2, Qa-4, and FcR for IgG2b on most effector cells. Anti-asialo-GMl or monoclonal anti-Qa-4 and complement treatment greatly diminished both the frequency of NK conjugates and the percentage of conjugates with detectable IgG2b FcR or asialo-GM1. These results confirm that NK cells co-express asialo-GMI and Fc receptors, at the single-celt level, and provide a simple method for greatly enriching NK populations at least 10-fold.     

Natural Killer and T-Cell Potentiation by Monoclonal IgG against Natural Killer Cell FcR(IgG) or the T3 Complex

Treatment of human natural killer (NK) cells with monoclonal antibodies of the IgG isotype against NK cell-FcR(IgG) increased lysis of most haematopoietic target cell lines with high or intermediate background NK susceptibility. Treatment of normal non-adherent lymphocytes with an IgG anti-T3 monoclonal antibody also increased lysis against the same target cells. Potentiating anti-FcR antibodies rapidly modulated FcR activity and the capacity of the cells to act as antibody-dependent killers, although such antibodies were demonstrable for a long time at the cell surface. Anti-FcR treatment did not influence concanavalin A (Con A)-dependent killing, in contrast to anti-T3 treatment, which suppressed lectin-dependent lysis but did not influence antibody-dependent killing. The data is compatible with a 'pro-receptor' theory for FcR in NK killing, stating that such receptors may function in the same way as the T3 complex interacts with specific T cell receptors.     

Comparison of receptors required for entry of Leishmania major amastigotes into macrophages.

We investigated the mechanisms of entry of amastigotes of Leishmania major from two different sources into macrophages by comparing their use of the Fc receptor (FcR), complement receptor type 3 (CR3), and mannose-fucose receptor (MFR). Amastigotes were obtained from BALB/c mice and SCID mice. FcR involvement was examined by opsonizing L. major with parasite-specific immunoglobulin G (IgG). Antiparasite IgG did not alter the uptake of amastigotes from BALB/c mice since these amastigotes had antibody bound to their surface: IgG1 was the most predominant antibody, followed by IgG2b, IgM, and IgG2a. However, opsonization with antiparasite IgG enhanced the entry of amastigotes that lacked antibody on their surface, namely, amastigotes obtained from SCID mice or from macrophages infected in vitro. These results indicate that the FcR is important for amastigote entry into macrophages. Down-modulation of FcRs onto immune complexes, however, did not reduce the entry of amastigotes containing surface-bound IgG into macrophages. Monoclonal antibodies against the CR3 inhibited the entry of amastigotes from either BALB/c or SCID mice into J774A.1 macrophage-like cells. Simultaneous blocking of FcR and CR3 further increased the inhibition of phagocytosis. Treatment of macrophages with soluble mannan or down-modulating the MFR onto mannan-coated coverslips had no effect on the entry of amastigotes from BALB/c or SCID mice. Thus, the MFR does not appear to be used by amastigotes of L. major. We show that ingestion of amastigotes appears to occur primarily through the FcR and CR3; however, additional receptors may also participate in the uptake of amastigotes.     

The binding of IgG1 containing immune complexes to the FcR of allogenically activated T cells induces changes in the membrane potential and the cell surface charge

The effect on membrane potential and cell surface charge of binding immune complexes containing IgG1 and IgG2a monoclonal antibodies to Fc receptors was studied in resting and allogenically activated murine T cells. IgG1 complexed by antigen or heat aggregation induced electrophysiological changes on activated T cells. A biphasic alteration of membrane potential was detected by measurement of the intra- and extracellular distribution of the fluorescent dye, DiOC6. A short-lived hyperpolarization, detectable for 4-6 min after adding the respective ligand, was followed by a longer lasting depolarization. The cell surface charge, measured by cell electrophoresis, was also changed. This alteration was detected 2-4 hr after addition of immune complexes and disappeared by the 8th hr of incubation. Monoclonal antibody 2.4.G2, reactive with mouse FcR, induced a similar membrane potential response on activated T cells, but did not affect the cell surface charge. Monomeric IgGs and complexes of IgG2a did not modify these parameters. FcR ligands had no effect on the studied characteristics of resting T cells.     

Characterization of Two Fc Receptors for Mouse Immunoglobulins on Human Monocytes and Cell Lines

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgG1 anti-CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgG1 present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by a recently developed EA rosette assay, using the erythrocyte-associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIgG1 is based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgG1 and does not bind mIgG2a or mIgG2b nor, surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG1 anti-CD3-induced T cell proliferation. mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR on human cell lines correlates well with their ability to support mIgG2a anti-CD3-induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specific for mIgG2a (which also binds human IgG), and a second specific for mIgG1.     

IgG3-mediated enhancement of the antibody response is normal in Fc gammaRI-deficient mice

Antibodies, administered together with their specific antigen, can feedback-regulate antibody responses to this antigen. IgG1, IgG2a and IgG2b enhance antibody responses to soluble protein antigens. This effect is primarily mediated by FcRs as enhancement is impaired in FcR gamma-/- mice, reported to lack Fc gammaRI and Fc gammaRIII because of deletion of the common FcR gamma chain. Also IgG3 can enhance antibody responses. However, this effect is unperturbed in FcR gamma-/- mice but severely impaired in complement-depleted animals and in animals lacking complement receptor 1 and 2. Although this argues against involvement of Fc gammaRs, FcR gamma-/- mice may express one-fifth of the normal levels of Fc gammaRI and, in addition, Fc gammaRI has been suggested to bind IgG3. We re-investigated the dependence of IgG3-mediated enhancement on Fc gammaRs using a mouse strain selectively lacking Fc gammaRI and found that IgG3-mediated enhancement is completely normal. Unlike IgE and IgG2a, which are both thought to enhance T-cell proliferation via FcR-mediated antigen presentation, IgG3 was a poor enhancer of T-cell proliferation both in vivo and in vitro. These findings argue against a significant involvement of Fc gammaRs in IgG3-mediated enhancement of antibody responses and support our previous conclusion that complement plays a major role.     

Separation of natural and antibody-dependent cytotoxicity by differential reactivity of human mononuclear cell Fc receptors with IgG.

Nylon wood non-adherent, human peripheral blood mononuclear cells which are reactive with the OKM1 monoclonal antibody could be separated into two subpopulations based on their Fc receptor reactivity with human monomeric IgG (FcR-IgG) using flow cytometry. The majority of natural killer cells was found primarily in the OKM1+ subset with low FcR reactivity. In contrast, effector cells for antibody-dependent cytotoxicity (K cells) were found in subsets with both high and low FcR-IgG reactivity.     

Standardization of antigen E in ragweed pollen extracts using a monoclonal antibody-based enzyme immunoassay

A hybridoma-derived monoclonal IgG antibody specific for ragweed AgE was used to develop a competitive binding enzyme immunoassay suitable for quantitation of antigen E levels in ragweed pollen extracts. The assay was capable of detecting as little as 30 ng/ml AgE in crude pollen extracts. The monoclonal antibody was shown to react with AgE present in commercial pooled pollen extracts from a number of ragweed species as well as a laboratory extract from a single species. In contrast to previous conventional xenoantisera, it could distinguish true ragweed (Ambrosia sp) from false ragweed (Franseria sp). The use of monoclonal antibodies in assay systems such as this offers a reproducible and widely applicable method for allergen standardization.     

IgG immune complex-binding in macrophages from arthritis-susceptible and arthritis-resistant mice following collagen type II immunization

Occupancy of Fc gamma receptors (FcgammaR) by immune complexes (IC) induces secretion of various inflammatory mediators and cytokines. Therefore, knowledge of the FcR function is fundamental for understanding inflammatory processes. Here, we report an alteration in the FcR function in collagen-induced arthritis (CIA). The FcgammaR-binding activity of peritoneal macrophages from arthritis-susceptible DBA/1 mice following collagen type II (CII)/CFA immunization was assessed by Fc rosetting of SRBC opsonized with different IgG subclasses. A progressive reduction of IgG1 IC-binding was observed after immunization, and by the time of arthritis onset, the IgG1 IC-binding was abolished. Binding of IgG2a or IgG2b IC, however, was not affected. The blocked IgG1 IC-binding was reversed by a prior mild acid wash of the CIA macrophages, indicating receptor occupancy as the cause of the blocked binding. The impaired IgG1 IC-binding was associated with arthritis development, as macrophages from CII/CFA-immunized, arthritis-resistant SWR mice or DBA/1 mice, immunized with CFA alone, did not show this effect. Normal DBA/1 macrophages, blocked with a monoclonal antibody to FcgammaRIIB/FcgammaRIII, and macrophages from FcgammaRIII-deficient mice did not bind IgG1 IC, indicating FcgammaRIII as responsible for IgG1 IC-binding. Our data suggest that an increased degree of saturation of FcgammaRIII precedes the development of CIA, which is reflected by a reduced IgG1 IC-binding in macrophages of CII/CFA-immunized DBA/1 mice.     

Immobilized IgG immune complex induces secretion of tumor necrosis factor-alpha by porcine alveolar macrophages.

Tumor necrosis factor-alpha (TNF-alpha) is an important inflammatory mediator produced by activated monocytes and macrophages. We have previously shown that porcine alveolar macrophages (PAM) mediate bystander cytotoxicity through hydrogen peroxide production following activation with immobilized IgG immune complex (IIC) (J. Immunol. 1983; 131:1438-1442). In this report, we have investigated whether IIC induces TNF-alpha secretion by PAM. Isolated PAM from Minnesota miniature swine were cultured for 18 h with and without recombinant human interferon-gamma (rhIFN-gamma). Cultured PAM were then incubated with IIC or IgG immune complex in suspension (SIC). The supernatants generated were assessed for cytotoxic activity using a TNF-alpha-sensitive WEHI-164 cell line. Anti-recombinant human TNF-alpha (rhTNF-alpha) monoclonal antibody neutralized the observed cytotoxicity of IIC-activated PAM supernatant completely, indicating that this cytotoxicity is mediated by TNF-alpha. IIC induced TNF-alpha secretion by PAM after 3 h of incubation, reaching a plateau from 6 to 12 h and decreasing thereafter. TNF-alpha release was enhanced by pretreatment of PAM with rhIFN-gamma. SIC did not induce significant levels of TNF-alpha secretion by PAM; however, SIC with cytochalasin B-pretreated PAM induced equivalent levels of TNF-alpha secretion as IIC-activated PAM. We conclude that IIC or SIC with cytochalasin B pretreatment, both of which prevent internalization of IgG immune complex-bound Fc receptor (FcR), provide a signal for PAM to generate TNF-alpha through FcR modulation. This suggests that in vivo, deposited (immobilized) IgG immune complexes-bound FcR may be a stimulus for activation of PAM to generate TNF-alpha rather than circulating (mobilized) immune complexes, which may contribute to the pathogenesis of diffuse interstitial fibrosis of the lung, especially in idiopathic pulmonary fibrosis.     

Characterization of natural killer (NK) cells and killer (K) cells in human blood: discrimination between NK and K cell activities.

Cell suspensions enriched and depleted for rosette-forming cells with sheep red blood cells (E-RFC) and depleted for RFC with antibody complexes were prepared. The isolated fractions were characterized by cell surface marker analysis and tested for their natural killer (NK) and killer (K) cell activity against K-562 cells and IgG-coated P-815 cells, growing in suspension, and against a number of monolayer tumor cell lines. It was found that the NK cells most likely belong to the T cell lymphocyte subpopulation. Furthermore, this study indicates that several subpopulations exist, e.g. NK cells that have no IgG Fc receptor (FcR) on their surface and NK cells that bear IgG FcR, indicating that for a proportion of NK cells the IgG FcR is not involved in the NK lytic process and hence antibody-independent. Moreover, monocytes and B lymphocytes appear not to be directly involved in the NK cell lytic process. Furthermore, cell separation procedures were used to obtain cell suspensions either bearing IgG FcR or lacking IgG FcR. Cells bearing IgG FcR were isolated in such a way that they lost their IgG FcR by shedding, as a result of the separation procedure. Again, all fractions were simultaneously characterized by cell surface marker analysis and tested for their NK and K cell lytic activity. The effect of immune complexes on the NK and K cell lytic activities was investigated. The data indicate that the IgG FcR is not involved in the NK lytic mechanism, although this receptor may be present on the NK cell. Moreover, prolonged culturing of lymphocytes increases and/or induces NK cell lytic activity.     

The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes

Herpes simplex virus type 1 (HSV-1) expresses a complex of two virally encoded glycoproteins, gE and gl, which is capable of binding nonimmune human IgG. The gE-gl complex has thus become known as an Fc receptor (FcR), which reportedly binds human IgG subclasses in the order IgG4 > IgG1 > or = IgG2 and does not bind IgG3 from many individuals. There is, however, allelic variation in the genes encoding the human IgG1 heavy chain constant region and this gives rise to allotypes of IgG1. Using recombinant monoclonal IgG molecules of known isotype and mutants thereof we have unexpectedly discovered that the HSV-1 FcR discriminates between IgG1 allotypes. This is evidence of functional differences between IgG1 allotypes that may account for their distribution in populations. Furthermore, these findings suggest HSV-1 FcR binding sites on the IgG molecule some distance from the proposed binding site in the CH2-CH3 domain interface.