IL-4 induces loss of B lymphocyte Fc gamma R II ligand binding capacity

Murine B lymphocytes cultured for 24 h with rIL-4 lost (mean reduction of 88%, range 81 to 96%) the capacity to bind Ag-IgG antibody complexes to B lymphocytes as assessed by flow microfluorometry. This effect was specific in that it was not seen with IL-1, IL-2, or IFN-gamma; IL-4 did not have a similar effect on other B lymphocyte membrane molecules; and the effect was completely prevented by anti-IL-4 (mAb 11B11). More than 60% inhibition of the binding of complexes was seen with as little as 1 U/ml of IL-4 although maximal inhibition was seen with greater than or equal to 30 U/ml. IL-4-induced inhibition of the binding of complexes was time dependent (the effect was first seen after 8 h and was not maximal until 24 h), temperature dependent (it did not occur at 4 degrees C), and reversible (B lymphocytes that had lost the ability to bind complexes due to IL-4 regained this capacity when re-cultured for 24 h in the absence of IL-4). The effect could be partially prevented by IFN-gamma. The inability to bind complexes appeared to be mainly due to an alteration of Fc gamma R (Fc Receptors) II rather than down-regulation of receptor expression because IL-4 induced only a moderate reduction in the binding of two Fc gamma R II specific mAb (20% for 2.4G2 and 32% for K9.361). The IL-4-induced loss of binding of complexes to B lymphocyte Fc gamma R II appears to be a novel form of receptor regulation (function rather than expression), and likely plays a role in the up-regulation of B lymphocytes by IL-4 by preventing Fc gamma R II-mediated inhibition of B lymphocyte responses.     

Interactions between murine B lymphocyte surface membrane molecules. Loaded but not free receptors for complement and the Fc portion of IgG co-cap independently with cross-linked surface Ig

We have investigated the possible physical interactions between CR, receptors for the Fc gamma R and surface Ig (sIg) on the surface membrane of murine B lymphocytes. We used the rat mAb to murine CR, 8C12, and 7G6, as CR ligands, and soluble Ag-antibody complexes as FcR ligands; and F(ab')2 fragments of rabbit antibodies specific for mouse IgM and IgD as sIg ligands. We have found that: 1) sIg, CR, and Fc gamma R are not directly linked, because capping of any one did not affect the expression of the others; 2) the mAb 8C12 and 7G6 failed by themselves to cross-link CR; 3) soluble Ag-antibody complexes crosslinked some, Fc gamma R on a minority of Fc gamma R+ lymphocytes; 4) once loaded with anti-CR mAb, CR co-capped with sIg when sIg was cross-linked; 5) once loaded with Ag-antibody complexes, Fc gamma R also co-capped with sIg when sIg was sIg was cross-linked; 6) loading of Fc gamma R did not affect the co-capping of surface CR with cross-linked sIg and conversely, loading of CR did not affect the co-capping of Fc gamma R with cross-linked sIg; only loaded CR or Fc gamma R co-capped with sIg regardless of the status of the other surface molecule; 7) neither loaded nor free CR co-capped with cross-linked Fc gamma R, and neither loaded nor free Fc gamma R co-capped with cross-linked CR. These results demonstrate that both Fc gamma R and CR independently become associated with sIg when either receptor is loaded and sIg is cross-linked.     

Characterization of murine lymphocyte IgE receptors by flow microfluorometry

A flow microfluorometric technique has been developed to analyze IgE receptors on splenic and mesenteric lymph node mononuclear cells from BALB/c mice. Our data show that 1) the binding of DIBADL cross-linked IgE dimers to IgE receptors is specific in that it is inhibited by monomeric rat and mouse IgE but not by mouse or rabbit IgG or by the monoclonal anti-Fc gamma R antibody 2.4G2, and conversely, the binding of DIBADL cross-linked IgG dimers is inhibited by monomeric IgG or 2.4G2 but not by rat or mouse IgE; 2) the binding of IgE dimers is saturable on cells from uninfected and Nippostrongylus brasiliensis (Nb)-infected mice; 3) IgE dimer binding is detectable on most splenic B lymphocytes from uninfected and Nb-infected mice, but not on T lymphocytes from uninfected mice, and on few, if any, T lymphocytes from Nb-infected mice; 4) Nb infection causes a parallel increase in the percentages of B lymphocytes and cells expressing IgE receptors and Fc gamma R; 5) Nb infection leads to a marked increase in B lymphocyte IgE receptor expression, has little if any effect on IgE receptor affinity, and causes only minor changes in Fc gamma R expression; and 6) in vivo activation of B lymphocytes by a goat antibody to mouse IgD decreases IgE receptor expression considerably, but has a minimal effect on Fc gamma R expression. Thus, there are separate receptors for IgE and IgG on murine B lymphocytes, and the effect of Nb infection or anti-IgD treatment on their expression is different.     

Functional association of CD9 with the Fc gamma receptors in macrophages

CD9, a member of the tetraspan family of proteins, is highly expressed on macrophages. Although a clear function for the molecule has yet to be described, we have found that the anti-CD9 mAb activates mouse macrophages. The rat anti-CD9 mAb, KMC8.8, but not the F(ab')(2), induced tyrosine phosphorylation of proteins including syk and cbl and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and Fc gamma R was required for the signal. Co-cross-linking of CD9-Fc gamma R with KMC8.8 on macrophages from three different FcR-deficient mice, FcR gamma-chain(-/-), Fc gamma RIIB(-/-), and Fc gamma RIII(-/-), revealed that Fc gamma RIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-Fc gamma RIIB/III mAb, 2.4G2, evoked similar phosphorylation patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 treatment led to reduce levels of TNF-alpha production and p42/44 extracellular signal-related kinase phosphorylation relative to 2.4G2 stimulation. Immunofluorescence staining showed that co-cross-linking of CD9-Fc gamma R with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, Fc gamma RIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of Fc gamma R-deficient macrophages revealed that the colocalization of CD9, Fc gamma RIII, Mac-1, and F-actin requires co-cross-linking of CD9-Fc gamma RIII, whereas co-cross-linking of CD9-Fc gamma RIIB induced the colocalization of only CD9 and Fc gamma RIIB. Our results demonstrate that co-cross-linking of CD9 and Fc gamma Rs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages.     

Monoclonal antibodies that bind to distinct epitopes on Fc gamma RI are able to trigger receptor function

The human monocyte and macrophage Fc receptor that binds human IgG with high affinity is a surface glycoprotein with a relative molecular mass of approximately 70 kDa. This receptor (Fc gamma RI) has been partially characterized using mAb 32 which binds outside the Fc binding domain of the receptor, but nonetheless triggers Fc receptor-dependent functions. In this study, we describe the properties of four new antibodies with specificity for Fc gamma RI. Based on additivity and cross-blocking studies, we conclude that two of these antibodies (mAb 22 and 44) define a third epitope which is distinct from the binding sites for both mAb 32 and the Fc portion of human IgG. Each Fc gamma RI-specific hybridoma was selected for stable sublines expressing high levels of mAb on the cell surface, and then tested for the ability of this surface mAb to trigger antibody-dependent cell cytotoxicity. All sublines were killed by human monocytes when used as targets in a 51Cr-release assay, whereas hybridomas specific for myeloid Ag other than Fc gamma RI were not killed. We conclude that Fc receptor function is triggered through binding to each of the three epitopes of Fc gamma RI that we have defined. These mAb will be useful for additional characterization of Fc gamma RI, and may, when incorporated into tumor-directed heteroantibodies, enhance tumor cell killing by human monocytes and macrophages.     

Modulation of the humoral immune response by antibody-mediated antigen targeting to complement receptors and Fc receptors

During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb.     

Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

Alterations of B lymphocyte Fc gamma R II expression and ligand binding capacity induced by various activators.

Murine B lymphocytes cultured with F(ab')2 anti-mouse mu or delta lost (85%) the capacity to bind antigen-IgG antibody complexes as assessed by flow microfluorometry. Anti-mu-induced loss of binding of complexes was concentration, time, and temperature dependent, reversible, and not due to decreased expression of the receptor because binding of monoclonal anti-Fc gamma R II to B lymphocytes cultured with anti-mu was unaffected. Activation of PKC and elevation of [Ca2+]i obtained by culturing B lymphocytes with the combination of PMA and Ca2+ ionophore induced a similar loss of binding of Cx. Since stimulation of B lymphocytes with anti-mu also activates PKC and elevates [Ca2+]i, these changes may be involved in the anti-mu-induced alterations in the binding of complexes to Fc gamma R II. In contrast to the effects of other activators, LPS caused increased expression (threefold) of B lymphocyte Fc gamma R II as measured by the binding of both complexes and monoclonal anti-Fc gamma R II. Thus, different B lymphocyte activators have distinct effects on Fc gamma R II expression or ligand binding capacity and can thereby affect Fc gamma R II-generated regulatory signals.     

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.     

Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo.

The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function.     

Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.     

Human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) belong to Fc gamma receptor-positive CD4-positive T cells

The phenotypic characteristics of human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) were investigated using dual-color surface immunofluorescence and cytofluorometric analysis of stained peripheral blood mononuclear cells (PBMC) from normal individuals. Two to ten percent of PBMC coexpressed CR1 and the CD5, CD2, or CD3 antigen. CR1 was detected on a subset of CD4+ T lymphocytes but not on CD8+ or on Leu-7+ lymphocytes. Costaining for CR1 and for the CD4 subpopulation markers anti-Leu-8, TQ1, OKT17, 2H4, and 4B4 indicated that CR1 on lymphocytes may be coexpressed with any of these phenotypic determinants. All CR1+ lymphocytes expressed Fc gamma receptors (Fc gamma Rs) as assessed by their ability to bind biotinylated dimeric human IgG. The expression of CR1 was increased in mixed lymphocyte reaction with kinetics similar to those of HLA-DR antigen expression. Coexpression of CR1 and Fc gamma R+ may provide a subset of CD4+ lymphocytes with an enhanced ability to bind and respond to C3-bearing complexes of IgG and antigen.     

Different membrane anchors of Fc gamma RIII (CD16) on K/NK-lymphocytes and neutrophils. Protein- vs lipid-anchor

Fc gamma RIII (CD16), the type three receptor for the Fc portion of IgG, is expressed on neutrophils, killer (K)/NK lymphocytes and macrophages. K/NK lymphocyte Fc gamma RIII, which plays a role in antibody-dependent cellular cytotoxicity, is an efficient signal transducing molecule, whereas neutrophil Fc gamma RIII, which plays a role in immune-complex clearance, seems less efficient in signal transduction. Neutrophil Fc gamma RIII has been reported to be a glycan-phosphatidylinositol-anchored membrane protein. Our studies suggest that K/NK lymphocyte Fc gamma RIII is protein-anchored rather than glycan-phosphatidylinositol-anchored. That is, K/NK lymphocyte Fc gamma RIII was resistant to phosphatidylinositol-specific phospholipase C and surface expression of Fc gamma RIII was not affected on K/NK lymphocytes from patients with paroxysmal nocturnal hemoglobinuria, a disorder of hemopoietic stem cells resulting in deficient expression of glycan-phosphatidylinositol-anchored proteins. Different membrane anchoring mechanisms of the Fc gamma RIII may account for different consequences of the ligand binding to two cell types.     

Evidence for cross-regulation of Fc gamma RIIIB (CD16) receptor-mediated signaling by Fc gamma RII (CD32) expressed on polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMN) express the low affinity receptors for the Fc domain of IgG (Fc gamma R), Fc gamma RII (CD32), and the glycosyl phosphatidylinositol-linked isoform of Fc gamma RIII (Fc gamma RIIIB, CD16) on their cell surface. Both of these receptors have been shown to be signal-transducing molecules. However, the mechanisms involved in such signaling are not clearly understood. In this report, we investigated intracellular Ca2+ ([Ca2+]i) signals triggered in PMN by both the receptors using aggregated human IgG (AggIgG) and specific mAb to Fc gamma RII (KuFc79) and Fc gamma RIII (3G8) as ligands. Addition of AggIgG as well as cross-linking of mAb KuFc79 and 3G8 bound to PMN induced [Ca2+]i flux. However, preincubation of PMN with mAb KuFc79 (whole Ig or Fab fragments) in the absence of cross-linking abrogated the [Ca2+]i flux induced by AggIgG and mAb 3G8, indicating that Fc gamma RII receptor occupancy by mAb KuFc79 can block signals mediated by Fc gamma RIIIB. KuFc79-isotype-matched control mAb (MOPC 195) did not abolish the signals generated by AggIgG and mAb 3G8. In addition, mAb KuFc79 did not abrogate [Ca2+]i responses elicited by the receptor for the chemotactic peptide FMLP indicating that modulation of signal transduction by Fc gamma RII-bound KuFc79 is selective for certain receptors. Immunofluorescence analysis of PMN initially treated with mAb KuFc79 followed by AggIgG showed that KuFc79 did not block the binding of AggIgG to PMN. Similarly, competitive binding studies revealed no stearic hindrance between mAb KuFc79 bound to Fc gamma RII and mAb 3G8 bound to Fc gamma RIIIB. Thus, the ability of mAb KuFc79 to modulate signals induced by AggIgG and 3G8 strongly suggests that Fc gamma RII may regulate Fc gamma RIIIB signaling. While previous studies on Fc gamma RII revealed a requirement for cross-linking of the receptor to induce its effector functions, the present study shows that binding of mAb KuFc79 to Fc gamma RII itself, even in a univalent form, results in cross-regulation of Fc gamma RIIIB-triggered signals. Treatment of PMN with protein tyrosine kinase inhibitors, genistein and herbimycin A, abrogated the [Ca2+]i signals elicited by both mAb KuFc79 and 3G8. These results suggest that tyrosine kinase enzyme(s) associated with these receptors may be crucial for positive/negative signals triggered by Fc gamma RII and Fc gamma RIIIB.     

A triggering structure recognized by G7 monoclonal antibody on porcine lymphocytes and granulocytes.

Monoclonal antibody (mAb) G7 has been developed and appears to recognize a triggering structure on porcine natural killer (NK) cells and granulocytes. G7 mAb binds to approximately 13% of lymphocytes, 70% of monocytes, and greater than 95% of granulocytes. G7 mAb does not react with B cells. G7 mAb immunoprecipitates a heterodispersed molecule of approximately 40 kDa. Functionally, whole but not F(ab')2 fragments of G7 mAb enhance NK killing of Fc receptor positive K562, U937, and MOLT-4 targets but not Fc receptor negative CEM, WEHI-164, or YAC-1 targets. Both whole and F(ab')2 fragments of G7 mAb inhibit lymphocyte-mediated antibody-dependent cellular cytotoxicity. Interestingly, G7 mAb induces dramatic levels of granulocyte killing against nucleated K562 targets. These results suggest that G7 mAb recognizes a trigger molecule involved in porcine cellular cytotoxicity.     

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.     

Monoclonal anti-Fc epsilon receptor antibodies with different specificities and studies on the expression of Fc epsilon receptors on human B and T cells

Three monoclonal antibodies, 1-7 (gamma 2b), 3-5 (gamma 1), and 8-30 (mu), specific to Fc epsilon receptors (Fc epsilon R) on human B cells were established. The two monoclonals (1-7 and 8-30) could inhibit the binding of IgE to Fc epsilon R in rosette formation assays, as well as FACS analysis, and were shown to recognize the same epitope of Fc epsilon R. The other monoclonal antibody (3-5) recognized the same molecule but a different epitope, and marginally inhibited the IgE binding. The molecules on RPMI 8866 cells recognized by these monoclonal antibodies had Mr of 46,000 and 25,000 to 30,000 daltons as determined by immunoprecipitation and SDS-PAGE analysis. By employing these monoclonal antibodies, the expression of Fc epsilon R on circulating lymphocytes was studied. Approximately 50% of B cells from normal, nonatopic individuals were found to express Fc epsilon R, and a remarkable increase in the expression of Fc epsilon R was observed in B cells of atopic patients. The expression of Fc epsilon R was not detected in T cells from atopic patients (including hyper IgE syndrome) as well as normal individuals. Incubation of B cells with PHA-conditioned medium plus IgE augmented the expression of Fc epsilon R in the Fc epsilon R+ B cell population but not in Fc epsilon R- population. PHA-conditioned medium plus IgE did not induce Fc epsilon R expression on T cells.     

Biochemical characterization of an Fc gamma receptor purified from human neutrophils

The Fc receptor identified by mAb 3G8 (Fc gamma RIII) was isolated by mAb affinity chromatography from 0.5 to 2 x 10(10) neutrophils yielding 33 to 149 micrograms of protein. Iodination of the purified protein identified a polypeptide of broad electrophoretic mobility from Mr 47 to 70 kDa and occasionally a fainter polypeptide at 100 to 130 kDa, which may be dimerized receptor. Two-dimensional isoelectric focusing gel electrophoresis illustrated multiple diffuse polypeptides ranging from a pI of less than 4.7 to 6.5. Treatment of the purified receptor with neuraminidase shifted the mobility of these polypeptides to a more basic pI, ranging from 6 to 8, illustrating the presence of sialic acid residues on Fc gamma RIII. The glycoprotein nature of Fc gamma RIII was characterized by several criteria. The receptor bound to Con A-Sepharose. Treatment of Fc gamma RIII with endoglycosidase H or F, which cleave high mannose and biantennary complex N-linked oligosaccharides, respectively, failed to alter the electrophoretic mobility of the Fc gamma R. Peptide N:glycosidase F, which cleaves all classes of N-linked oligosaccharides, reduced the Mr of Fc gamma RIII by 60% to reveal two poorly resolved polypeptides centered at Mr 25 kDa and ranging from Mr 16 to 28 kDa. Chemical deglycosylation with trifluoromethanesulfonic acid, which cleaves O- and N-linked oligosaccharides except for the asparagine-linked N-acetylglucosamine, reduced the Mr of Fc gamma RIII to 21 to 36 kDa. These results demonstrate that Fc gamma RIII is an acidic complex sialoglycoprotein and suggest that there may be 8 to 15 N-linked oligosaccharide chains on Fc gamma RIII.