Monoclonal antibody to human IgG Fc receptors. Cross-linking of receptors induces lysosomal enzyme release and superoxide generation by neutrophils

The monoclonal antibody KuFc79 binds to a determinant on the Fc receptors (Fc gamma R) of human leukocytes. We examined the biologic effects of the interaction of this antibody with Fc gamma R on human neutrophils (PMNL). The univalent Fab fragment of KuFc79 inhibits the formation of rosettes with IgG-sensitized sheep erythrocytes by as much as 91.7%. In other experiments in which PMNL were washed after exposure to Fab of KuFc79, phagocytosis of IgG-sensitized sheep erythrocytes was inhibited by 36%. Fab fragments of other mouse IgG2b monoclonal proteins did not have these effects. When PMNL are exposed to coverslips coated with univalent Fab fragments of this antibody, the Fc gamma R are removed from the surface of the PMNL. Under these conditions, rosetting could be inhibited by 85.4%. We examined cross-linking of receptor bound monoclonal antibody or its Fab fragment by either Protein A or F(ab')2 of an anti-mouse Ig. As much as 31.7% of beta-glucuronidase, a marker for lysosomal enzymes, is specifically released by cross-linking the Fc gamma R on PMNL. The generation of O2- is also induced by specifically cross-linking Fc gamma R with Fab and anti-Fab. The data constitute the first formal demonstration that cross-linking of Fc gamma R on PMNL leads to enzyme release and superoxide generation.     

Selective modulation of two human monocyte Fc receptors for IgG by immobilized immune complexes

Two types of IgG FcR, FcRI and FcRII, are constitutively expressed by human monocytes. FcRI (identified by mAb 32.2) binds human (h) IgG, FcRII (identified by mAb IV.3) has a low affinity for hIgG but interacts strongly with murine (m) IgG1. These receptors can be assayed by using indicator E sensitized by hIgG (EA-hIgG) or mIgG1 (EA-mIgG1), respectively. We further characterized these two FcR by modulation studies by using substrate-immobilized immune complexes containing rabbit IgG, goat IgG, or one of the mouse Ig classes or subclasses. After incubating monocytes in microtiter wells containing such immune complexes, binding of the two types of indicator red cells on the apical surface of the monocytes was quantitated using a photometric assay employing the pseudoperoxidase activity of E. No effect on the binding of sensitized E was observed after incubation of monocytes with immune complexes containing mouse IgE, IgA, or IgM, or F(ab')2 fragments of rabbit IgG. High concentrations of immune complexes containing IgG of mouse, rabbit, or goat, however, were able to induce a decrease in binding of both types of sensitized E, suggestive of modulation of both FcRI and FcRII. At lower concentrations of immune complexes, more selective patterns of modulation emerged. Under these conditions, immune complexes containing mIgG1 or mIgG2b, or, surprisingly, goat IgG induced a selective decrease in the binding of EA-mIgG1 (FcRII modulation), while immune complexes containing mIgG2a or rabbit IgG mainly affected the binding of EA-hIgG (FcRI modulation). By using anti-FcR mAb IV.3, it was confirmed that FcRII was modulated from the apical surface of monocytes after incubation on immune complex coated substrates. Selectivity of FcR-modulation was demonstrated by showing that under these conditions binding of anti-C receptor mAb, and several other anti-monocyte mAb did not decrease.     

Analysis of the monocyte Fc receptors and antibody-mediated cellular interactions required for the induction of T cell proliferation by anti-T3 antibodies

The induction of human T cell proliferation by antibodies that cross-link T3 antigens is dependent on functional interactions of anti-T3 antibodies with monocyte Fc receptors. In this report, we used a panel of anti-T3 antibodies of differing heavy chain isotype and a variety of other monoclonal antibodies to analyze several features of the antibody-mediated interactions between T cells and monocytes that are required for mitogenesis. Whereas three IgG2a anti-T3 antibodies were mitogenic for cells from all individuals, IgM and IgG2b anti-T3 antibodies did not induce T cell proliferation in any donor and could block the proliferative responses induced by other mitogenic anti-T3 antibodies. Dose-response analyses with four IgG1 anti-T3 antibodies demonstrated donor heterogeneity as reported by other investigators. However, in contrast to these previous reports, high concentrations of IgG1 anti-T3 antibodies were found to be mitogenic for all donors, indicating that this heterogeneity is based on relative rather than absolute defects in low responder monocytes. Cell mixing experiments in which monocytes from two different low responder donors were co-cultured with T cells and IgG1 anti-T3 antibodies did not identify any complementary defects, suggesting that the low responder phenotype results from a relatively restricted polymorphism. To assess the nature of the signals required for inducing T cell proliferation, nonmitogenic anti-T3 antibodies were co-cultured with other pan-T cell antibodies having the IgG2a isotype. The combination of signals from T3 antigen cross-linkage and those independently generated by other IgG2a antibodies bound to monocyte Fc receptors did not induce T cell proliferation. Hence, it appears that the T3 antigen or closely associated structures must be clustered at the monocyte membrane for mitogenesis. Finally, in competitive inhibition experiments, the isotype specificity of monocyte Fc receptors involved in the induction of T cell proliferation was examined. Two distinct Fc receptor sites, one that binds murine IgG2a and IgG3 antibodies and a second that binds murine IgG1 antibodies, were identified. Murine IgM or IgG2b did not appear to bind either of these receptor sites. Taken together, these data indicate that human monocytes have two distinct Fc receptor sites, which must specifically and directly interact with T cell-bound anti-T3 antibodies for mitogenesis.     

Protein tyrosine phosphorylation induced via the IgG receptors Fc gamma Ri and Fc gamma RII in the human monocytic cell line THP-1.

We have investigated the role of protein tyrosine phosphorylation in transmembrane signaling via the IgG receptors Fc gamma RI and Fc gamma RII in the human monocytic cell line THP-1. Fc gamma RI and Fc gamma RII were selectively engaged using the anti-Fc gamma RI mAb 197 (IgG2a) and the anti-Fc gamma RII mAb IV.3 (IgG2b). Addition to cells of mAb 197, but not addition of IgG2a mAb of irrelevant specificity, resulted in the rapid induction of cytoplasmic protein tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting. A similar pattern of tyrosine phosphorylation was induced by mAb IV.3, but not by control IgG2b mAb. The induction of tyrosine phosphorylation by anti-Fc gamma R mAb was not dependent on antibody Fc region-FcR interactions, because tyrosine phosphorylation was also induced by cross-linked anti-Fc gamma RI F(ab')2 fragments and by cross-linked anti-Fc gamma RII Fab fragments. To investigate the relationship of Fc gamma R-induced tyrosine phosphorylation and activation of phospholipase C, which is known to follow Fc gamma R engagement, we assessed the effect of the tyrosine kinase inhibitor herbimycin A on Fc gamma R-induced Ca2+ flux. Herbimycin A strongly inhibited cellular Ca2+ flux induced by mAb 197, but did not inhibit Ca2+ flux induced by aluminum fluoride, suggesting that tyrosine phosphorylation may be important in regulating Fc gamma R-mediated activation of phospholipase C. Consistent with this, mAb 197 induced rapid phosphorylation of the gamma-1 isoform of phospholipase C. Finally, herbimycin A strongly inhibited the induction of TNF-alpha mRNA accumulation by Fc gamma R cross-linking. These results suggest that protein tyrosine phosphorylation may play an important role in the activation of phospholipase C and in the induction of monokine gene expression that follows engagement of Fc gamma R in human monocytes.     

Two distinct classes of IgG Fc receptors on a human monocyte line (U937) defined by differences in binding of murine IgG subclasses at low ionic strength

We have defined two distinct classes of IgG Fc receptors (FcR) on cells of a human monocytic line (U937) by analyzing the direct binding of murine IgG subclasses in medium of low ionic strength. Four lines of evidence support this contention. The binding of aggregated murine IgG2b (AggmIgG2b) to U937 and Daudi cells was enhanced at low ionic strength, whereas monomeric murine IgG2a (mIgG2a) did not bind to Daudi cells and its high affinity binding to U937 cells was unaffected by changes in ionic strength. Double reciprocal inhibition experiments with U937 cells indicated that the binding of both ligands was inhibited 30 to 135 times more efficiently by the homologous ligand than by the heterologous one. That is, the binding of 125I-AggmIgG2b was inhibited 50% by 3.5 micrograms/ml of AggmIgG2b and 100 micrograms/ml of mIgG2a. Similarly, the binding of 125I-mIgG2a was inhibited 50% by 2.5 micrograms/ml of mIgG2a and only 44% by 243 micrograms/ml of AggmIgG2b. A monoclonal antibody of the IgG2b subclass raised against an IgG FcR on K562 cells inhibited binding to U937 cells of AggmIgG2b but not of mIgG2a. Trypsinization of U937 cells abrogated by 32% the binding of mIgG2a but did not affect the binding of AggmIgG2b. Human IgG inhibited binding of both AggmIgG2b and mIgG2a to U937 cells. We propose that the newly recognized FcR that binds AggmIgG2b is the human homologue of the murine macrophage IgG2b/1 FcR (FcRII), and that the previously described 72,000 dalton high-affinity FcR on U937 cells that binds mIgG2a is the human equivalent of the murine macrophage IgG2a FcR (FcRI).     

Dependence of cell survival on DNA repair in human mononuclear phagocytes.

Mononuclear phagocytes play a central role in the pathogenesis of chronic inflammatory diseases. It is therefore important to define chemotherapeutically exploitable metabolic pathways that distinguish monocytes from other cell types. Blood monocytes do not synthesize deoxynucleotides de novo, and their transformation to macrophages occurs without cell division. Whether or not monocytes can repair DNA damage, and whether or not DNA repair is necessary for their survival, is unknown. The present experiments demonstrate that normal human monocytes, unlike neutrophils, rapidly repair DNA strand breaks induced by gamma-irradiation. Monocyte extracts contain functional immunoreactive DNA polymerase-alpha. DNA repair synthesis in normal monocytes is blocked by aphidicolin, an inhibitor of DNA polymerase-alpha with respect to dCTP. Aphidicolin is also directly toxic to normal monocytes, but has no effect on nondividing lymphocytes or fibroblasts. Compared to most other cell types, monocytes and macrophages have very low dCTP pools, but abundant deoxycytidine kinase activity. This suggests that dCTP derived from salvage pathways is important for DNA repair in these cells. Consistent with this notion, exogenous deoxycytidine could partially protect monocytes from aphidicolin killing. The unexpected toxicity of aphidicolin toward normal human monocytes may be attributable to their high rate of spontaneous DNA strand break formation, to the importance of DNA polymerase-alpha for DNA repair in these cells, and to their minute dCTP pools.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.     

Evaluation of production and characterization of monoclonal antibodies to human IgG of four subclasses

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.     

Modulation of Fc gamma receptors on the human macrophage cell line U-937

Macrophage receptors for the Fc portion of IgG play an important role in host defense, inflammation, and the pathophysiology of autoimmune disorders. We studied one important function of Fc gamma receptors--the ability to bind IgG ligand. Direct binding experiments analyzed by nonlinear regression were consistent with monomeric and trimeric IgG binding to a single class of receptors. Indirect binding experiments were also consistent with this interpretation and revealed that both IgG ligands completely inhibited the binding of the other. In addition, we used an anti-Fc gamma RII monoclonal antibody known to compete for the Fc gamma RII ligand binding site and known to inhibit IgG trimer binding to other cells. At concentrations of antibody which saturated all Fc gamma RII sites, no inhibition of IgG trimer binding to U-937 was observed. This was evident despite the observation that the numbers of Fc gamma RI and Fc gamma RII, determined by equilibrium binding of monomeric IgG and anti-Fc gamma RII antibody, respectively, were similar on U-937. Monoclonal antibodies were used to compare the expression and modulation of Fc gamma receptor proteins with their ability to bind monomeric and trimeric IgG ligands. Dexamethasone and gamma-interferon regulated U-937 Fc gamma RI protein expression and IgG ligand binding to a similar degree. In contrast, the expression of Fc gamma RII was not altered by dexamethasone. Interferon-gamma primarily stimulated Fc gamma RI, as determined both by reactivity with monoclonal antibody (227 +/- 26%) and by monomeric IgG ligand binding (350 +/- 151%). In addition, dexamethasone inhibited by 33% the gamma-interferon effect on Fc gamma RI protein and by 56% the effect on Fc gamma RI binding of monomeric IgG. Preincubation of U-937 with anti-Fc gamma RII antibody did not alter the effect of dexamethasone or gamma-interferon on IgG trimer binding. These data indicate that on U-937 cells Fc gamma RII does not function in the recognition of small molecular weight immune complexes and that Fc gamma RI is the Fc gamma receptor responsible for the binding of both monomeric and trimeric human IgG. Furthermore, Fc gamma RI is the major Fc gamma receptor on U-937 that is modulated by both gamma-interferon and glucocorticoids.     

C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI.

C-reactive protein (CRP) is an acute-phase protein that binds to components of damage tissue, activates C, and stimulates phagocytic cells. CRP binding to receptors on monocytic and polymorphonuclear phagocytes has been shown. Recently, CRP-binding proteins of 38 to 40 kDa and 57 to 60 kDa have been identified on the human promonocyte cell line U-937 and the mouse macrophage cell line PU5 1.8, respectively. However, analysis of CRP binding to these cells and to peripheral blood leukocytes suggests that additional CRP receptor sites may be present. Because many studies have shown interactions between CRP binding and IgG binding to leukocytes, we have examined further the CRP binding sites on U-937 cells and determined their relationship to the FcR for IgG (Fc gamma R) expressed on these cells. Our results demonstrate specific saturable binding of CRP to peripheral blood monocytes and U-937 cells, which is readily inhibited by aggregated IgG. Monomeric IgG, which binds specifically to Fc gamma RI, inhibited a maximum of 20% of CRP binding to these cells. mAb 197 and mAb IV.3, which block IgG binding to Fc gamma RI and Fc gamma RII, respectively, failed to inhibit CRP binding to U-937 cells. Two CRP-binding molecules were identified by precipitation of lysates from surface-labeled U-937 cells and cross-linking experiments. One of these had a molecular mass of 43 to 45 kDa, similar to the molecule previously described as the CRPR on U-937 cells. The other had the same mobility by SDS-PAGE as Fc gamma RI. The identity of this protein with Fc gamma RI was confirmed by the ability of both IgG-Sepharose and CRP-Sepharose to preclear the protein from cell lysates and by inhibition of binding to both IgG-Sepharose and CRP-Sepharose by anti-Fc gamma RI mAb 197.     

Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types

A 90-kD lymphocyte surface glycoprotein, defined by monoclonal antibodies of the Hermes series, is involved in lymphocyte recognition of high endothelial venules (HEV). Lymphocyte gp90Hermes binds in a saturable, reversible fashion to the mucosal vascular addressin (MAd), a tissue-specific endothelial cell adhesion molecule for lymphocytes. We and others have recently shown that the Hermes antigen is identical to or includes CD44 (In[Lu]-related p80), human Pgp-1, and extracellular matrix receptor III-molecules reportedly expressed on diverse cell types. Here, we examine the relationship between lymphoid and nonlymphoid Hermes antigens using serologic, biochemical, and, most importantly, functional assays. Consistent with studies using mAbs to CD44 or Pgp-1, mAbs against five different epitopes on lymphocyte gp90Hermes reacted with a wide variety of nonhematolymphoid cells in diverse normal human tissues, including many types of epithelium, mesenchymal elements such as fibroblasts and smooth muscle, and a subset of glia in the central nervous system. To ask whether these non-lymphoid molecules might also be functionally homologous to lymphocyte homing receptors, we assessed their ability to interact with purified MAd using fluorescence energy transfer techniques. The Hermes antigen isolated from both glial cells and fibroblasts--which express a predominant 90-kD form similar in relative molecular mass, isoelectric point, and protease sensitivity to lymphocyte gp90Hermes--was able to bind purified MAd. In contrast, a 140-160-kD form of the Hermes antigen isolated from squamous epithelial cells lacked this capability. Like lymphocyte binding to mucosal HEV, the interaction between glial gp90Hermes and MAd is inhibited by mAb Hermes-3, but not Hermes-1, suggesting that similar molecular domains are involved in the two binding events. The observation that the Hermes/CD44 molecules derived from several nonlymphoid cell types display binding domains homologous to those of lymphocyte homing receptors suggests that these glycoproteins represent a novel type of cell adhesion/recognition molecule (H-CAM) potentially mediating cell-cell or cell-matrix interactions in multiple tissues.     

Monoclonal antibodies to cytochrome P-450 immunopurify a 45-kDa protein from a human lymphoblastoid cell line

Monoclonal antibodies (MAbs) to rat liver cytochromes P-450 have previously been used for successful immunopurification of cytochromes P-450 from animal tissues. We now report application of this MAb-based immunopurification technique to the human lymphoblastoid AHH-1 cell line. Immunopurification carried out with 3 different MAbs each yielded a 45-kDa polypeptide. The purified protein contains an MAb-specific epitope present on cytochromes P-450, and may therefore be a human cytochrome P-450.     

Effect of protease inhibitors on human monocyte IgG Fc receptor II. Evidence that serine esterase activity is essential for Fc gamma RII-mediated binding

We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Regulation of antibody production mediated by Fc gamma receptors, IgG binding factors, and IgG Fc-binding autoantibodies.

Fc receptors (FcRs) are immunoglobulin-binding structures that enable antibodies to perform a variety of functions by forming connections between specific recognition and effector cells. Besides eliciting cytotoxicity, inducing secretion of mediators and endocytosis of opsonized particles, FcRs are involved in the regulation of antibody production, both as integral membrane proteins and as soluble molecules released from the cell surface. Most FcRs belong to the same family of proteins as their ligands (immunoglobulin superfamily). This review contains recent data obtained by use of monoclonal antibodies and cloning studies on FcRs and FcR-like molecules. The importance of fine specificity of receptor binding site(s)--that of the conformation of FcRs and their ligands in triggering signaling mechanisms--is analyzed. The regulatory function of membrane-bound and -released FcRs; the correlation between cell cycle, FcR expression, and release; as well as the possible mechanisms of these phenomena are discussed.     

Mast cell membrane antigens and Fc receptors in anaphylaxis: II. Functionally distinct receptors for IgG and for IgE on mouse mast cells

The present work was aimed at analyzing the functional relationships between mouse mast cell receptors for IgG and IgE antibodies. It was based on a study of the inhibition of IgG1-and IgE-induced passive mast cell degranulation produced by various immunoglobulin preparations capable of interfering with Fc receptors. Rat myeloma IgE, a high-affinity ligand for IgE receptors, was used to search for a possible participation of IgE receptors in IgG1-dependent degranulation. Mouse myeloma IgG, which inhibited only weakly IgG1-mediated reactions, had no chance to compete successfully with high-affinity IgE antibodies, but aggregated HGG was found to behave as a high-affinity ligand for IgG receptors. This enabled us to search for a possible participation of IgG receptors in IgE-dependent degranulation. The results show that rat myeloma IgE and aggregated HGG specifically inhibited IgE-induced and IgG1-induced reactions, respectively, but failed to inhibit reactions not requiring free Fc receptors. The conclusion was that receptors for IgG and for IgE are functionally independent on mouse mast cells, and are both expressed on the same cells.     

Human monocyte spreading in vitro--inducers and effects on Fc and C3 receptors.

Human peripheral blood monocytes undergo cytoplasmic spreading following attachment to a glass surface. The extent of spreading is greater in the presence of antigen-antibody complexes, manganese, subtilisin and dithiothreitol. The human blood monocyte spreads more rapidly than the mouse peritoneal macrophage and is not inhibited by serum. Fc receptor activity is diminished when spreading is induced by antigen-antibody complexes and is not affected by other inducers. The binding of erythrocytes coated with C3 and the ingestion of latex particles are not inhibited during cytoplasmic spreading.