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.     

Molecular interactions between human IgG, IgM rheumatoid factor and streptococcal IgG Fc receptors

Group A streptococci type M15 were previously shown to bind both human IgG via the Fc component and a purified monoclonal IgM kappa rheumatoid factor (IgM RF). Using 125I-labelled IgG and 125I-labelled IgM RF, the present study gave association constants of 2.2 x 10(7) and 2.9 x 10(8) M-1, respectively. The binding of 125I-IgG to the streptococci was inhibited by unlabelled IgG, IgG Fc and fragment D of staphylococcal protein A but not by the IgM RF or F(ab')2 of anti-idiotype antibodies to RF (anti-Id RF). Inversely, unlabelled IgM RF and anti-Id RF inhibited the binding of 125I-IgM RF markedly and unlabelled human IgG and IgG Fc only slightly or moderately, respectively. Thus, group A streptococci type M15 showed different binding sites for IgG Fc and the antibody combining sites of a human monoclonal RF. The findings were still more complex on a background of previous reports showing that streptococcal IgG Fc receptors and RFs bind to the same amino acids on the Fc molecule. This complex pattern may play a role in the pathogenesis of rheumatoid arthritis.     

Cell surface expression of the varicella-zoster virus glycoproteins and Fc receptor

Varicella-zoster virus (VZV) specifies the synthesis of viral glycoproteins which are important antigens for induction of the host immune response. In this report the technology of laser-activated flow cytometry has been employed to measure the membrane expression of VZV glycoproteins gpI, gpII, gpIII, and gpIV. By use of biotinylated monoclonal antibodies as probes, all four glycoproteins were demonstrated on the infected cell surface. The temporal appearance of the viral glycoproteins was defined in a time course experiment and shown to be maximal about 24 hr postinfection. The issue whether VZV induces the cell surface expression of an Fc receptor (FcR) was investigated with biotinylated nonimmune human IgG, followed by streptavidin-phycoerythrin. By this technique a 10-fold increase in fluorescence intensity was seen in the VZV-infected cells as compared to the mock-infected controls. When the experiment was repeated with purified human Fc fragment rather than whole IgG, a similar degree of binding was seen. Both the VZV glycoproteins and the VZV FcR were exquisitely sensitive to trypsin treatment (1 mg/ml); likewise, the cell surface expression of these VZV products was diminished by treatment of the infected cultures with monensin, an inhibitor of glycoprotein transport. In order to prove that VZV infection was not causing the induction of a cellular Fc gamma R, the VZV-infected and mock-infected cells were stained with monoclonal antibodies directed against each of the three human cellular IgG FcR, but no differences were observed. Therefore, the FcR activity seen in the infected culture was not due to one of the known cellular Fc gamma R.     

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.     

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.     

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.     

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.     

IgG Fc receptors on epithelial cells of distal tubuli and on endothelial cells in human kidney

IgG Fc receptors (FcR) in cryostat sections of human kidney were studied using functional assays and monoclonal antibodies (MoAbs). Using functional assays, FcR were detected on cells in glomeruli and distal convoluted tubuli, on endothelial cells and on interstitial cells. These cells were also stained with B1D6, a MoAb reactive with a 40-kD molecule with low affinity FcR activity. Using MoAbs against leukocyte FcR, a different pattern of reactivity was obtained. MoAbs against FcR I (32.2), FcR II (IV.3 and C1KM5) and FcR III (3G8 and Leu11b) stained interstitial macrophages only. The data confirm the presence of FcR on renal glomeruli and interstitial cells and also indicate that epithelial cells of distal tubuli and endothelial cells express FcR. Furthermore, renal FcR appear to be structurally different from the FcR I, II and III defined on leukocytes.     

The Interaction of Ethanol with Human Monocyte IgG-Fc Receptors, Characterized by Monoclonal Antibodies Raised against Two Distinct Receptor Subpopulations

Human blood monocytes (Mo) in medium containing 10% autologous serum were exposed to ethanol (160 mM) at 37 degrees C for 15 min. After being washed, the cells were incubated with murine monoclonal antibodies (MoAb), one binding to the 40 kDa Fc receptor (FcR) (MoAb IV3) and the other to the 72 kDa FcR (MoAb 32). The incubation was performed with and without excess of human or rabbit IgG. The amount of receptor-bound MoAb was evaluated by fluorescein isothiocyanate (FITC)-labelled anti-mouse IgG. Most control Mo bound MoAb IV3 (90 +/- 8% stained cells) while only 52 +/- 2% Mo were positively stained by MoAb 32. The staining increased in the presence of human IgG in the assay mixtures. Pre-incubation with 160 mM ethanol reduced the MoAb IV3 binding (61 +/- 2% stained cells), but had no significant effect on the binding of MoAb 32. Wash-out experiments indicated normalization of receptor function (MoAb IV3 binding) after 4 h. Treatment of medium or serum with ethanol before incubation of the Mo had no effect. It was concluded that a brief exposure of human Mo to ethanol leads to changes in a subpopulation of IgG FcR. Since these changes appeared when MoAb were used against the receptors, they probably represent a decrease in functional receptors and not changes in affinity.     

Expression of Fc mu receptors on human natural killer cells

Fc receptors for IgG (CD16) have been described as the only type of immunoglobulin receptor on large granular lymphocytes (LGL). However, the ability of natural killer (NK) cells to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of monoclonal or polyclonal IgM and the inhibition of NK activity by highly purified IgM could not be explained on the basis of FcR for IgG. In order to directly assess the expression of Fc receptors for IgM (Fc mu R), NK cells were treated with human polyclonal IgM, and its binding was visualized by a direct anti-globulin rosette assay with identification of rosette-forming LGL on Giemsa-stained smears. The data indicated that a high proportion of LGL (up to 68%) were Fc mu R-positive cells. However, this percentage varied depending on the IgM preparation (polyclonal or monoclonal), the indicator reagent used for the rosette assays, and the cell preparations studied. Two-color flow cytometry of human nonadherent lymphocyte preparations confirmed the presence of CD56+IgM+ cells, which represented from 43 to 78% of CD56+ cells. Flow cytometry was also performed using highly enriched preparations of human NK cells (the mean percentage of CD3-CD56+ cells was 84%). Up to 88% of purified NK cells bound FITC-labeled monoclonal IgM at a saturating concentration. By indirect immunofluorescence, from 34 to 62% of NK cells purified from the peripheral blood of normal donors were able to bind polyclonal IgM. Similar results were obtained with LGL from a patient with NK lymphoproliferative disease. Thus the presence of Fc mu R on a majority of human NK cells was demonstrated by different techniques, using unseparated peripheral blood mononuclear leukocytes, purified normal NK cells, and also LGL from a patient with NK lymphoproliferative disease.     

Receptors for immunoglobulin isotypes (FcR) on murine T cells. II. Multiple FcR induction on hybridoma T cell clones

In the preceding report (Eur. J. Immunol. 1985. 15: 662), we described a variety of receptors for the Fc portion of the different isotypes of mouse immunoglobulins (FcR), that were found to be expressed on hybridoma T cell clones. In the present work, we wondered whether the expression of these T cell FcR would be regulated by environmental influences such as the presence of corresponding ligands. We found that exposing the cells to the bulk of serum immunoglobulins in vivo, or to purified monoclonal immunoglobulins in vitro both resulted in FcR induction. The expression of all constitutive receptors, i.e. Fcgamma 1/2bR, Fcgamma3R, FcalphaR and FcepsilonR, could be increased upon incubation with IgG1, IgG2b, IgG3, IgA and IgE, respectively. After induction, the specificity of FcR was not modified. Two FcR were detectable only upon induction. These were Fcgamma2a/2b/1R, induced by IgG2a and FcmuR, induced by IgM. Interestingly, FcR detectable after induction only were short-lived at the membrane. Ten to 15 h after induction they were not detected any more, whereas the expression of constitutive FcR remained elevated for at least 24 h following induction. Therefore, depending on the concentration of immunoglobulins in the environment, and depending on whether they are short lived or long lived, FcR can modulate their expression on the membrane of T cells. Such a versatility might be an efficient means to contribute to isotypic regulation through the release of regulatory immunoglobulin-binding factors. factors.     

Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein.

Two murine monoclonal antibodies (immunoglobulin M) evoked against human kidney tissue were examined for cross-reactivity with group A streptococci. A glomerulus-specific antibody, PMII.40.H.2, cross-reacted in an enzyme-linked immunosorbent assay with purified pepsin-extracted M proteins from type 6 and 12 streptococci, but not type 1, 3, 5, 19, and 24 M proteins. The cross-reactive monoclonal antibody also opsonized type 6 and 12 streptococci, indicating that it was directed against protective M protein epitopes that were exposed on the surfaces of these organisms. A control antibody, which was tubule specific, did not cross-react with any of the purified M proteins, nor did it opsonize any of the serotypes of streptococci tested. Western immunoblot experiments identified the glomerular cross-reactive antigen as a 43-kilodalton protein. The results demonstrate that M protein of group A streptococci and glomerular antigens of the human kidney possess cross-reactive determinants.     

Binding of IgM rheumatoid factor to group A, C and G streptococci with IgG Fc receptors

The possibility that IgM rheumatoid factors bind to streptococci was studied. Using a sequence of Sephadex G200 gel filtration, protein A-Sepharose CL-4B chromatography and preparatory electrophoresis, IgM was isolated from the sera of 2 patients with rheumatoid arthritis and then radiolabelled with 125I. There was significant binding of radiolabelled IgM to group-A streptococci types M1, M15 and M22, and to a group-C and a group-G strain, all expressing IgG Fc receptors, but none to Staphylococcus aureus, Escherichia coli or to 11 other strains of streptococci without IgG Fc receptors. The radiolabelled IgM was separated by affinity chromatography on a column containing human IgG. Types M1 and M15 bound the fraction retained on the column, whereas M22 bound both this fraction and the non-retained fraction. Commercial human IgG, even at high concentrations, did not inhibit binding. The binding reaction, which is perhaps triggered either by the IgM rheumatoid factor or by IgG complexed with rheumatoid factor, could be a useful tool for removal of anti-immunoglobulin from the blood of patients with rheumatoid arthritis.     

Binding of monoclonal IgM rheumatoid factor to streptococci via the antibody combining site

Radiolabelled monoclonal IgM rheumatoid factors, four from patients with type II essential cryoglobulinaemia and one originating from a patient with rheumatoid arthritis, were tested for binding to group A, B, C and G streptococci and Escherichia coli. Two of the preparations exhibited different binding patterns for the streptococci, whereas the remaining three were not reactive. The binding of one of the factors to group A streptococci type 15 was inhibited by F(ab')2-fragments of anti-idiotypic antibodies but not anti-IgM Fc or F(ab')2 of normal rabbit IgG, indicating that the reaction was mediated via the antibody combining sites.     

Specificity and protective activity of murine monoclonal antibodies directed against the capsular polysaccharide of type III group B streptococci.

We have obtained 41 monoclonal antibodies directed against type III group B streptococci by immunizing Balb/c mice with formalin-killed bacteria. All of these antibodies reacted with purified type-specific carbohydrate by enzyme-linked immunosorbent assay and immunoprecipitation tests. The epitope recognized by all of these antibodies was associated with terminal sialic acid residues, as indicated by abrogation of immune reactions by treatment of the type-specific carbohydrate with neuraminidase. Two purified monoclonal antibodies (the IgM P9D8 and the IgG3 P4F12) were further characterized for their protective activity in a neonatal rat model of infection. P9D8 and P4F12 antibodies were significantly protective when administered in a dose of 0.5 and 2.5 mg/kg, respectively, at the same time as 3 x 10(5) colony forming units of type III streptococci. Protection was still observed when the antibodies were given up to 9 h after challenge. No protection was afforded against infections with type Ia/c and II streptococci. Similarly, both antibodies effectively opsonized type III, but not Ia, Ib or II bacteria, in an in vitro assay. These and similar, previously described, monoclonal antibodies may be useful, possibly after "humanization" by genetic engineering, for the therapy of neonatal group B streptococcal infections.     

Solubilization of human peripheral nerve Fc gamma receptors and purification of a functional 40 kDa receptor

Extracts from myelinated and unmyelinated nerves, prepared using Tris-HCl buffer with EDTA and ME, contained functionally active receptors for the Fc region of IgG (FcR). This was evident from the results obtained in indirect haemagglutination and rosette inhibition assays. Using a monoclonal antibody, a functional active 40-kDa FcR was purified from the nerve extracts. The receptor was a single-chained glycoprotein with low affinity for native IgG, apparently belonging to the FcRII family. In addition, peripheral nerve extracts contain FcR not recognised by the monoclonal antibody.     

Extraction and characterization of IgG Fc receptors from group C and group G streptococci

Immunoglobulin G (IgG) Fc receptors (FcRs) were extracted by proteolytic digestion of four strains each of group C and group G streptococci. The solubilized proteins were analyzed in Western blots and multiple IgG-binding bands were obtained. The banding patterns of some of the strains were very similar, but this property was independent of which streptococcal group the strains belonged to. Highly purified FcRs were prepared from one group C and one group G strain. The 13 N-terminal amino acids were determined, and found to be identical, whereas comparison with the sequence of staphylococcal protein A did not reveal any homology. The isolated streptococcal FcRs also appeared closely related antigenically and functionally. Thus, both molecules were capable of inhibiting each others binding to immobilized IgG, and the radiolabelled group G FcR was completely inhibited from binding to IgG by an antibody to the group C FcR. Finally, in a direct binding assay both proteins were capable of reacting to a similar degree with a wide variety of IgGs, thereby demonstrating the great potential of streptococcal FcRs as tools for binding and detection of IgG antibodies.     

Comparative study with monospecific and monoclonal antibodies gainst a 65 K human cytomegalovirus protein

Summary Immunofluorescence assay using monospecific and monoclonal antibodies to the 65K major protein of human cytomegalovirus (HCMV) was carried out to monitor the expression of this protein in infected cells. Regardless of differences in the reactivity of the monoclonal antibodies, as determined by immunoblotting and immunofluorescent staining, all stained cytoplasmic inclusion bodies localized to the site of the HCMV-induced receptor for the Fc portion of IgG, suggesting that most of the 65K major protein of HCMV colocalizes with the HCMV-induced FcR.     

Agonistic Effects of Anti-CD2 and Anti-CD16 Antibodies on Human Natural Killer Killing

Two monoclonal antibodies (MoAb), 9-1 (anti-CD2) and 3G8 (anti-CD16), were previously shown to enhance the cytotoxic activity of human natural killer (NK) cells. The present study examined the effect of 9-1 and 3G8 with different effector and target cells to determine whether they activate NK cells through a common mechanism. Analysis of purified lymphocyte subpopulations demonstrated that the CD3+CD16+CD3- NK effector cell population is enhanced by both antibodies, while purified CD2+CD16-CD3+ T cells are not activated by either antibody. Although both antibodies enhance killing of K-562 and Daudi, killing of T-cell lines is enhanced by 9-1 and inhibited by 3G8. In contrast, killing of the promyelocytic cell line, U-937 is inhibited by 9-1 and enhanced by 3G8. On NK-susceptible cells the pattern of enhancement with 3G8, an IgG1 MoAb, is consistent with the pattern of target cell expression of an Fc receptor, FcR II, known to bind IgG1 antibodies. This suggests that 3G8 may cross-link effector and target cells through CD16 on the effectors and FcR II on these targets. This could activate NK killing by a mechanism similar to antibody-dependent cellular cytotoxicity reactions (ADCC) with the MoAb in the reverse orientation. The failure of 3G8 F(ab')2 fragments to enhance NK killing, further supports the reverse ADCC mechanism of enhancement by 3G8. The pattern of enhancement mediated by 9-1, an IgG3 MoAb, is not correlated with any target cell Fc-receptor known to bind IgG3 MoAb. The effect of 9-1 may result, instead, from its binding to the unique 9-1 epitope on the CD2 molecule involved in CD2-mediated T-cell activation, as previously described. Alternative mechanisms, including activation of NK killing by 9-1 mediated cross-linking of CD2 and CD16 on the effector cells, have also been discussed.     

Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes.

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG (2-10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgG1 and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.