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).     

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

The interaction of murine IgG subclass proteins with human monocyte Fc receptors

The use of murine monoclonal antibodies in the immunotherapy of human disease has prompted interest in the interactions of murine IgG with Fc receptors (FcR) expressed on human effector cells. We examined the heterocytophilic interactions between monomeric murine IgG subclass proteins and the FcR expressed on human monocytic cells (peripheral blood monocytes and interferon (IFN)-gamma-induced U937 cells). All four murine IgG2a antibodies and both murine IgG3 antibodies that were tested bound to human monocyte FcR with high affinity (10(8) to 10(9) M-1). By contrast, the affinities of four murine IgG1 and four IgG2b monomers were 100-fold to 1000-fold lower than the affinity of the human IgG1-FcR interaction. A 68,000 to 72,000 dalton protein was isolated by affinity chromatography from blood monocytes and from IFN-gamma-induced U937 cells on murine IgG2a, IgG3, and human IgG immunoadsorbents. In binding assays with IFN-stimulated U937 cells, murine IgG2a and IgG3 antibodies showed complete cross-blocking with a human IgG1 myeloma protein, indicating that murine and human IgG interact with the same population of Fc-binding proteins. No evidence for heterogeneity of cross-reactive FcR was observed. The ability of murine IgG2a and IgG3 monomers to compete with human IgG1 monomers for binding to human monocyte FcR suggests the potential usefulness of antibodies of these isotypes in the immunotherapy of diseases in which monocyte- or macrophage-mediated, antibody-dependent cellular cytotoxicity may play a role in the modification or remission of disease.     

Binding and catabolism of aggregated immunoglobulins containing C3b by U937 cells

We studied Fc receptor and C3b receptor (CR1) function on U937 cells, a human monocyte cell line. C3b was incorporated into stable soluble heat aggregates of 125I-IgM (A-IgM) and 125I-IgG (A-IgG) by using functionally pure classical pathway components. C3b incorporation was verified by the ability of aggregates to bind to human red cells and by cosedimentation of 125I and 131I during ultracentrifugation. Cell uptake and degradation of A-IgG X C3b was increased up to twofold compared with A-IgG not containing C3b molecules. However, A-IgG X C3b bound to CR1 after Fc receptors were blocked with nonradiolabeled A-IgG were also not endocytosed and catabolized. Moreover, A-IgM X C3b was bound but not degraded by U937 cells. As expected, uptake of A-IgM without C3b was negligible. CR1-mediated binding of A-IgM X C3b was specifically inhibited both by a murine monoclonal antibody against the human CR1 that blocks C3b binding and by C3b oligomers generated by trypsin activation of C3, but not by monoclonal antibodies against the iC3b receptor (CR3). We conclude that CR1 on U937 cells cause increased binding of A-IgG, and this increased binding leads to increased Fc-mediated endocytosis and catabolism of model immune complexes. However, binding of soluble ligands by CR1 alone, even when binding is multivalent, does not lead to endocytosis and degradation of soluble ligands bearing C3b.     

MLC-conditioned media stimulate an increase in Fc receptors on human macrophages

Macrophage Fc receptors (FcR) are essential for antibody-dependent cellular cytotoxicity and for optimal phagocytosis of opsonized particulate antigens. Culture in the presence of conditioned medium from mixed leukocyte cultures (MLC-CM) resulted in a dose- and time-dependent increase (up to 10-fold) in FcR-dependent binding of 125I-labeled IgG1 to promyelocytic HL-60 cells, macrophage-like U-937 cells, and normal cultured human monocytes. FcR increase in HL-60 cells was blocked by cycloheximide (100 microM) and was accompanied by a slight decrease in binding affinity. Since cell volume did not change, the increase in FcR probably represents an increase in the surface density of FcR sites. MLC-CM prepared with or without serum were equally effective in augmenting FcR sites, whereas only serum-containing MLC-CM caused morphologic change of U-937 and HL-60 cells.     

Fc receptor-mediated binding and endocytosis by human mononuclear phagocytes: monomeric IgG is not endocytosed by U937 cells and monocytes

Fc receptor-mediated endocytosis of monomeric IgG1 by human mononuclear phagocytes was evaluated under conditions where aggregated IgG and insulin readily undergo receptor-mediated internalization. U937 cells or normal human peripheral blood monocytes were incubated at 37 degrees C in the absence of free radioligand after having first bound 125I-IgG1 at 0 degrees C. To determine the amount of cell-associated IgG1 internalized after varying periods of 37 degrees C incubation, surface-bound IgG1 was removed by sequential exposure of cells at 0 degrees C to a nonspecific proteinase for 1 h and to acetic acid at pH 3.2 for 3 min. The failure to develop a proteinase- and acid-resistant fraction, similar to that seen over time at 37 degrees C in parallel experiments with 125I-insulin and 125I-aggregated IgG, and the lack of degradation of the IgG1 released into the medium from the same cells over time show that these cells do not endocytose and degrade monomeric IgG by an Fc receptor-specific mechanism and suggest that constitutive recycling without degradation is unlikely to be occurring. These data fulfill one prediction of the hypothesis that receptor-receptor interaction triggers Fc receptor-mediated endocytosis.     

Increased T gamma and T mu cells in BALB/c mice with IgG and IgM plasmacytomas and hybridomas

The results of previous studies in our laboratory have shown that mice bearing plasmacytomas and hybridomas that secrete IgA or IgE are accompanied by increased frequencies of Lyt-1-2+ T lymphocytes bearing Fc receptors (FcR) for IgA (T alpha) or IgE (T epsilon), respectively. The present study was undertaken to examine whether IgG- or IgM-secreting tumors influenced the frequency of T lymphocytes that express FcR for IgG or IgM. We studied mice bearing IgG- and IgM-secreting plasmacytomas and hybridomas. BALB/c mice injected subcutaneously with the IgG-secreting hybridoma HDP1 (gamma 1 kappa, anti-TNP) were sequentially examined for the frequencies and Lyt phenotypes of splenic lymphocytes bearing FcR for IgG (T gamma), IgM (T mu), and IgA (T alpha). A threefold increase in the frequency of T gamma lymphocytes that were Lyt-1-2+, L3T4- was seen. The frequencies of T mu and T alpha lymphocytes in these mice were not significantly altered. Similarly, mice injected subcutaneously with the IgM-secreting plasmacytoma MOPC 104E (mu lambda, anti-dextran) or the IgM-secreting hybridoma C1D1 (mu kappa, anti-ox RBC) were examined sequentially for the frequencies of T gamma, T mu, and T alpha lymphocytes. Mice with established IgM subcutaneous tumors showed a twofold increase in splenic, nylon wool-nonadherent T mu lymphocytes. This was associated with a relative increase in Lyt-2+ splenic T lymphocytes and a relative decrease in Lyt-1+ splenic T lymphocytes. No changes were observed in the frequencies of either T gamma or T alpha lymphocytes. These studies extend to IgG and IgM the observation that plasmacytomas and hybridomas secreting immunoglobulins of a specific isotype cause an expansion of T lymphocytes bearing FcR specific for the corresponding isotype. The expansion of FcR+ Lyt-1-2+ T lymphocytes likely represents an exaggerated, but otherwise normal, immunoregulatory response of the host. These cells may be an important element in the regulation of isotype expression.     

Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937

Human C-reactive protein (CRP) is an acute phase reactant that is opsonic and an activator of macrophage tumoricidal function. CRP also activates the classical C cascade. These activities suggest that CRP might interact with monocytes/macrophages via specific receptors in a manner analogous to the interaction of IgG with FcR. With the use of radio-labeled human CRP, we have observed specific binding of CRP to human blood monocytes and the human monocytic cell line U-937. Binding was saturable at a pathophysiologic concentration of CRP, with an estimated KD of 9.5 x 10(-8) M and 3.6 x 10(5) binding sites/cell. Specific binding was inhibited by polyclonal human IgG as well as an IgG1 myeloma. In the converse experiment, CRP failed to inhibit specific [125I]IgG binding. The mAb IV.3, which inhibits binding of IgG immune complexes to FcRII, did not inhibit CRP binding. A 100-fold excess of phosphorylcholine or the phosphorylcholine binding peptide of CRP (residues 47-63) failed to inhibit binding. Although human rIFN-gamma and PMA increased FcRI expression, these reagents had no affect on CRP receptor expression. A single membrane protein of 38 to 41 kDa from U-937 cells was chemically cross-linked to [125I]CRP; the cross-linking was inhibited by human IgG1 but not the IV.3 mAb. Furthermore, two membrane proteins with a Mr of 38 to 40 kDa and 58 to 60 kDa were isolated by CRP ligand-affinity chromatography. These proteins were of a distinct size from those isolated for FcRI from an IgG ligand matrix. These studies demonstrate specific binding of human CRP to a human monocytic cell line via receptors that are distinct from the IgG FcR and implicate CRP in nonspecific, preimmune host defense reaction mediated by cells of the monocytic lineage.     

Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.     

Interaction between hybrid mouse monoclonal antibodies and the human high-affinity IgG FcR, huFc gamma RI, on U937. Involvement of only one of the mIgG heavy chains in receptor binding

Here we have used hybrid mouse IgG1-2a and IgG2a-2b mAb to demonstrate that the interaction between the human high-affinity IgG FcR (huFc gamma RI) and monomeric mouse IgG2a mAb requires only one of the mIgG2a H chains. Recently, we reported a method for the generation and isolation of hybrid hybridomas, producing hybrid mouse mAb. Using this method we have obtained hybrid mouse (m)IgG1-2a and mIgG2a-2b mAb reacting with either horseradish peroxidase or human IgA1 (monospecific mAb) or with both Ag (bispecific mAb). Using protein A- or Ag-affinity chromatography purified hybrid mAb, we demonstrate here the binding of monomeric hybrid mIgG1-2a and mIgG2a-2b mAb to huFc gamma R on U937 cells, whereas no binding could be observed to the K562 cell line. Monomeric mouse IgG2a mAb and human IgG1 were found to be capable of inhibiting the binding of these hybrid mIgG1-2a and mIgG2a-2b mAb in a manner similar to the way they inhibited binding of monomeric mIgG2a mAb to U937 cells; this is in contrast to our findings for mIgG1 and mIgG2b mAb which did not inhibit the binding of both hybrid mAb. In addition, the binding of the hybrid mIgG1-2a and mIgG2a-2b mAb could be blocked by mAb TB-3, which is known to block huFc gamma RI-mediated binding by the "Kurlander phenomenon" and not by the anti-Fc gamma RII mAb CIKM5 and IV.3. These results indicate that both types of monomeric hybrid mAb are bound by the huFc gamma RI. Scatchard plots of mIgG2a, hybrid mIgG1-2a, and mIgG2a-2b mAb binding revealed similar numbers of binding sites and similar affinity constants of huFc gamma RI for these mAb (0.9 to 3.6 x 10(8) M-1). These results suggest that huFc gamma RI, present on the U937 cell line, are capable of binding monomeric hybrid mIgG1-2a and mIgG2a-2b mAb, and that this interaction requires only one of the mIgG2a H chains.     

The saturable high affinity association of factor X to ADP-stimulated monocytes defines a novel function of the Mac-1 receptor

Initiation of the coagulation protease cascade as it assembles on cell surfaces requires limited proteolytic activation of the zymogen factor X. Not previously suspected to be the ligand of an organizing receptor on cell surfaces, we now describe that factor X specifically associates with cells of monocyte lineage and we identify the high affinity receptor for this zymogen. Following stimulation with ADP (10 microM), or with the ionophore ionomycin (1 microM), isolated human monocytes bind 125I-factor X in a saturable fashion with a dissociation constant (Kd) of 21.8-44.9 nM. Equilibrium binding analyses indicate that the reaction is optimal at room temperature, requires Ca2+ ions, and saturates at 128,500 +/- 21,300 molecules of 125I-factor X specifically associated with the cell surface. Molar excess of unlabeled factor X inhibits and reverses the binding, whereas the homologous gamma-carboxylated coagulation proteins factors II, VII, IX, IXa, and Xa are without effect. Similarly, chelation of divalent ions immediately dissociates bound 125I-factor X. The monoblast cell line U 937 and the monocytic cell line THP-1 when stimulated with ADP or ionomycin, bind 125I-factor X with characteristics similar to monocytes. Receptor identity was explored using antibodies to the leukocyte adhesive receptors Mac-1, LFA-1, and p150.95. Monoclonal antibodies specific for the alpha subunit of Mac-1 (M 1/70, LM 2/1) or for the common beta subunit (TS 1/18, 60.3) bound equally to resting and ADP- or ionomycin-stimulated cells and also completely blocked the binding of 125I-factor X to stimulated monocytes, U 937, or THP-1 cells. To distinguish between modulatory effects of the monoclonal antibodies and direct spatial hindrance binding of 125I-factor X to Mac-1 was analyzed directly. OKM10 anti-alpha subunit of Mac-1 monoclonal antibody immunoprecipitated 125I-factor X chemically cross-linked to its receptor on stimulated cells. In addition, the complement protein fragment C3bi, which is a recognized ligand for Mac-1, competitively inhibited the association of 125I-factor X. These findings indicate that human blood monocytes and less differentiated cells of this lineage possess an inducible receptor specific for factor X; and also support the conclusion that the heterodimeric leukocyte adhesive receptor Mac-1 functions as the specific receptor structure. We suggest that the novel properties of this receptor may be of importance in the organization and regulation of certain coagulation protease cascades on the monocyte surface.     

Binding of recombinant-produced interferon beta ser to human lymphoblastoid cells. Evidence for two binding domains

Human interferon beta (IFN beta ser), produced by recombinant DNA technology, was radiolabeled to approximately one atom of iodine-125/molecule of interferon without detectable loss of antiviral activity. At 37 degrees C, binding of 125I IFN beta ser occurred rapidly (t1/2max less than or equal to 15 min) followed by internalization and degradation of bound ligand. Kinetic analysis at 4 degrees C indicated diffusion-limited association kinetics independent of 125I IFN beta ser concentration. Dissociation of bound 125I IFN beta ser from Daudi cells was slow (t1/2 = 1.2 h) of bound radiolabeled ligand was observed in the presence of unlabeled IFN beta ser, naturally produced IFN beta, and IFN alpha 6, but was not observed with unlabeled IFN gamma or nonspecific proteins. Concomitantly, equilibrium analysis indicated heterogeneous binding of 125I IFN beta ser to six cell lines of lymphoid origin consistent with either negative cooperativity or two populations of receptors. Analysis of binding of 125I IFN beta ser to Daudi cell receptors in the presence of unlabeled IFN alpha 6 suggested that one receptor served both ligands. The latter conclusion was supported by results of chemical cross-linking experiments in which an 125I IFN beta ser/receptor complex (Mr 120,000-130,000) was observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This complex was absent when binding occurred in the presence of either excess unlabeled IFN beta ser or IFN alpha 6.     

Differential binding of histidine-rich glycoprotein (HRG) to human IgG subclasses and IgG molecules containing kappa and lambda light chains.

In previous studies we showed that the plasma protein histidine-rich glycoprotein (HRG) binds strongly to pooled human IgG. In the present work myeloma proteins consisting of different human IgG subclasses were examined for their ability to interact with human HRG. Using an IAsys optical biosensor we found initially that IgG subclasses differ substantially in their affinity of interaction with HRG. However, the most striking finding was the observation that the kinetics of the HRG interaction was dramatically affected by whether the IgG subclasses contained the kappa or lambda light (L)-chains. Thus, the on-rate for the binding of HRG to the kappa L-chain containing IgG1 and IgG2 (IgG1kappa and IgG2kappa) was approximately 4- and approximately 10-fold faster than that for the binding of HRG to lambda L-chain containing IgG1 and IgG2 (IgG1lambda and IgG2lambda), respectively, with the dissociation constants (K(d)) in the range 3-5 nM and 112-189 nM for the kappa and lambda isoforms, respectively. In contrast, the on-rate for the binding of HRG to IgG3kappa and IgG4kappa was found to be 9- and 20-fold slower than that for the binding of HRG to IgG3lambda and IgG4lambda, respectively, with the K(d) in the range 147-268 nM and 96-109 nM for the kappa and lambda isoforms, respectively. The binding of HRG to immunoglobulins containing the kappa L-chain (particularly IgG1kappa) was generally potentiated in the presence of a physiological concentration (20 microM) of Zn(2+) (K(d) decreased to 0.60 +/- 0.01 for IgG1kappa), but Zn(2+) had no effect or slightly inhibited the binding of HRG to immobilized IgG subclasses possessing the lambda L-chain. Interestingly, HRG also bound differentially to Bence Jones (BJ) proteins containing kappa and lambda L-chains, with HRG having a 14-fold lower K(d) for BJkappa than for BJlambda when 20 microM Zn(2+) was present. HRG also bound to IgM (IgMkappa), but the affinity of this interaction (K(d) approximately 1.99 +/- 0.05 microM) was markedly lower than the interaction with IgG, and the affinity was actually decreased 4-fold in the presence of Zn(2+). The results demonstrate that both the heavy (H)- and L-chain type have a profound effect on the binding of HRG to different IgG subclasses and provide the first evidence of a functional difference between the kappa and lambda L-chains of immunoglobulins.     

Interaction of IgG3 anti-streptococcal group A carbohydrate (GAC) antibody with streptococcal group A vaccine: enhancing and inhibiting effects of anti-GAC, anti-isotypic, and anti-idiotypic antibodies

Enhancement of binding of one monoclonal antibody to an antigen in the presence of a second monoclonal antibody (specific for an independent epitope on the same antigen) has been observed for several antigen-antibody systems involving primarily protein, or glycoprotein, antigens. We have analyzed the interaction between radiolabeled IgG3 kappa anti-streptococcal group A carbohydrate (GAC) antibody (125I-HGAC 39) and streptococcal group A vaccine (GAV; traditionally used to elicit anti-GAC antibody) in the absence and presence of unlabeled anti-GAC antibodies, anti-isotypic antibodies, or anti-idiotypic antibodies, respectively. A variety of significant enhancing or inhibiting effects on the binding of 125I-HGAC 39 to solid-phase GAV (GAVsp) were noted. First, high concentrations of IgG3 anti-GAC antibodies specifically inhibit binding of 125I-HGAC 39 to GAVsp, but the presence of lower concentrations of IgG3 anti-GAC antibodies is associated with markedly increased (up to 300 to 400%) binding of 125I-HGAC 39 to GAVsp. In contrast, with the concentrations used, IgM anti-GAC antibodies only inhibit binding of 125I-HGAC 39 to GAVsp. A monoclonal anti-gamma 3 antibody (2E.6) also enhances binding (up to 700%) of 125I-HGAC 39 to GAVsp, whereas another high-affinity anti-isotypic antibody, anti-C kappa (187.1), only inhibits binding of 125I-HGAC 39 to GAVsp. In a similar manner, an antiidiotypic antibody (anti-IdX) specific for a framework idiotope located near the C kappa domain inhibits the interaction between 125I-HGAC 39 and GAVsp. Evidence is presented to suggest that neither anti-C kappa nor anti-IdX blocks the HGAC 39 paratope, and therefore, the inhibition of binding mediated by these antibodies must be on some other basis. An alternative explanation for this effect, on the basis of the impairment of functional bivalency of 125I-HGAC 39, is discussed. Finally, anti-idiotypic antibodies (anti-IdI-3a and anti-IdI-1) that bind closer to the antigen-binding site of HGAC 39 inhibit binding of 125I-HGAC 39 to GAVsp in a manner that is most readily interpreted as competition for the GAC-binding site (or nearby sites) on the HGAC 39 variable domain. These effects are shown to require specific immunologic recognition of either GAVsp or 125I-HGAC 39.     

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.     

Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.     

Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937.     

Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface

We recently reported the preparation and characterization of a monoclonal antibody, 32.2, specific for the high-affinity Fc receptor (FcR) for IgG on human monocytes. We have utilized the hybridoma cell line producing this antibody as a target for monocyte-mediated cytotoxicity. The hybridoma was selected for stable sublines that expressed high quantities of surface 32.2 immunoglobulin (Ig) through flow cytometry. Monocyte-mediated cytotoxicity, with these sublines used as targets, was evaluated with the use of a 51Cr-release assay. It was found that monocytes could efficiently lyse the hybridoma cells (HC 32.2) bearing surface Ig directed to the high-affinity FcR. Consistent with the specificity of the 32.2 antibody for an epitope on the high-affinity receptor outside of the ligand binding site, human IgG did not block monocyte killing of HC 32.2. In contrast, monocytes could not mediate lysis of hybridoma cells bearing high levels of antibody directed to other monocyte cell surface molecules, in particular, class I MHC molecules, the C3bi receptor, and the My 23 antigen. The effect of IFN-gamma on the ability of monocytes to mediate lysis of the 32.2 Ig-bearing hybridomas was also assessed. Monocytes cultured in the absence of IFN-gamma could lyse the hybridoma line expressing high levels of 32.2 Ig as efficiently as monocytes cultured in the presence of IFN-gamma. However, untreated monocytes were less able than IFN-gamma-treated monocytes to kill HC 32.2 expressing lower levels of Ig. Thus, IFN-gamma may enhance the efficiency of monocyte-mediated antibody-dependent killing under conditions where limited antibody is available on the target. These studies demonstrate that the high-affinity FcR on monocytes can act as a cytotoxic trigger molecule for killing of tumor cell targets and that this trigger does not require specific binding to the Fc binding epitope. These results further encourage possible clinical application of the 32.2 monoclonal antibody in tumor therapy.