Arachidonic acid release is closely related to the Fc gamma receptor-mediated superoxide generation in macrophages

Stimulation of macrophages with IgG2 immune complexes induced dose-dependently the O2- generation and the release of arachidonic acid and its metabolites. This Fc gamma R-mediated O2- generation was inhibited by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB), in parallel to the dose-dependent inhibition of arachidonic acid release. The main arachidonic acid metabolites released were shown to be prostaglandin E2 and thromboxane B2 and blocking of the production of these metabolites by indomethacin did not inhibit the O2- generation. Inhibition of the Fc gamma R-mediated O2- generation and the arachidonic acid release by the C-kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), was less intense than by 4-pBPB. These results support the previously proposed hypothesis that arachidonic acid acts as an intracellular activator of the Fc gamma R-mediated O2- generation in macrophages. Although the C-kinase activation may also contribute to the activation of the O2--generating system, arachidonic acid release appears to play a major role in Fc gamma R-mediated O2- generation. In contrast, activation of C-kinase seems to be contributing mainly in the induction of both the arachidonic acid release and O2- generation by 12-o-tetradecanoylphorbol 13-acetate (TPA). Furthermore, suboptimal concentrations of TPA and arachidonate were found to act synergistically to stimulate O2- generation and the inhibition study suggested a positive synergism between C-kinase and arachidonic acid release to induce O2- generation. This synergistic action may have general importance in receptor-mediated O2- generation.     

Trapping antigen-antibody complexes within the human placenta

The study was designed to evaluate possible mechanisms for the specific depletion of antifetal antibodies within the human placenta. The principal observations were that endogenous IgG is bound to the same cells to which exogenous antigen antibody complexes attach, i.e., placental macrophages. Moreover, the IgG was demonstrated to be attached to Fcγ receptors on the placental macrophages. Endogenous IgG was completely and specifically displaced by high concentrations of heterologous nonimmune IgG. That the IgG was present as antigen-antibody complexes was evidenced by macrophage receptor affinity, IgG size profile, and C1q binding. The results strongly support the theory that antifetal antibodies are removed by macrophages in the placenta as antigen-antibody complexes in much the same manner as macrophages filter immune complexes from the blood in the liver and spleen.     

Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes.

Two herpes simplex virus type 1 glycoproteins, gE and gI, have been shown to form a complex that binds the Fc domain of immunoglobulin G (IgG). We demonstrate that this complex is required for the binding of monomeric nonimmune IgG but that gE alone is sufficient for binding polymeric IgG in the form of IgG complexes. Evidence that gE but not gI is required for binding IgG complexes is as follows. IgG complexes bound equally well to cells infected with gI-negative mutants or with wild-type virus, whereas cells infected with gE-negative mutants did not bind IgG complexes. Furthermore, L cells transiently transfected to express gE bound IgG complexes. Additional evidence that gI fails to augment binding of IgG complexes comes from experiments in which the gI gene was inducibly expressed in cells after infection. Inducible gI expression failed to increase binding of IgG complexes to infected cells in comparison with cells not capable of inducible gI expression. In contrast, expression of both gE and gI was necessary for binding of monomeric IgG, as demonstrated by flow cytometry using cells infected with gE-negative and gI-negative mutants. These observations demonstrate that herpes simplex virus type 1 Fc receptors (FcRs) have different binding characteristics for monomeric IgG and IgG complexes. Furthermore, it appears that gE is the FcR for IgG complexes and that gE and gI form the FcR for monomeric IgG.     

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. Continuous oxidase activation requires continuous de novo receptor cross-linking

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.     

Triggering of the superoxide generation of macrophages by crosslinking of Fc gamma receptor

Crosslinking of monomeric IgG2 molecules bound to the Fc gamma receptors on the cell surface of guinea pig macrophages generated the triggering signal for the superoxide-generating system. A binding experiment indicated that macrophages have saturable binding sites for monomeric IgG2. Scatchard analysis of the binding data showed that macrophages have an average of 4 X 10(5) binding sites per cell and the association constant for the binding was 4.2 X 10(6) M-1. Binding of monomeric IgG2 to macrophages could be detected by subsequent reaction with the 125I-labeled F(ab')2 fragment of rabbit antibody specific for guinea pig Fab. Although binding of IgG2 monomer to Fc receptor did not stimulate superoxide release, further addition of the F(ab')2 fragment of anti-guinea pig Fab antibody did induce generation and release of superoxide, and the amount released was dependent on the dose of cell-bound IgG2. When macrophages were bound with a constant dose of IgG2 monomer in the first step, the superoxide release triggered by the addition of the F(ab')2 of anti-guinea pig Fab was dependent on the dose of the F(ab')2 fragment added. These results show that crosslinking of Fc receptors triggers the superoxide generation.     

Structural studies of Fc receptors. III. Isolation and molecular weight analysis of the component chain of Fc gamma receptor of macrophage

Surface receptors of guinea pig peritoneal macrophages specific for the Fc region of IgG (Fc gamma receptor) were isolated and identified as a surface-radioiodinated component with a molecular weight of 44,000 that bound in an Fc-specific manner to IgG2 of guinea pig immunoglobulin immobilized in any of the following three different ways: IgG2 antibody in insoluble immune complex, IgG2 antibody bound to antigen-coupled Sepharose, and IgG2 covalently coupled to Sepharose. In order to obtain the Fc gamma receptor retaining the binding activity, the Fc-binding component was isolated by IgG2 affinity chromatography in which mild acidic buffer (pH 5.0-4.0) was chosen to elute the component bound to the affinity column. Forty-five to sixty-two percent of the eluted radioactivity was shown to rebind to the IgG2-affinity column. The bound fraction showed a single radioactive peak of 44,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Fc-binding component isolated by the affinity chromatography behaved similarly in gel filtration in the presence of a detergent, as did the detergent-solubilized Fc gamma receptor before isolation by affinity chromatography. These results suggested that the Fc gamma receptor was isolated in a native form. Furthermore, it was confirmed that the isolated Fc gamma receptor is distinct from actin or the actin-like protein (DNase I-binding protein) which had been reported to bind to IgG-affinity column.     

Regulation of the Fc-receptor-mediated respiratory burst: treatment of primed murine peritoneal macrophages with lipopolysaccharide selectively inhibits H2O2 secretion stimulated by immune complexes

The effect of bacterial lipopolysaccharide on the Fc-receptor-mediated respiratory burst in murine peritoneal macrophages has been examined. After treatment overnight with small quantities of LPS, macrophages exhibited dramatic diminution of their capacity to generate and secrete H2O2 when triggered with immune complexes. The effect of LPS treatment was dependent on the state of macrophage functional activation; only cells that were primed or fully activated in vivo or were treated with interferon-gamma in vitro were sensitive to this effect of LPS. The LPS-mediated loss of secretory function was both dose and time dependent and could be reproduced with the lipid A moiety of LPS. The effect was selective for H2O2 secretion triggered through the Fc receptor; the respiratory burst stimulated by phorbol diesters remained unaltered. Furthermore, LPS treatment did not alter either binding or ingestion of radiolabeled immune complexes in parallel with the change in H2O2 secretion, indicating that the suppressive effect was not due to compromised endocytic function. These results indicate that LPS treatment of primed macrophages regulates the function of Fc receptors and may uncouple receptor occupancy from generation and secretion of H2O2.     

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.     

Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes

We have studied the Fc receptor-mediated pinocytosis of immunoglobulin G (IgG)-containing immune complexes by mouse macrophages. IgG complexes were formed from affinity-purified rabbit dinitrophenyl IgG and dinitrophenyl modified BSA at molar ratios of 2.5-10:1. Both the specificity of binding and the fate of internalized receptors were analyzed using monoclonal and polyclonal anti-Fc receptor antibodies. Based on the susceptibility of surface-bound ligand to release by proteolysis, we have found that at 37 degrees C, 125I-labeled IgG complexes were rapidly internalized (t1/2 less than 2 min) and delivered to lysosomes; acid-soluble 125I was detectable in the growth medium within 5-10 min of uptake. However, kinetic evidence indicated that Fc receptors were not efficiently re-used for multiple rounds of ligand uptake. Instead, macrophages that were exposed continuously to saturating concentrations of IgG complexes exhibited a selective and largely irreversible removal of Fc receptors from the plasma membrane. This loss of surface receptors correlated with an increased rate of receptor turnover, determined by immune precipitation of Fc receptors from 125I-labeled macrophages. Thus, in contrast to the results obtained in the accompanying paper (I. Mellman, H. Plutner, and P. Ukkonen, 1984, J. Cell Biol. 98:1163-1169) using a monovalent ligand, these data indicate that the interaction of Fc receptors with polyvalent complexes leads to the degradation of both ligand and receptor following their delivery to lysosomes.     

Characterization of the 2:1 complex between the class I MHC-related Fc receptor and its Fc ligand in solution.

The neonatal Fc receptor (FcRn) facilitates the transfer of maternal immunoglobulin G (IgG) to offspring and prolongs the half-life of serum IgG. FcRn binds IgG in acidic intracellular vesicles and releases IgG upon exposure to the basic pH of the bloodstream. The crystal structure of an FcRn/Fc complex revealed FcRn dimers bridged by homodimeric Fc molecules to create an oligomeric array with two receptors per Fc [Burmeister et al. (1994) Nature 372, 379-383], consistent with the 2:1 FcRn:Fc stoichiometry observed in solution [Huber et al. (1993) J. Mol. Biol. 230, 1077-1083; Sánchez et al. (1999) Biochemistry 38, 9471-9476]. Two distinct 2:1 FcRn/Fc complexes were present in the cocrystal structure: a complex containing an FcRn dimer interacting with an Fc and a complex in which single FcRn molecules are bound to both sides of the Fc homodimer. To determine which of the two possible 2:1 FcRn/Fc complexes exists in solution, we generated recombinant Fc molecules with zero, one, and two FcRn binding sites and studied their interactions with a soluble form of rat FcRn. The wild-type Fc with two FcRn binding sites binds two FcRn molecules under all assay conditions, and the nonbinding Fc with no FcRn binding sites shows no specific binding. The heterodimeric Fc with one FcRn binding site binds one FcRn molecule, suggesting that the 2:1 FcRn/wild-type Fc complex formed in solution consists of single FcRn molecules binding to both sides of Fc rather than an FcRn dimer binding to a single site on Fc.     

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.     

Antigen-antibody complexes bound to B-lymphocyte Fc gamma receptors regulate B-lymphocyte differentiation

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.     

Macrophage Fc receptors control infectivity and neutralization of canine distemper virus-antibody complexes.

Dogs that are persistently infected or that become moribund after exposure to canine distemper virus (CDV) have antibody that neutralized CDV when tested in dog lung macrophage cultures but failed to neutralize CDV when tested in epithelial, fibroblastic, or lymphatic cells. The antibody attached to protein A and was found in the immunoglobulin G fraction. The antibody bound complement and lysed CDV-infected target cells. The neutralizing activity in macrophages could be abolished (i) by pepsin digestion and removal of Fc portions from the antibody, (ii) by blocking the Fc receptors of macrophages with heat-treated normal dog serum, and (iii) by binding of protein A to Fc portions of the antibody. It was concluded that attachment of the CDV-antibody complex to Fc receptors of macrophages was essential for virus neutralization. If this attachment was hindered, the CDV-antibody complex became infectious for macrophages. In contrast, serum from recovering dogs neutralized CDV when tested in epithelial, fibroblastic, or lymphatic cells as well as in macrophages.     

Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.     

Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcγ receptors

The in vitro binding of monomeric, dimeric and multimeric forms of monoclonal IgG1 molecules, designated mAb1 and mAb2, to the extracellular domains of Fcγ receptors RI, RIIA and RIIIB were investigated using a surface plasmon resonance (SPR) based biosensor technique. Stable noncovalent and covalent dimers of mAb1 and mAb2, respectively, were isolated from CHO cell expressed materials. The dissociation constants of monomeric mAb1 and mAb2 were determined to be 1 nM for the FcγRI-binding and 6–12 μM for the FcγRIIA- and FcγRIIIB-binding. Dimeric mAb1 and mAb2 exhibited increased affinities, by 2-3 fold for FcγRI and 200-800 fold for FcγRIIA and FcγRIIIB. Further increases in binding were observed when the antibodies formed large immune complexes with multivalent antigens, but not in a linear relation with size. The binding properties of monomeric mAb2 were identical with and without a bound monovalent antigen, indicating that antigen-binding alone does not induce measurable change in binding of antibodies to Fcγ receptors. Dimerization is sufficient to show enhancement in the receptor binding. Given the wide distribution of the low-affinity Fcγ receptors on immune effector cells, the increased affinities to aggregated IgG may lead to some biological consequences, depending on the subsequent signal transduction events. The SPR-based in vitro binding assay is useful in evaluating Fcγ receptor binding of various species in antibody-based biotherapeutics.     

Arachidonic acid acts as an intracellular activator of NADPH-oxidase in Fc gamma receptor-mediated superoxide generation in macrophages

A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), inhibited phorbol ester (12-o-tetradecanoylphorbol 13-acetate)-induced and Fc gamma receptor-mediated superoxide anion (O2-) generations in guinea pig macrophages, but the inhibitory effect on Fc gamma receptor-mediated O2- generation was only partial. Both O2- generations were inhibited extensively by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB). It was confirmed in control experiments that H-7 and 4-pBPB had no direct inhibitory effect on NADPH-oxidase activity. Dose-dependent stimulation of O2- generation was induced by arachidonate in macrophages, and the arachidonate-induced O2- generation was not inhibited by H-7. Arachidonate could also induce NADPH-oxidase activation in a post-nuclear fraction obtained from unstimulated macrophages and this activation was not inhibited by H-7, indicating that protein kinase C activation was not involved in this cellfree system. These results support the hypothesis that the O2- generation induced by Fc gamma receptor stimulation is mainly mediated by arachidonic acid which is released by the action of phospholipase A2 activated by receptor stimulation. Arachidonic acid seems to be acting rather directly in activating the NADPH-oxidase system of macrophage membrane. Protein kinase C may have a significant role in Fc gamma receptor-mediated O2- generation but it is not obligatory, and protein kinase C seems to activate NADPH-oxidase rather indirectly, probably by inducing the arachidonic acid release.     

Structural differences among guinea pig Fc gamma 1/gamma 2 receptors on macrophages, polymorphonuclear cells, and lymphocytes.

Using the cDNA, D-3, coding for Fc gamma 1/gamma 2 receptor of guinea pig macrophages that binds IgG1 and IgG2 (Fc gamma 1/gamma 2R), we examined the cell distribution of this receptor by RNA blot analysis. The Fc gamma 1/gamma 2R mRNA was expressed in polymorphonuclear cells and B cells as well as in macrophages, but not at the detectable level in T cells. The cDNA amplified from RNA of polymorphonuclear cells in the polymerase chain reaction was the same as D-3. The cDNA of B cells was found to have about 140 bp cDNA segment inserted to the cytoplasmic tail of D-3. We found that the cDNA amplified from T cell RNA differed in signal peptide and extracellular domain sequence from cDNAs of other cell types. This cDNA does not seem to be amplified from the mRNAs of contaminating other cell types.     

Activating and inhibitory Fc gamma receptors in rheumatoid arthritis: from treatment to targeted therapies

Fc gamma receptors (Fc gammaRs) bind the constant Fc region of IgG molecules. IgG/antigen-containing immune complexes elicit a variety of effector functions in cells that express activating Fc gammaRs. Because activating Fc gammaRs are present on cells from the innate immune system, such as dendritic cells, monocytes/macrophages and granulocytes, these IgG receptors form a crucial link between the innate and the acquired immune systems. Recently, the ability to detect the inhibitory Fc gammaRIIb on cells has indicated an imbalance between activating and inhibitory Fc gammaRs in rheumatoid arthritis. This progress offers an opportunity to study modulation of Fc gammaR balance and could stimulate development of Fc gammaR-directed immunotherapy.     

An avidin-biotin-peroxidase method for Fc receptors on macrophages isolated from and in sections of rat lung

Receptors for the Fc region of immunoglobulin G (Fc receptors) were detected on pulmonary macrophages by adapting an avidin-biotin-peroxidase technique to isolated cells and sections of rat lung. After incubation with soluble rabbit immunoglobulin G (IgG), surface bound IgG was identified consistently and reproducibly on glass-adherent pulmonary macrophages and on macrophages in tissue sections made from incubated lung slices. Control experiments indicated that binding was specifically mediated by surface Fc receptors. This method may be useful for identifying macrophages in intact tissues.     

Binding characteristics of dimeric IgG subclass complexes to human neutrophils

Immune complexes were prepared by incubation of human IgG paraproteins with F(ab')2 fragments of the mAb K35 against the kappa-L chain of human IgG. The composition of these complexes was analyzed by centrifugation over sucrose gradients, by gel filtration, by RIA with either IgG Sepharose or K35 Sepharose and by double-labeling studies. The results indicated that the complexes consist of saturated tetramers composed of two IgG molecules cross-linked by two F(ab')2 fragments of the mAb. These complexes were used to study the binding of the different IgG subclasses to human neutrophils at 4 degrees C. Human neutrophils bound IgG3 complexes approximately three times faster than IgG1 complexes. Binding of IgG2 or IgG4 dimers to the neutrophils was undetectable. The same number of IgG1 complexes and IgG3 complexes bound to the neutrophils, but considerable inter-donor variation was found (mean number of Fc gamma R per neutrophil: 190,000, range 120,000 to 400,000). The Ka for the binding of IgG1 complexes to neutrophils (median 11 x 10(7) M-1) was lower than the Ka for the binding of IgG3 complexes (median 47 x 10(7) M-1). Competition studies between labeled IgG1 complexes or IgG3 complexes and unlabeled complexes showed that the Fc gamma R of human neutrophils do not display an IgG subclass specificity. Incubation of neutrophils with a mAb against the FcRIII completely blocked the binding of IgG1 complexes and IgG3 complexes. Incubation with a mAb against the FcRII reduced the affinity of the complexes for the neutrophils but had no effect on the maximum number of complexes bound. This indicates that one complex may bind simultaneously to one FcRIII and to one FcRII.