Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer

Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer. Cells were stained with fluorescently-labeled RTX and mAbs specific for C3b/iC3b fragments or for human IgG, and then imaged using the ImageStream cytometer and analyzed with an algorithm (Similarity Bright Detail Score, SBDS) which tests for co-localization of fluorescent probes. SBDS, calculated on 10,000 cells, verified that the majority of deposited C3b/iC3b is co-localized with bound RTX. In contrast, when cells were first opsonized in serum alone, washed and then reacted with RTX, SBDS confirmed that RTX and C3b/iC3b are poorly co-localized, thus demonstrating that cell-bound RTX directs deposition of C3b. In addition, a sulfhydryl-specific probe, maleimide conjugated to AF488, exhibited substantial co-localization with an anti-C3b/iC3b mAb on Raji cells opsonized with RTX and serum, thus validating maleimide labeling as an alternative for detecting cell-bound C3b/iC3b. The digital imaging method described should have wide applicability for quantitative analysis of co-localization.     

Monoclonal anti-human C3d antibodies: stabilization of the alternative pathway C3 convertase.

IgG mouse monoclonal antibody (mAb) was prepared by fusion of spleen cells from mice immunized with human C3d (mAb:C3d) using syngeneic thymocytes as feeder cells. mAb:C3d was assessed for its effect on the stabilization of the cell-bound alternative pathway C3 convertase EAC3bBb. It bound to cell-bound C3b and stabilized C3bBb at 30 degrees in the presence of EDTA-GVB. The plasma protein H reduced the stabilization effect of the stabilized C3 convertase. These results suggest that binding of antibody to C3d may stabilize C3bBb. It seems likely that such antibody induces in C3b conformational change, which increases the C3bBb complex stability.     

Murine monoclonal anti-Ba antibody that enhances haemolytic activity of factor B.

A murine monoclonal antibody (mAb 20-ET) (IgG1, kappa) was selected from a panel of stable hybridomas produced by fusion of P3-X-63-Ag8-U1 (P3UI) myeloma cells with spleen cells from a BALB/c mouse immunized with human factor B. This antibody was shown by the immune blotting method to be directed against the Ba domain of factor B. The haemolytic activity of factor B was enhanced dose-dependently by mAb 20-ET when it was incubated with factor B and EAC4b, 3b cells (sensitized erythrocytes bearing complement fragments C4b and C3b). However, when the antibody was added after factor B had been bound to EAC4b,3b cells and the cells had been washed, it caused little enhancement of the haemolytic activity. The enhancing effect of this antibody was not due to its stabilization of the C3b-B complex, because EAC4b,3b dissociated from mAb 20-ET-bound factor B complexes rather more readily than from uncomplexed factor B. The presence of mAb 20-ET in the reaction mixture caused and maintained a much higher steady-state level of binding of factor B with EAC4b,3b cells than that in its absence. Factor P caused delayed dissociation of mAb-bound factor B from EAC4b,3b cells, thus enhancing the haemolytic activity of factor B bound with the mAb.     

Application of human erythrocytes to a radioimmune assay of immune complexes in serum.

An immune adherence receptor exists on the surface of primate erythrocytes, and has been characterized as a receptor for the activated third component of complement (C3b). We have applied human red blood cells (RBCs, blood group O) to a sensitive determination of complement-fixing, soluble immune complexes in serum. The method involved the binding of immune complexes with RBCs in the presence of complement and the detection of cell-bound IgG molecules by radiolabelled anti-human IgG antibodies. Since the binding of RBCs with monomeric IgG was minimal, cell-bound IgG molecules were taken as representing immune complexes. When aggregated human gammaglobulin (AHG) was used as a model of immune complexes, as little as 5 μg dissolved in 1 ml of normal human serum were detected. The binding of RBCs with AHG was inhibited in EDTA solution where the classical complement pathway could not be activated. The RBC radioimmune assay was successfully applied to the determination of soluble immune complexes in pathological serum samples obtained from the patients with systemic lupus erythematosus and those with fulminant Type B hepatitis. False-positive results by autoantibodies against RBCs could be excluded by performing a Coombs test and by comparing the binding in the presence of complement with that in EDTA solution. The ubiquitous availability of RBCs coupled with a high sensitivity would allow the RBC radioimmune assay to be added to the battery of previous methods to determine immune complexes in the serum.     

The fluid-phase binding of human C4 and its genetic variants, C4A3 and C4B1, to immunoglobulins

Covalent binding of the fourth complement protein, C4, to immune complexes is an important first step in the complement mediated processing of the complexes. Many of the initial encounters between the proteins of the complement system and antigen and antibody occur in solution, and prior to this report, studies of the interactions between them have focused on complement binding to preformed immune precipitates that most likely are not found in vivo. We have characterized the covalent binding of C4b to immunoglobulin molecules in a fluid-phase system consisting only of antibody in solution and purified C4 and C1s. We demonstrate that human C4b binds to IgG in the fluid phase, that its covalent binding is predominantly to the heavy chain of IgG, and that the covalent linkage is by either amide or acyl ester bonds. In addition, we compare the covalent binding efficiencies of two genetic variants of C4, C4A3 and C4B1, to IgG. C4A3 binds 3-4 times more IgG than C4B1 over a range of C4 concentrations, and C4A3 has a higher binding efficiency than C4B1 for IgM, IgA, IgG2a and F(ab')2 as well as for a protein antigen, BSA. Furthermore, we found that whereas C4A3 is bound to immunoglobulins in the fluid-phase predominantly by amide linkage, C4B1 is bound by either amide or acyl ester bonds. The results presented here suggest that the covalent binding efficiency of C4A3 and C4B1 to IgG is similar to that reported for their covalent binding to small molecules.     

Induction of transient arthritis by the adoptive transfer of a collagen II specific Th1 clone to HLA-DR4 (B1*0401) transgenic mice

Collagen II arthritis (CIA) represents an animal model of human RA that can be induced in DBA/1J (H-2(q)) but not in C57BL/6 mice (H-2(b)). A vigorous CII specific CD4 Th1-cell response but not IgG2 anti-CII antibody or CIA could be induced in C57BL/6 mice made transgenic for the RA shared epitope DR4 (B1*0401). We developed CD4 Th1-cell clones specific for CII from these transgenic (tg) mice in order to determine if the adoptive transfer of these clones into syngeneic tg C57BL/6 recipients could induce CIA. Three bovine CII specific (bCII) CD4 Th1-cell clones and one T-cell line specific for an immunodominant region of bCII (p261-273) were generated. Among these only one clone that could up-regulate anti-CII, IgG2 antibody in the recipient mice was able to induce transient arthritis. However, this level of IgG2 anti-CII antibody was only one-third of that seen in CII immunized DBA/1J mice that develop persistent arthritis. These results confirm our previous observations that the induction of CIA requires a sustained IgG2 antibody response to CII, an effect difficult to achieve even in DR4 (B1*0401) tg mice reconstituted with CD4 Th1 cells. This suggests that a rate limiting step in the development of human RA among those individuals expressing the RA shared epitope may be the requirement to generate sustained levels of complement fixing antibody to arthritogenic antigens.     

Activation of hemolytic complement by mouse monoclonal IgG1 antibody: comparison with rabbit IgG

We investigated the ability of a mouse anti-hapten monoclonal IgG1 antibody (Ab) to bind to cell-bound specific hapten and to fix and activate C1 and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind C1 and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity. We also found that even though monoclonal IgG1 Ab was able to bind strongly to a protein A-Sepharose column and could be eluted only by a low-pH buffer, the purified Ab, when bound to cell surface hapten, showed a weak ability to react with free protein A.     

Heparin prevents formation of the human C3 amplification convertase by inhibiting the binding site for B on C3b

Fluid-phase heparin prevents generation of the C3 amplification convertase of human complement, C3b, Bb most likely by inhibiting the formation of the bimolecular complex between cell-bound C3b and B. The effects of heparin on the binding of B to C3b was examined using ¹²⁵I-labelled B and C3b-bearing sheep erythrocytes (E^sC3b). In the absence of heparin, B found to E^sC3b with an affinity of 0.5–1 × 10⁶ M⁻¹ in the presence of 5 mM Mg²⁺. Incremental amounts of heparin (100–700 μg/107 E^sC3b) inhibited the binding of ¹²⁵I-B to C3b in a dose-dependent manner. Scatchard analysis of the binding data in the presence of four inhibitory concns of heparin revealed that heparin did nor affect the binding affinity of B for C3b but decreased the number of C3b sites recognized by B on the cells. No inhibition of binding occurred in the presence of totally (N- and O-) desulfated heparin which has no anticomplementary activity. These results demonstrate that heparin prevents generation of the C3 amplification convertase by binding to cell-bound C3b and masking the binding site for B on C3b.     

Complement, membrane glycoproteins, and complement receptors: their role in regulation of the immune response

To determine the effect of complement on the normal antibody response we studied seven patients with genetically determined complement component deficiencies, guinea pigs deficient of C4 and C2, respectively, and two patients whose neutrophils and monocytes lack the C3bi receptor. Patients deficient of early complement components (C4, C2, C3) have abnormal antibody responses to the T-cell-dependent antigen, bacteriophage phi X 174. Complement-deficient guinea pigs (C4, C2) produce less antibody than normal guinea pigs and are unable to maintain measurable antibody levels; during secondary immunization they fail to develop amplification and to switch from IgM to IgG. This defect can be overcome by increasing the antigen dose or by injecting normal guinea pig serum at the time of the primary (but not the secondary) immunization. Patients with deficiency of the C3bi receptor were shown to have a significantly depressed antibody response to T-dependent antigens. We postulate that the contribution of complement to the mature humoral immune response is related to activation of C3. The initial production of IgM following antigen injection leads to antigen-antibody complexes which interact with complement, to be nonspecifically trapped by C3b and C3bi receptors on B cells or macrophages. Thus antigen is selectively accumulated within the lymphoid organs and in turn may entrap antigen-specific B cells by interaction of the trapped antigen with surface immunoglobulin. As a result, close approximation between antigen, antibody, and a network of specific and nonspecific lymphoid cells is initiated, allowing generation of specific memory cells and initiation of a prompt mature antibody response on subsequent exposure to antigen.     

Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune – three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20⁺ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.     

Relationship between complement activation and renal deposition of immune complexes made with IgG2a monoclonal antibodies

Previous studies identified a single murine monoclonal IgG2a anti-dinitrophenyl antibody that, when combined with the antigen, formed immune complexes (IC) that were preferentially deposited in glomeruli. The present study examined the clearance and organ localization in Balb/c mice of expanded panels of radiolabeled IC containing murine monoclonal antibodies. The results identified a second IgG2a antibody that formed IC with a predilection for renal deposition. IC made with the two IgG2a antibodies that were preferentially deposited in the kidney were the least efficient binders of human C1q or homologous murine C3b and C4b within the IgG2a panel. These observations suggest a new model of IC-mediated renal disease initiation in which relatively weak complement activation leads to inefficient IC clearance by complement receptor-bearing circulating cells and consequent IC deposition in tissues susceptible to IC-mediated injury.     

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigenpresenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,χ and IgG2a,χ murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and puritied in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of χ light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigenpresenting cells.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Antibody-mediated neutralization of cytomegalovirus: modulation of efficacy induced through the IgG constant region

Antibodies can neutralize the infectious properties of human cytomegalovirus (CMV). In vivo, the major neutralization determinants are located on glycoprotein B (gB). Recombinant human antibodies, that carry different constant regions (IgG1, IgG3 and the synthetic variant IgG3mA) against two of these epitopes were investigated for their ability to recruit the complement cascade for destruction of the virus. It was shown that all variants of an antibody against the antigenic domain (AD)-2 epitope displayed a similar neutralization activity despite the fact that improved C1q binding was observed for IgG3 and IgG3mA over the IgG1 variant. In contrast, an antibody against the AD-1 epitope carrying the normal IgG3 constant region, was less efficient than its IgG1 counterpart in neutralizing the virus in the absence of complement. However, it restored its activity in the presence of complement to the level of the naturally occurring IgG1 version. The same antibody was substantially more potent in neutralizing the virus in the presence of complement if it carried the IgG3mA constant region. This demonstrates the importance of the constant domain for the biological activity of AD-1 specific antibodies, a factor that should be taken into account when using antibody-based therapeutics or when inducing antibodies by vaccination.     

The covalent interaction of C3 with IgG immune complexes

Antigens (Ags) are converted into immune complexes (antigen-antibody complexes, IC) as soon as they encounter their specific antibodies (Abs). In fluids containing complement, the process of IC formation and fixation of complement components occur simultaneously. Hence, the formation of Ag-Ab-complement complexes is the normal way of eliminating Ags from a host. C3b-C3b-IgG covalent complexes are immediately formed on interaction of serum C3 with IgG-IC. These C3b-C3b dimers constitute the core for the assembly of C3/C5-convertase on the IC, which are subsequently converted into iC3b-iC3b-IgG by the complement regulators. These complexes are detected on SDS-PAGE by two bands of molecular composition, C3alpha65-C3alpha43 (band A) and C3alpha65-heavy chain of the Ab (band B), which correspond to C3b-C3b and C3b-IgG covalent interaction respectively, and that identify opsonized IC (C3b-IC). C3b can attach to Fab and Fc regions of the Ab molecule with similar efficiency. The presence of multiple C3b binding regions on IgG is considered an advantageous characteristic that facilitates the elimination of Ags in the form of C3b(n)-IC. Ab molecules on the IC recognize the Ag, and also serve as a very good acceptor for C3b binding. In this way, Ags, even if they have no acceptor sites for C3b, can be efficiently processed and removed. When C3 is activated in serum by IC or other activators, secondary C3b-IgG covalent complexes are generated, with bystander monomeric circulating IgG, and thus constitute, physiological products of complement activation. These complexes gain importance when IgG concentration is extremely high as in cases of infusion of intravenous IgG (IVIG) in several pathologies. The covalent attachment of activated complement C3 (C3b, iC3b, C3 d,g) to Ags or IC links innate and adaptative immunity by targeting Ags to different cells of the immune system (follicular dendritic cells, phagocytes, B cells). Hence C3b marks Ags definitively, from the earliest contact with the innate immune system until their complete elimination from the host.     

C1q binding to antibody-coated cells: Predictions from a simple multivalent binding model

We have derived a simple mathematical model describing the multivalent binding of Clq to IgG antibody-coated target cells. The model contains the assumptions that the Clq is hexavalent for IgG, that the cell-bound antibody is mobile in the plane of the plasma membrane, and that the six Clq subunits bind sequentially to the cell-surface antibody. We have calculated how Clq would bind at equilibrium to cells coated with different amounts of antibody, and how free monomeric IgG would inhibit this interaction. The model predicts that the degree of binding of Clq to the cells will be strongly enhanced by increasing the cell surface antibody density, but that even small amounts of cell-bound antibody will render the target cells capable of binding Clq.Scatchard plots of Clq binding invariably show negative curvature due to a fall in the density of free antibody sites as the cell-bound antibody becomes saturated with Clq. Over a wide range of cell-bound antibody densities little of the cell-associated Clq is bound pentavalently or hexavalently. While Clq binding is easily inhibitable by free IgG at low levels of cell-bound antibody, this inhibition is progressively overcome by increasing the density of antibody on the cell. This reflects the inherent property of multivalent ligands such as Clq to seek out regions of high site density.     

Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS.     

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD)

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.     

Phagocytosis of target particles bearing C3b-IgG covalent complexes by human monocytes and polymorphonuclear leucocytes.

Immunoglobulin G (IgG) provides an efficient acceptor site for nascent C3b, and complement activation on the surface of IgG-coated bacteria has been shown to generate significant numbers of C3b-IgG complexes. We have studied the relative efficiency of IgG alone, C3b-IgG complexes, and similar densities of IgG and C3b residues deposited independently, in mediating ingestion of sheep erythrocyte (E) targets by human phagocytes. Human 125I-C3b covalently bound to rabbit anti-Forssman IgG was generated as described elsewhere (Fries et al., 1985). E,EIgMC4b, or EIgMC4b3b (prepared with IgM antibody and purified complement components) were sensitized with radiolabelled anti-Forssman IgG or C3b-IgG heterodimers to generate targets bearing IgG alone, C3b-IgG covalent complexes, or C3b and IgG in equivalent numbers but not bound to each other. Phagocytosis by monocytes and polymorphonuclear leucocytes (PMN) of targets bearing C3b-IgG was markedly enhanced relative to those bearing IgG alone, especially at levels of less than 2000 opsonin residues/target cell. Uptake of C3b-IgG-bearing targets was also significantly more resistant to competitive inhibition by ambient monomeric IgG. Phagocytosis of EIgMC4b + C3b-IgG by monocytes was superior to the uptake of either EAC4b + IgG or EAC4b3b + IgG bearing equivalent amounts of C3b and IgG not in covalent complex (P less than 0.05, n = 10). Similar results were obtained with PMN. Thus, generation of C3b-IgG complexes in vivo may not only promote complement activation and enhance C3b deposition, but also produce a compound opsonic residue which is a more potent promoter of phagocytosis than an equal number of C3b and IgG residues randomly distributed relative to each other.     

Studies on the mechanism of C3b inhibition of immune complex induced C1 activation

We had previously demonstrated that in normal human serum (NHS) nascent C3b inhibited C1 activation by immune complexes (IC). We have now investigated the mechanism of this feedback inhibition. For these studies, EA-IgG were added to solutions containing physiological concns of purified C1, C1-In, C2, C3 and C4. Mixtures were then incubated at 37° C for 30 min. Western blot and autoradiographic analyses revealed that almost half of the IgG molecules had become covalently linked to C3b in a 1:1 complex with the C3' chain of C3b being bound to the heavy chain of IgG. IgG-C3b and free IgG were separated by ion exchange chromatography and immune complexes were formed with each. The consumption of complement in NHS by EA-IgG and EA-(IgG-C3b) were then compared. The results indicate that binding of C3b to IgG did not significantly inhibit the C1 activating potential of the IgG. Thus feedback inhibition is not due to the binding of C3b to IgG. An alternative mechanism was next explored. After incubation of EA-IgG with C1 through C3, EA were separated from supernantant fluid by centrifugation. It was determined that one-third to one-half of the IgG had been released from the erythrocytes. Release appears not to have been due to C3b binding to IgG, since the released IgG-C3b readily bind to fresh sheep erythrocyte (E), and since IgG that was free of C3b was also released from EA by complement, it is more likely that C3b binding to the E caused the dissociation of antibody.

These results indicate that under physiological conditions, the C1 activating potential of an immune complex is greatly reduced as the result of the binding of nascent C3b to the antigen moiety of the IC, thereby causing the displacement of complement activating antibody. In addition to IgG, IgG-C3b is also released from the IC.