Stimulation of complement amplification by F(ab')(2)-containing immune complexes and naturally occurring anti-hinge antibodies, possible role in systemic inflammation

A systemic inflammatory response syndrome follows excessive complement amplification, but how complement amplification is stimulated is unknown. Immune complexes containing IgG (IgG-IC) rarely stimulate complement amplification in human plasma. IgG molecules doing so may have an affinity for C3 within their framework and therefore preferentially generate C3b(2)-IgG complexes, potent C3 convertase precursors. However, immune complexes generated from F(ab')(2) fragments of any homologous and heterologous IgG antibody (F(ab')(2)-IC), which have been studied over 35 years, stimulate complement amplification in human plasma. Stimulation of complement amplification by F(ab')(2)-IC is not simply due to the lack of the Fc portion, but unexpectedly requires a naturally occurring antibody (NAb) directed to the hinge region, exposed on F(ab')(2)-IC. Anti-hinge NAbs and bound antigen stabilize F(ab')(2), allowing dimeric C3b molecules to deposit to one of its shortened heavy chains. Complement amplification can also be stimulated by this mechanism in vivo, because neutrophilic elastase can generate F(ab')(2) from IgG molecules. The concentrations of F(ab')(2) and of factor Bb correlated linearly with that of elastase in septic patients at the onset of SIRS and F(ab')(2) fragments migrated on gel filtration columns as immune complexes, also containing anti-hinge NAbs in septic patients at the onset of SIRS.     

Quantitative immunoassay for IgA class circulating immune complexes using solid phase Facb fragment of anti-C3

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab')2 antibodies that caused false positive results when the F(ab')2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab')2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.     

Antibodies against C1q in anti-glomerular basement membrane nephritis.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.     

Parallelism of serum anti-F(ab')2 and anti-cationic IgG reactivities in patients with systemic lupus erythematosus

Cationic anti-DNA antibodies may be related to glomerular injury in murine lupus nephritis or in patients with systemic lupus erythematosus (SLE). Therefore, anti-cationic antibodies in SLE could include antibodies with regulatory function on such pathogenic cationic molecules. Since anti-F(ab')2 antibodies may be involved in the idiotype control of anti-DNA antibodies in some patients with inactive SLE, the present study was aimed to determine if SLE patients with significant serum levels of anti-F(ab')2 produce antibodies reacting with cationic IgG molecules. Three SLE sera with high titers of anti-F(ab')2 antibodies were individually adsorbed by sequential affinity chromatography on three Sepharose columns coupling normal IgG from Cohn Fraction II, pooled cationic IgG myeloma paraproteins displaying idiotypic anti-DNA markers (F4 and 8.12), and F(ab')2 fragment from allogeneic IgG, respectively. Eluates obtained from cationic IgG adsorption showed predominant anti-F(ab')2 reactivity. A similar profile was also detected in a serum from a normal control donor with high levels of anti-F(ab')2. Biotinylation of anti-cationic eluates showed that such antibodies were significantly more reactive with cationic than anionic or neutral IgG, confirming their apparent affinity for positively charged antigens on IgG molecules. Since anti-cationic absorptions were able to remove the anti-F(ab')2 activities in the SLE sera studied, it is possible that anti-cationic antibodies could function as immunoregulatory antibodies in the idiotypic control of some SLE autoreactive phenomena, including glomerular anti-DNA deposition.     

Homeostatic roles of naturally occurring antibodies: an overview

Immunoglobulins may have been developed in evolution to provide specificity for clearing body waste in the first animals with three germ layers. Tissue homeostasis in vertebrates comprises clearance of proteins released from lysed cells, elimination of altered plasma proteins, of senescent and apoptotic cells. Rather specific IgM and IgG naturally occurring antibodies (NAbs) to cytoplasmic and cytoskeletal proteins bind to proteins released from lysing cells and the IgG NAbs are slightly upregulated upon demand. Some of these NAbs along with complement have devastating effects when massive amounts of intracellular proteins are released during an infarct or an ischemia/reperfusion experiment. IgM NAbs to neoepitopes on plasma proteins/lipids help clear denatured proteins and are protective. IgG NAbs to an exposed protein, band 3 from red blood cells, bind to oligomerized band 3 and due to an affinity for C3 within their framework preferentially form C3b2-IgG complexes from nascent C3b. Thus, anti-band 3 NAbs gain potency by using avidity and generating a potent precursor of the amplifying C3 convertase. IgM NAbs to neoepitopes, which are generated by oxidized lipids forming Schiff bases with proteins, are protective and help clear this waste in atherosclerosis, but IgG antibodies (NAbs?) of the same specificity promote disease.     

Human polyclonal and monoclonal IgG and IgM complement 3 nephritic factors: evidence for idiotypic commonality

Complement 3 nephritic factors (C3NeF) were isolated from the sera of patients with membranoproliferative glomerulonephritis (MPGN) and the supernatants of pokeweed mitogen-stimulated mononuclear cells from patients with MPGN. Three human monoclonal C3NeF antibodies (two IgGs, CK and PH, and one IgM, K3C4) were established. Using an exhaustive series of affinity columns, we isolated anti-C3NeF idiotypic antibodies (anti-IdNeF) (three from normal and two from patient sera). Anti-IdNeF preparations bound to F(ab')2-NeF and prevented its ability to stabilize C3bBb convertase. We have used the above reagents to address questions on the genesis and the diversity of C3NeF antibodies. The following results were obtained: All anti-IdNeF preparations bound to C3NeF isolated from patient sera, cell culture supernatants, and IgG and IgM monoclonal C3NeF. None of the monoclonal C3NeF bound to an extensive battery of common antigens, including Fc portion of IgG, TNP, beta-galactosidase, DNA, and bacterial products. These data indicate that C3NeF express one common idiotype and that these antibodies are not raised in response to an obvious antigen.     

SLE患者血清抗F(ab′)_2片段抗体的测定及其临床意义

用酶解法制备正常人 IgG-F(ab')_2片段及 SLE 患者血清抗 ds-DNA-F(ab')_2片段,ELISA 法检测同一组(46例)SLE 患者血清中抗 F(ab')_2片段抗体,两者检测结果呈高度相关性.同时发现,缓解期 SLE 患者血清中抗 F(ab')_2片段抗体水平明显高于活动期 SLE 患者,而抗 ds-DNA 抗体水平与之相反.提示,正常人 IgG-F(ab')_2片段与 SLE 患者血清抗 ds-DNA-F(ab')_2片段可能有共同的结构表位,其抗 F(ab')_2片段抗体测定可作为临床观察 SLE 活动性的一个指标.     

Monoclonal antibodies against human anti-F(ab')2 antibodies react with light chain epitopes.

Anti-F(ab')2 antibodies affinity isolated from sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or normal SLE relatives were used to produce monoclonal antibodies (mAbs) in Balb/c and NZB mice. Four of five mAbs showed only primary light chain specificity. Only one mAb produced in an NZB mouse against anti-F(ab')2 from a single SLE patient showed anti-mu-chain specificity. Parallel identical control immunizations with IgG or a single human IgG kappa myeloma produced mAbs with a predominant gamma-chain/Fc fragment specificity. Anti-light chain specificity of mAbs was demonstrated to involve epitopes requiring tertiary structure of the entire light chain instead of antigens confined to Ckappa/lambda or Vkappa/lambda fragments. Anti-kappa specificity of three mAbs was extremely similar but not identical to that defined by anti-Km1 allotyping systems. No evidence was obtained with any of the mAbs produced for antigens unique to SLE or RA anti-F(ab')2 antibodies. The light chain antigenic prominence of many anti-F(ab')2 antibodies may reflect structural features shared by this group of immunoglobulins somehow important for their biologic function.     

Anti-C1q autoantibodies from active lupus nephritis patients could inhibit the clearance of apoptotic cells and complement classical pathway activation mediated by C1q in vitro

Anti-C1q antibodies are prevalent in patients with active lupus nephritis and were found to be closely associated with renal involvement and predictive for a flare of nephritis. However, the pathogenesis of anti-C1q antibodies involved in human lupus nephritis remains unclear. C1q, which plays a key role in apoptotic cell and immune complex removal, is a very important functional molecule in the pathogenesis of SLE. The aim of this study was to investigate the influence of anti-C1q autoantibodies from active lupus nephritis patients on the bio-functions of C1q in vitro. We purified IgG autoantibodies against C1q from lupus nephritis patients, and found that they could recognize C1q bound on early apoptotic cells at 30 μg/ml, and could significantly decrease the phagocytosis by macrophages of early apoptotic cells opsonized by 50 μg/ml C1q in comparison with normal IgG. Levels of circulating immune complexes of the ten patients were measured by a circulating immune complexes (CIC)-C1q Enzyme Immunoassay Kit. Anti-C1q autoantibodies affinity purified by microtiter plates could significantly inhibit the deposition of C3c on CIC-C1q in a dose dependent manner in comparison with IgG from 10 healthy blood donors. The binding of opsonized immune complexes to RBCs was significantly inhibited by anti-C1q autoantibodies purified by microtiter plates in a dose dependent manner. Our observations suggest that serum anti-C1q autoantibodies from active lupus nephritis patients could interfere with some biological function of C1q in vitro.     

Enhancement of human B cell proliferation by an antibody to the C3d receptor, the gp 140 molecule

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.     

Human Anti-F(ab'')2 Antibodies and Pepsin Agglutinators React with Fv Determinants

Affinity-purified human IgG anti-tetanus antibody was subjected to papain and then pepsin digestion, and residual fragments retaining antibody activity were re-isolated by adsorption and elution from Sepharose-tetanus toxoid columns. Both Fab' and Fv fragments isolated by gel filtration showed strong reactivity with anti-F(ab')2 antibodies. Failure of tetanus toxoid to completely block reactivity of anti-F(ab')2 antibody with anti-tetanus Fv fragments indicates that these antibodies react with framework antigens within variable antibody regions.     

Mechanism of activation of the classical pathway of complement by monoclonal IgE (DES). Restricted regulation of C4b by C4b-binding protein

A human monoclonal IgE from patient DES, IgE (DES), has been shown to activate the classical pathway of complement. The mechanism of this activation has been investigated and can be summarized as follows: (a) IgE (DES) is able to bind and activate C1 in a dose-dependent fashion. This activation increases with the size of the aggregates used, but the affinity of C1 for IgE (DES) is weaker than for IgG. (b) A classical pathway C3 convertase can be assembled on IgE (DES) using purified C1, C4 and C2. The formation decay of this convertase is similar to that formed on IgG with an half-life of 9 min at 37 degrees C. (c) The extrinsic regulation of the C3 convertase by C4bp is restricted on IgE (DES) as compared to IgG. This restriction is shown on both the formation and the decay of the convertase. The mechanism of activation of the classical pathway of complement by IgE (DES) thus present some similarities with the assembly of the C3 convertase by the alternative pathway.     

In vitro inhibition of anti-DNA producing cells from systemic lupus erythematosus patients by autologous anti-F(ab')2 antibodies

We have previously documented an inverse relationship between serum levels of anti-F(ab')2 antibodies and disease activity in systemic lupus erythematosus (SLE). The present study focused on the specific in vitro inhibition of anti-DNA producing cells from SLE patients by autologous anti-F(ab')2 antibodies. Peripheral blood lymphocytes (PBL) from eleven inactive SLE patients with no apparent disease activity were cultured in vitro to evaluate anti-DNA antibody secretion. Low levels of synthesis of anti-DNA antibody were detected in 3 of 11 SLE patients using unstimulated PBL; on the contrary, pokeweed mitogen stimulation of cultured cells increased production of anti-DNA in all SLE subjects. Parallel cultures were also performed in the presence of heterologous and autologous anti-F(ab')2 antibodies and results on production of anti-DNA evaluated. Lymphocytes from SLE patients in remission showed inhibition of synthesis of anti-DNA antibodies when autologous anti-F(ab')2 antibodies were added to the cultures, whereas production of anti-tetanus toxoid IgG by the same cells was not significantly altered under the same conditions. These data suggest that a functional anti-idiotypic role may be assigned to anti-F(ab')2 antibodies during clinical remission of SLE.     

Immune deposition of C1q and anti-C1q antibodies in the kidney is dependent on the presence of glomerular IgG

Anti-C1q autoantibodies are found frequently in patients with Systemic Lupus Erythematosus (SLE) and several studies indicate that these autoantibodies are associated with renal involvement. We have shown earlier that administration of anti-C1q antibodies to normal BALB/c mice results in the deposition of these antibodies and C1q in the kidney. In the present study we have investigated which factors are essential for this C1q-anti-C1q deposition. Injection of anti-C1q antibodies in C57BL/6 mice results in deposition of both C1q and anti-C1q in glomeruli, while administration of equal concentrations of anti-C1q to immunoglobulin deficient Rag2-/- mice did not result in deposition of anti-C1q antibodies. Analysis of renal sections of naive Rag2-/- mice revealed absence of mouse IgG and C1q in the glomeruli, while circulating C1q was within normal levels. Reconstitution of Rag2-/- mice with IgG, either by injection with purified mouse IgG or by splenocyte transfer, resulted in restored localization of mouse IgG together with C1q in the kidney. Subsequent injection of anti-C1q antibodies in these IgG reconstituted mice resulted in clear deposition of C1q together with anti-C1q in the kidneys comparable to that found in C57BL/6 mice receiving anti-C1q. We propose that the continuous presence of serum derived non-immune IgG in the glomerulus serves as a target for low affinity interactions with C1q, which then can serve as antigen for anti-C1q antibodies. Therefore we hypothesize that high and fluctuating levels of IgG as observed in patients with SLE may contribute to flares of renal inflammation in those patients with anti-C1q autoantibodies.     

Characterization of C1q-binding IgG complexes in systemic lupus erythematosus

The molecular size of C1q-binding immunoglobulin (Ig) G complexes in systemic lupus erythematosus (SLE) sera was studied by gel filtration using C1q solid-phase radioimmunoassay (C1q SPRIA). All 15 SLE sera tested contained predominantly small-sized IgG complexes, cofractionating with monomeric IgG. In contrast to heat-aggregated IgG, these small-sized IgG complexes retained C1q-binding activity even after pepsin digestion, exposure to low pH, or reduction and alkylation, suggesting that the F(ab')2 region is involved in C1q-binding activity of these complexes. To see whether anti-C1q antibodies or small antigen-IgG complexes, which bind to C1q via their antigens, are responsible for C1q-binding activity via the F(ab')2 region, the pepsin-digested Ig fractions of SLE sera were fractionated at high salt. C1q-binding activity in the fractions corresponding to the F(ab')2 region increased 2.5- to 3.9-fold at high salt. These results suggest that the C1q-binding, small-sized IgG complexes may be comprised mostly of anti-C1q antibodies and that some of the antibodies, which are dissociated with their antigens at high salt, might be cross-reactive with C1q.     

C1q autoantibodies in HIV infection: correlation to elevated levels of autoantibodies against 60-kDa heat-shock proteins

Antibodies to solid phase C1q (C1qAb) were determined in 295 serum samples from 132 HIV-infected subjects and in sera from 140 HIV-seronegative healthy individuals as control. An ELISA method applied for the determination of C1qAb in other diseases was used. In part of these sera, other autoantibodies (antibodies reacting with 60-kDa human heat shock protein (hsp60) or mycobacterial hsp65; IgA and IgG class antibodies against the Fab and F(ab')2 moieties of IgG) as well as complement-mediated antibody-dependent enhancement/neutralization (C'-ADE) were also determined. Increased amount of C1qAb was found in HIV-infected subjects as compared with HIV-seronegative controls (P = 0.0138). In 17 of 132 (13.0%) seropositive individuals but only in 7/140 (5.0%) samples from the controls, the amount of C1qAb exceeded the upper limit (95th percentile) of the normal values (P = 0.031). The amount of C1qAb significantly decreased during a follow-up period of 65 months. C1qAb levels were found to strongly correlate to hsp60/65 autoantibodies but did not correlate or only weakly correlated to the amount of anti-Fab or anti-F(ab')2 autoantibodies measured in the same serum samples. Anti-C1q antibodies recognized the solid phase hsp60/65. Three predicted epitope regions of M. paratuberculosis hsp65 were able to bind efficiently C1q antibodies. An inverse correlation was found between C1qAb and C'-ADE, neutralization was more frequent in the sera with detectable C1qAb, whereas sera without C1qAb more likely enhanced HIV infection in vitro.     

Detection of circulating immune complexes with polyethylene glycol precipitation F(ab')2 anti-C3 ELISA

A polyethylene glycol (PEG) precipitation F(ab')2 anti-C3 ELISA for the detection of complement-fixing IgG circulating immune complexes (CIC) is described. For this assay, test sera were treated with 3.5% PEG and then measured with F(ab')2 anti-C3 ELISA. The lower detection limit was 4 micrograms/ml of heat aggregated human IgG (HAHG). Intra-assay coefficient of variation (CV) was 4.9-8.3%. High levels of CIC are found in the sera of patients with systemic lupus erythematosus (SLE), hepatitis B and stomach cancer.     

Endocytosis of the C3b receptor of complement within coated pits in human polymorphonuclear leukocytes and monocytes

The distribution and endocytosis of the C3b receptor by human polymorphonuclear leukocytes and monocytes were visualized by both fluorescent and electron microscopic examination of cells that had been labeled with monospecific F(ab')2 anti-C3b receptor and anti-F(ab')2 conjugated with rhodamine or ferritin. When prefixed or unfixed cells that were labeled at 0 to 4 degrees C were examined, the receptor was distributed within clusters on the plasma membrane. After the cells had been warmed to room temperature or to 37 degrees C for 5 minutes, the fluorescently labeled receptors appeared to enter the cells, and the ferritin-tagged receptors often occurred within coated endocytic pits and coated vesicles within the cytoplasm. After incubation at 37 degrees C for 20 minutes, the C3b receptor-antibody complexes were largely cleared from the cell surface, and much of the label was found within lysosomes. These results indicate that C3b receptors may directly mediate endocytosis within coated pits, thus utilizing a mechanism shared by a variety of other receptors for the rapid, efficient, and selective internalization of extracellular ligands.     

Specificity of IgM Antibodies to Pooled Human F(ab')2 Fragments

An isotope specific immunoassay which minimizes interference by endogenous rheumatoid factors was used to determine the specificity of IgM anti-F(ab')₂ antibodies in human serum. We underscore the heterogeneity of these antibodies. While one subset of IgM anti-F(ab')₂ antibodies reacts only with intact F(ab')₂, another recognizes determinants present following reduction and alkylation of F(ab')₂ and separation of Fd' fragments from light chains. IgM anti-F(ab')₂ antibodies in sera from rheumatoid arthritis patients do not react significantly with intact pooled IgG and, therefore, probably are not anti-idiotypic antibodies. Some sera, but not all, contain elevated levels of antibodies that are crossreactive with rabbit F(ab')₂. Such crossreactive antibodies may interfere with assays which utilize F(ab')₂ fragments of rabbit antibodies specific for antigens of clinical relevance.     

Suppression of anti-erythrocyte autoantibody-producing B cells by a physiological IgG-anti-F(ab')2 antibody and escape from suppression by tumour transformation; a model relevant for the pathogenesis of autoimmune haemolytic anaemia.

We showed previously that broadly reactive IgG anti-immunoglobulin autoantibodies produced by rats during the immune response suppress the B cell response. We report here on the effect of a similar human antibody on self-reactive human B cells. IgG anti-F(ab')2 was added to cultures of anti-erythrocyte autoantibody-producing B cells derived from healthy donors. A dose-dependent suppression of the antibody response was obtained (maximum at 1.3 ng IgG/10(6) cells). This effect was competitively inhibited by F(ab')2 gamma. Autoimmune haemolytic anaemia can be caused by chronic monoclonal B cell proliferation. To reproduce this condition in vitro we immortalized B cells with Epstein-Barr virus (EBV) and raised a B cell population with anti-erythrocyte autoantibody activity. These cells were electrically fused with CB-F7 tumour cells and an IgG1 cold-reactive anti-erythrocyte autoantibody-producing B cell line was established. Surprisingly, the tumour cells were not suppressed by IgG anti-F(ab')2. It is known that anti-immunoglobulins selectively suppress antigen-receptor (AgR)-occupied B cells by a Fc gamma-receptor (Fc gamma R)-mediated mechanism. To occupy their AgR, we preincubated the tumour cells with anti-AgR antibody. In spite of this, their susceptibility to suppression was not restored. As shown by rabbit IgG-sensitized ox erythrocyte (EA)-rosetting, this refractoriness was not due to a loss of Fc gamma R. Our experiments delineate a mechanism of peripheral B cell suppression to autoantigens, and show a way of escape from control relevant for the pathogenesis of autoimmune haemolytic anaemia.