The reaction between the complement subcomponent C1q, IgG complexes and polyionic molecules.

The strength of the bond between 125I-labelled C1q and immune complexes, Fc piece, dextran sulphate, polyglutamic acid and polylysine has been investigated. The binding of C1q to Fc piece, small molecular weight (less than 10,000) dextran sulphate, polyglutamic acid and polylysine have value; for the functional affinity constant (Ko) in the range of 0.2-1.5 X 10(4) M-1. In contrast the binding of C1q to immune complexes and large molecular weight polyions (greater than 100,000 is much greater and lies in the range 3 X 10(7)--4 X 10(8) M-1. The differences in the binding constants between the two groups can be explained if the Fc piece and small molecular weight compounds bind to only 1 head of the C1q molecule but the immune complexes and large molecules bind to 2 heads. There are probably 6 binding sites on the C1q molecule for dextran sulphate. The enhancement of the binding affinity of C1q by reduction in ionic strength and the reaction with polyions, indicate that ionic groups are present near or within the binding sites.     

Iron ions and haeme modulate the binding properties of complement subcomponent C1q and of immunoglobulins

The complement system and circulating antibodies play a major role in the defence against infection. They act at the sites of inflammation, where the harsh microenvironment and the oxidative stress lead to the release of free iron ions and haeme. The aim of this study was to analyse the consequences of the exposure of C1q and immunoglobulins to iron ions or haeme. The changes in target recognition by C1q and in the rheumatoid factor activity of the immunoglobulins were investigated. The exposure of C1q to ferrous ions increased its binding to IgG and to IgM. In contrast, haeme inhibited C1q binding to all studied targets, especially to IgG1 and C-reactive protein. Thus, the haeme released as a result of tissue damage and oxidative stress may act as a negative feedback regulator of an inappropriate complement triggering as seen in ischaemia-reperfusion tissue injury. The results also show that iron ions and haeme were able to reveal rheumatoid factor activity of IgG. The modulation of the C1q-target binding as well as the revealing of rheumatoid factor activity of IgG by exposure to redox-active agents released at the sites of inflammation may have important consequences for the understanding of the immunopathological mechanisms of inflammatory and autoimmune diseases.     

C1q binding to human vascular smooth muscle cells mediates immune complex deposition and superoxide generation

Evidence was obtained for the binding of Clq to the membrane of cultured vascular smooth muscle cells derived from human umbilical cord veins. Clq was fixed to the cell membrane at 4°C, whereas it was ingested into the cytoplasm, as a cytoplasmic inclusion, when tested at 37°C. The addition of Clq in advance inhibited the subsequent binding of Clq. Neither fibronectin nor laminin was detected on the cell membrane. Aggregated IgG bound to vascular smooth muscle cells in the case of preincubation with Clq at 4°C, whereas aggregated IgG did not bind to the cells in the absence of Clq. The addition of Clq molecules to the cells in suspension enhanced Superoxide generation by vascular smooth muscle cells. There was no effect of Clq on Superoxide generation by the cells in monolayer. These results suggest that Clq binds on the membrane of vascular smooth muscle cells via its specific receptor that mediates immune complex binding to the cells and Superoxide generation. These properties elucidate the mechanisms by which circulating immune complexes deposit in the vascular wall, and subsequent degradation of tissue components surrounding vascular smooth muscle cells occurs through oxidative burst of the cells.     

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.     

Effects of soluble aggregates of IgG on the binding, uptake and degradation of the C1q subcomponent of complement by adherent guinea pig peritoneal macrophages

Earlier studies have indicated that C1q, the first subcomponent of complement component C1, is bound to lymphocytes via specific C1q receptor sites. We have recently shown that adherent guinea pig peritoneal exudate macrophages express specific receptors for C1q (Veerhuis, R. et al., Immunology 1985. 54: 801). The present studies were performed to determine whether binding of 125I-labeled human C1q (125I-C1qhu) to adherent guinea pig peritoneal exudate macrophages would also result in ingestion and subsequent degradation of 125I-C1qhu. The binding of 125I-C1qhu to adherent peritoneal macrophages at 4 degrees C is inhibited fully not only by C1qhu and guinea pig C1q (C1qgp) but also by pepsin fragments of C1qhu. The amount of trichloroacetic acid nonprecipitable radioactivity that appeared in the supernatant was used as a measure for the degradation of 125I-C1qhu. 125I-C1qhu is degraded initially into fragments of 25 kDa, after which it is degraded further into small molecular weight peptides. Ingestion of 125I-C1q by the macrophages occurs before the 125I-C1q is degraded. In the presence of limited amounts of soluble aggregates of guinea pig IgG2 (AIgG), a known activator of C1, part of the C1q is bound to the AIgG and all of the AIgG in turn is bound to the cellular Fc receptors leading to an enhanced binding of 125I-C1q to the cells, a binding that was maximal at near equimolar concentrations of 125I-C1qhu and 131I-AIgG. In the presence of a 30-fold excess of AIgG, however, only a small percentage of the AIgG binds to cellular Fc receptors and the interaction of C1q with its receptor is decreased due to competitive inhibition. The results presented in this report thus suggest that free C1q may be eliminated by specific interaction with C1q receptors present on circulating and tissue phagocytoses and, in addition, that in the presence of immune complexes modulation of elimination of C1q may be encountered.     

Enhancement of the binding of C1q to immune complexes by polyethylene glycol

The binding of 125I-labelled human C1q to insoluble rabbit IgG:ovalbumin immune complexes was enhanced by polyethylene glycol (PEG, Mr 8 x 10(3)) in the concn range 0-2.5% (w/v). C1q with native immunoglobulin bindings sites rendered inactive by diethylpyrocarbonate treatment did not bind to immune complexes in the presence of PEG. The ionic strength dependence of the binding was independent of the presence of PEG. There was a linear relationship between the logarithm of the apparent affinity constant of the C1q:immune complex interaction and PEG concn.     

Direct binding of C1q to apoptotic cells and cell blebs induces complement activation

Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus-like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell-derived blebs. In addition, for the first time, we demonstrate that surface-bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.     

The subcomponent C1q of the first component of guinea pig complement: Purification and characterization

Guinea pig C1q was purified, in a highly active hemolytic form, by a combination of precipitation with chelating agents, CM-cellulose and Sepharose 6B. Yields ranged from 30 to 35% protein, and the activity of final preparations was in the range of 2 × 10¹³– 3 × 10¹³ C1q effective molecules/mg. The molecular weight of C1q was approximately 430,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). C1q was shown to be composed of two non-covalently linked subunits of approximate molecular weights 46,500 and 45,000 in a molar ratio 2 : 1. On reduction, the higher molecular weight subunit gave two chains having approximate molecular weights of 24,500 and 23,000 in equimolar ratio, and the lower weight subunit gave one chain with a molecular weight of approximately 22,300. C1q contained hydroxyproline, hydroxylysine and a high percentage of glycine. Thus, the overall molecular structure of guinea pig C1q appears similar to that of human C1q.The antiserum against the purified C1q showed only one precipitation band with guinea pig whole serum of purified C1q on immunodiffusion analyses and was found to be monospecific.     

Region of the C1q molecule involved in the interaction between platelets and subcomponent C1q of the first component of complement

The interaction between platelets and collagen can be inhibited by intact C1q or by a large, collagen-like fragment derived from C1q by limited proteolysis with pepsin (Cazenave et al, 1976; Wautier et al., 1977). C1q is composed of 6A, 6B and 6C polypeptide chains, each of which is approximately 200 amino acid residues long and contains collagen-like regions of approximately 80 amino acid residues (Reid, 1979). Samples containing all, or portions, of the collagen-like regions from each of the three types of polypeptide chains were obtained by either separation of the A, B and C chains after performic oxidation, or by the isolation of large peptides after cyanogen bromide digestion of the intact molecule. These samples were then tested for their ability to inhibit platelet adhesion and platelet aggregation in the presence of type I and type III collagen. It appears probable that the A chain of C1q contains the collagen-like amino acid sequences which are important in the interaction between C1q and platelets.     

Kinetics of the biosynthesis of complement subcomponent C1q by murine macrophages: LPS, immune complexes, and zymosan alone and in combination with interferon-gamma.

We investigated the effects of bacterial lipopolysaccharide (LPS), immune complexes (IC), and C3b opsonized zymosan (AZ) alone and in combination with interferon-gamma (IFN-gamma) priming on macrophage synthesis and secretion of C1q. Our results indicated that LPS, IC, and AZ alone stimulated C1q mRNA and secretion in the absence of IFN-gamma. The increase in mRNA accumulation was detectable after 3 h, peaked at 6 h and was maintained at constitutive levels for 24 h. There was a corresponding early burst of increased secretion of functional C1q after 3 to 6 h which declined rapidly after 9 to 24 h culture of LPS-stimulated macrophages. Priming of macrophages with IFN-gamma and simultaneous triggering with LPS, IC, or AZ produced additive rather than synergistic increases in C1q mRNA accumulation. These same agents inhibited constitutive secretion of C1q in the absence of IFN-gamma priming as determined by autoradiographic analysis of metabolically radiolabeled secretory C1q. Triggering of IFN-gamma primed macrophages with LPS, IC, or AZ also markedly suppressed the increased rate of C1q secretion induced by IFN-gamma in a dose-related fashion. A corresponding dose-dependent increased accumulation of endogenous C1q in cell lysates was detected by Western blot analysis of macrophages which had been stimulated by LPS, IC, or AZ alone or in combination with IFN-gamma. Our findings indicate that LPS as well as FcR and C3bR triggering agents stimulate early and sustained C1q synthesis accompanied by an early and short-lived burst of C1q secretion which rapidly diminished and results in an increased intracellular accumulation of C1q due to ongoing synthesis. IFN-gamma appeared to further amplify the same kinetics of increased C1q mRNA accumulation and decreased extracellular accumulation mediated by LPS, IC, and ZM. Our results suggest that LPS, IC, and AZ alone or in combination with IFN-gamma stimulate early C1q production to modulate macrophage effector functions followed by an inhibition of C1q secretion when the activation process has been culminated.     

The use of [125I]C1q subcomponent for the measurement of complement binding antibodies on cell surfaces.

An assay using 125I-labelled human C1q has been developed for the measurement of complement fixing antibodies bound to cell monolayers or cell suspensions. The method has been adapted for use either during or after sensitisation of the cells with antiserum, is simple to perform and does not require require prelabelling of the target cells.     

Antibody-independent binding of the first component of complement (C1) and its subcomponent C1q to the S and R forms of Salmonella minnesota.

Strong bactericidal effects of normal guinea pig and human sera against the Salmonella minnesota S form and an R form (Re) depend on Ca2+, complement component C4, and subcomponent C1q of complement component C1. Therefore, the interaction of C1 and C1q with these forms was investigated. The bacteria directly bound subcomponent C1q, as demonstrated by fixation and transfer tests and by fluorescent methods. Binding of macromolecular C1 was shown by fixation and transfer tests and by C4 consumption. C1 fixation and transfer tests provide evidence that C1 and C1q were bound more tightly to the Re form than to the S form. At physiological ionic strength, all cell-bound molecules were released from the S form, whereas at least 60% remained on the cell surface of the Re form. The Re form showed another binding behavior for C1: preincubation of bacteria with purified C1q totally prevented C1 uptake by the S form, compared to only 10% inhibition of the uptake by the Re form. Therefore, we conclude that macromolecular C1 is bound differently by the S form than by the Re form. The analysis of five other core-deficient mutants of S. minnesota (Ra, Rb, Rc, Rd1, and Rd2) revealed that the difference could be explained by a deficiency of the O-specific polysaccharide. In contrast, all the C1q bound to Ra, Rb, and Rc mutants was detectable by the transfer test. Therefore, we postulate that binding of macromolecular C1 to these mutants must be due to an additional C1 subcomponent besides C1q.     

C1q binding and Raji immune complex assays: a comparison using defined immunoglobulin aggregates

Aggregated IgG is frequently employed as a standard in systems for the measurement of immune complexes in man and animals. In this paper aggregates prepared by heat or alkali denaturation of human IgG were fractionated by column chromatography through LKB AcA 22 Ultrogel. Heat aggregation yields preparations containing considerably more monomer than alkali treatment (47% and 6.3% respectively). The bulk of aggregated material prepared by both methods was of size 19 S or greater. Smaller aggregates were present in assayable quantities only in the alkali aggregated material. The sized fractions of aggregates IgG were tested in the presence of a human complement source for their efficiency in the C1q binding and Raji radioimmunoassay for immune complexes. Both techniques efficiently measured large aggregates (greater than or equal to 19 S) but the C1q binding assay measured smaller material with greater efficiency than did the Raji cell assay. Neither technique detected monomeric IgG. The date presented is relevant to the binding characteristics of the 2 assay systems studied and suggests that when used together they are capable of measuring immune complexes present over a wide range of sizes.     

Electron microscopic study showing antibody-independent binding of C1q, a subcomponent of the first component of complement, to serum-sensitive salmonellae.

Effective serum-mediated killing of sensitive gram-negative bacteria requires all the complement components. In the preimmune phase the antibody-independent interaction of the first component of complement, C1, with the bacteria might be especially important. Electron microscopic studies showed that the C1 subcomponent C1q binds only to the serum-sensitive R form of Salmonella minnesota and not to the serum-resistant S form.     

C1q binding and complement activation by prions and amyloids

C1q binds to many non-self and altered-self-materials. These include microorganisms, immune complexes, apoptotic and necrotic cells and their breakdown products, and amyloids. C1q binding to amyloid fibrils found as extracellular deposits in tissues, and subsequent complement activation are involved in the pathology of several amyloid diseases, such as Alzheimer's disease. Prion diseases, such as scrapie also involve formation of amyloid by polymerization of the host prion protein (PrP). Complement activation is likely to contribute to neuronal damage in the end stages of prion diseases, but is also thought to participate in the initial infection, dissemination and replication stages. Infectious prion particles are likely to bind C1q and activate the complement system. Bound complement proteins may then influence the uptake and transport of prion particles by dendritic cells (DCs) and their subsequent proliferation at sites such as follicular DCs.     

Precipitin reactions of the C1q component of complement with aggregated gamma-globulin and immune complexes in gel diffusion

A gel diffusion method for demonstrating precipitin reactions of C1q with aggregated γ-globulin and immune complexes is described. Optimal precipitin lines were found to occur with 0.6 per cent agarose in 0.01 M EDTA at pH 7.2 and ionic strength 0.1. Reduction and alkylation of γ-globulin aggregates destroyed the precipitability with C1q. Precipitation occurred only with aggregates greater than 19S and with soluble immune complexes formed in two to twenty times antigen excess. γ-Globulin complexes were detected by this procedure in hypocomplementaemic sera from patients with systemic lupus erythematosus and hypocomplementaemic joint fluids from patients with rheumatoid arthritis. The relevance of this in vitro system to in vivo complement consumption in various disease states is discussed.     

In vivo reduction of circulating C1q binding immune complexes by intravenous gammaglobulin administration

Six patients with systemic lupus erythematosus were treated with high-dose intravenous gammaglobulin. Immunological parameters were studied and included solid-phase immune complex determinations, quantitative immunoglobulins G, A, and M, as well as C3 and C4 concentrations. Pretreatment values of circulating immune complex concentrations as measured by either C1q binding or anti-C3 binding assays were elevated in all patients. Posttreatment values showed reductions in all C1q binding immune complexes (p less than 0.01) and anti-C3 binding immune complexes also decreased in 5 out of 6 patients. These assays are described in detail and were also used to define in vitro interactions between the intravenous gammaglobulin preparation and heat-aggregated IgG or sera containing elevated circulating immune complexes. No reduction of immune complex levels were observed when IgG was incubated in vitro with either heat-aggregated IgG or sera with elevated immune complex concentrations. The duration of the in vivo effect and the patients' clinical responses are described. These findings show that high-dose intravenous gammaglobulin administration can reduce certain types of immune complexes in patients with elevated levels of these substances.     

Nucleosomes and C1q bound to glomerular endothelial cells serve as targets for autoantibodies and determine complement activation

Various studies indicate a role for both anti-nucleosome and anti-C1q autoantibodies in glomerulonephritis in patients with systemic lupus erythematosus. However, a causal relationship between these autoantibodies and the development of lupus nephritis has not been fully established. Since injury of the endothelium is a major target in lupus nephritis we assessed the interaction of C1q and nucleosomes with glomerular endothelial cells in vitro in the presence or absence of autoantibodies against these antigens. We demonstrate a direct and dose-dependent binding of both nucleosomes and C1q to immortalized human glomerular endothelial cells (GEnC) in vitro, which in part is mediated by cell surface heparan sulfate. We demonstrate that nucleosomes and C1q serve as targets for monoclonal and polyclonal antibodies as well as for anti-nuclear autoantibodies from patients with systemic lupus erythematosus. An additive effect of anti-C1q autoantibodies on anti-nucleosome mediated complement activation was observed. Furthermore, we showed that the activation of complement on glomerular endothelial cells is mediated by the classical pathway since the deposition of C3 on GEnC is abrogated by MgEGTA and does not occur in C1q-depleted serum. Taken together, our studies demonstrate a direct binding of both nucleosomes and C1q to glomerular endothelial cells in vitro. The subsequent binding of autoantibodies against nucleosomes in patients with systemic lupus erythematosus is potentially pathogenic and autoantibodies against C1q seem to have an additional effect.     

DNA levels in circulating immune complexes decrease at severe SLE flares-correlation with complement component C1q.

The objective of this study was to investigate the relationship of DNA content in circulating immune complexes with disease course and activity in SLE. The DNA content in circulating immune complexes containing anti-DNA antibodies of IgG class was determined in serial samples from 28 patients with SLE by a quantitative immunochemical assay. The patients presented various active disease manifestations over 5-55 months. Disease activity (SLEDAI-score), drug treatment and ACR-criteria were recorded. Levels of anti-dsDNA, CRP, leukocytes, complement components C3, C4 and C1q were measured. Patients with severe flares and high SLEDAI scores had low Clq levels at onset of active disease manifestations. The patients with low C1q serum levels during flare (n=13) had significantly lower amounts of DNA in immune complexes than patients with normal Clq (P=0.001). Levels of DNA in immune complexes correlated with Clq at flares (r=0.62, P<0.0001) and correlated inversely with SLEDAI scores (r=-0.47, P=0.012). In conclusion, the low levels of DNA in circulating immune complexes found in severely ill SLE patients with concomitantly low serum concentrations of Clq prior to flares might be related to tissue deposition of immune complexes.