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Affinity of IgG to the first complement factor C1q was found out to increase in 10-30% glycol solutions. Analytical ultracentrifugation and turbidity data showed that IgG molecules do not aggregate at such concentrations of glycol. The complement-binding effect may be caused by a conformational transition in the IgG molecules.
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Collectins are a group of soluble proteins, each of which has collagenous and non-collagenous globular domains, and may be complement associated and/or have lectin activity. The complement protein C1q is structurally and functionally related to the collectins. Members of the collectin family are mannan-binding protein (MBP), lung surfactant protein A, CL-43, conglutinin and lung surfactant protein D. All except the last of these proteins have been shown to interact with a single, widely distributed receptor, named collectin receptor (also called C1q receptor). It has been proposed that collectins play an important role as opsonins in innate immunity. MBP is a serum protein and has been shown to activate the classical complement pathway. MBP has also been shown to opsonize bacteria and enhance clearance of bacteria by phagocytosis. In this article we demonstrate the presence of MBP in amniotic fluid and the localization of collectin receptor on the surface of amniotic epithelium. MBP levels in amniotic fluid were found to increase sharply from about 32 weeks of gestation. Collectin receptor was purified from a cell line, FL, derived from amniotic epithelium and was shown to interact with MBP. These results suggest that MBP may play a role in the antibody-independent recognition and clearance of pathogens in the amniotic cavity, towards term.
In this report we have investigated the association of fibronectin with antigen-antibody-C1q complexes incubated in fibronectin-depleted and C1q-depleted plasma. When BSA--anti-BSA immune aggregates are incubated in plasma depleted of both fibronectin and C1q to which 125I-fibronectin has been reconstituted, little radio-activity is bound to the immune complexes. However, pre-incubation of immune complexes with purified C1q prior to incubation in the plasma causes an approximately 10-fold increase in the amount of radioactivity bound. The binding of 125I-fibronectin to preformed antigen-antibody-C1q complexes is specific, since the reaction is inhibited by the addition of unlabelled fibronectin but not by ovalbumin. When antigen-antibody-C1q complexes are incubated in C1q-depleted plasma containing physiological concentrations of fibronectin, and analysed by immunoblotting, fibronectin antigens are detected on the immune complexes. Identical results are obtained using immune complexes composed of sheep erythrocyte rabbit anti-sheep erythrocyte C1q (EAC1q) cells. There is no specific requirement for preformed antigen-antibody-C1q complexes, since fibronectin can be detected on antigen-antibody complexes after incubation in normal human serum or in C1q-depleted ethylenediamineteraacetic acid (EDTA) serum reconstituted with purified C1q prior to incubation with the complexes. Finally, we also demonstrate that in the presence of C1q, 125I-fibronectin will associate with soluble antigen-antibody complexes.
To characterize the mechanisms of complement activation by human immunodeficiency virus type 1 (HIV-1)-infected cells, Cl-4 cells stably expressing the envelope glycoproteins of HIV-1 and the parent African green monkey cell line CV-1 were tested for C1q binding and complement activation. While the parent cell line CV-1 only showed a weak spontaneous activation of the alternative pathway, Cl-4 cells additionally triggered the classical pathway of complement activation independent of anti-HIV antibodies by direct C1q binding. Earlier studies had shown different complement activating potential of cells infected with various HIV isolates. Recombinant soluble CD4-induced shedding of gp120 from the surface of HIV-1-infected cells converted a weak activator isolate (MVP-899) into a strong complement activator. The increase in complement activation was paralleled by the concomitant unmasking of a previously hidden gp41 epitope comprising the major complement-activating domain of gp41 (aa. 601-613). Our results strongly suggest that the transmembrane protein gp41 induces the activation of complement on the surface of infected cells as has been described previously for purified HIV-1 virions. Furthermore, we present evidence that the different potential of HIV isolates to activate the complement system on the cell surface is caused by different degrees of spontaneous gp120 shedding by various HIV isolates.
SLE is a disease characterized by the presence of multiple autoantibodies and high levels of circulating immune complexes. We studied the presence and functional relevance of autoantibodies directed against a receptor for the collagen-like stalks of the first subcomponent of complement, also known as calreticulin (cC1qR/CaR), in patients with SLE. In a cross-sectional study it was found that higher titres of antibodies against cC1qR/CaR are present in sera of SLE patients compared with normal donors. No association between anti-cC1qR/CaR titres and SLE disease activity was found. Following gel filtration of SLE serum it was found that anti-cC1qR/CaR reactivity is associated with the peak of monomeric IgG. Purified IgG from patients was able to specifically immunoprecipitate cC1qR/CaR. Since we have shown previously that cC1qR/CaR is able to inhibit the haemolytic activity of Clq, we determined a possible pathogenic role for anti-cC1qR/CaR on complement regulation. IgG derived from SLE serum reversed the inhibitory capacity of cC1qR/CaR in a dose-dependent fashion up to 63%, whereas IgG from normal donors had no significant effect. With respect to the capacity of anti-cC1qR/CaR antibodies to activate neutrophils, it was found that incubation of normal neutrophils with F(ab')2 anti-cC1qR/CaR resulted in a very limited oxidative burst. However, cross-linking of F(ab')2 anti-cC1qR/CaR on the neutrophils clearly induced neutrophil activation. Pre-incubation of the SLE-derived F(ab')2 with cC1qR/CaR prevented activation of neutrophils up to 81+/-5%. These results suggest that the presence of anti-cC1qR/CaR antibodies in patients with SLE may modulate complement and neutrophil activation.
The amyloid plaque is the hallmark of Alzheimer's disease (AD). The transmembrane domain and a portion of the C-terminus (A beta) of the amyloid precursor protein, are known to form the nucleus of the amyloid plaque. It has been demonstrated recently, using in vitro assays, that the A beta peptide can activate both the classical (antibody-independent) and alternate pathways of complement activation. The proposed complement activation is due to the binding of A beta to the complement components C1q and C3, respectively, which initiate formation of the proinflammatory C5a and C5b-9 membrane attack complex. In this report, we have investigated the in vitro findings for the likely complement-dependent proinflammatory properties of the Alzheimer's disease A beta peptide. We have performed experiments using congenic C5-deficient and C5-sufficient mice injected with synthetic A beta and recombinant polypeptide (C-100) containing A beta. Injection of C-100 into C5-sufficient mice induced a clear increase in the number of polymorphonuclear cells (neutrophils) at the site of injection due to complement activation and the subsequent release of proinflammatory chemtoactic factors. In sharp contrast, the C5-deficient mice did not show any increase in cellular influx. The vaccinia virus complement control protein, an inhibitor of both the classical and alternate pathway can down-regulate the biologically significant activation of complement by A beta, as demonstrated by an in vitro immunassay. The therapeutic down-regulation of A beta-caused complement activation could greatly alleviate the progression of some of the chronic neurodegeneration characteristic of Alzheimer's disease.
We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.
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A possible role for C1q in antibody-dependent granulocyte-mediated killing of nonphagocytosable targets was investigated utilizing IgG-dependent granulocyte cytotoxicity directed against microfilariae of Dirofilaria immitis. Granulocyte-mediated killing of microfilariae is enhanced by addition of fresh serum. Lack of C4 did not significantly reduce the observed increase in cytotoxicity. The addition of highly purified monomeric human Clq (0.2 microgram/ml) in the presence of immune IgG resulted in a two- to fivefold enhancement of killing (P less than 0.025). C1q enhancement of killing occurred in the absence of fluid-phase IgG, but killing was significantly less than when both fluid-phase IgG and C1q were present. The effect of C1q was inhibited by the addition of solubilized type I collagen (44-92% inhibition of killing, P less than 0.05). Significant 125I-Clq binding to microfilariae occurred only in the presence of immune IgG. In addition, C1q in concentrations ranging from 0.5 to 2.0 micrograms/ml resulted in a dose-dependent increase in binding of 125I-immune IgG to microfilariae. Finally, when purified C1q was added to preopsonized, washed microfilariae, granulocyte production of superoxide was increased from 0.25 +/- 0.07 to 0.68 +/- 0.07 nm/10(6) cells.10 min (P less than 0.01). These results describe a novel functional role for C1q in enhancement of antibody-dependent cellular cytotoxicity towards nonphagocytosable targets.
Although the existence and ubiquity of the C1q receptor is well established, its role in health and disease is largely unknown. Interleukin 1 (IL 1) is the major immunoregulatory molecule produced by macrophages and B lymphoblastoid cell lines. In this paper we present evidence that occupancy of C1q receptors by C1q on B lymphoblastoid cell lines inhibits "IL 1-like" activity, which is constitutively produced by these cells. When 5 X 10(6) Raji cells were cultured (24 hr at 37 degrees C) in the presence or absence of various concentrations of C1q (final concentration; 5 to 50 micrograms/ml) and the cellfree supernatants were analyzed for their effect on thymocyte proliferation by using the concanavalin A co-mitogenesis assay for IL 1, a statistically significant dose-dependent reduction of 3H-TdR incorporation was observed. Similar results were obtained when Daudi and Wil2WT cells were used, whereas Molt4, which is a T cell lymphoblastoid cell line, neither produced "IL 1-like" activity nor was affected by C1q. When immune complex-bound C1q was used instead of free C1q, inhibition of B cell IL 1 activity was also observed. Mixing experiments showed that Raji cells incubated with C1q released an inhibitor of IL 1-induced thymocyte proliferation. C1q alone had no effect on thymocyte proliferation in the presence or absence of human IL 1. In addition, the effect of C1q on IL 1 activity was abrogated either when the C1q was heat inactivated (56 degrees C, 60 min) or when the C1q was incubated with the cells in the presence of F(ab')2 anti-C1q. On the basis of these data, it is concluded that B cell receptor-bound C1q may play an important role in immunoregulation.
The current studies were designed to determine the relevance of T cell antigen density, besides antibody isotype, with regard to the success of antibody serotherapy. We compared the immunosuppressive effects of two rat IgG2b monoclonal anti-Thy-1 antibodies, RmT1 and 30-H12, with distinct binding sites in a graft-vs.-host disease (GVHD) model of fully H-2 and I-A region-mismatched bone marrow transplantation, making use of the difference in Thy-1.2 antigen density between homozygous (BALB/c) and heterozygous (BALB/c X AKR/J)F1 GVHD-promoting donor cells. Antibodies RmT1 (directed against a monomorphic determinant on mouse Thy-1) and 30-H12 (reactive with the Thy-1.2 allele-specific determinant) did not differ in their anti-GVHD activity with regard to Thy-1.2 homozygous grafts. However, in the region of a critical number of binding sites a small difference in the amounts of the two antibodies bound (about 8 X 10(3) IgG molecules/cell) obviously accounts for a great difference in anti-GVHD activity. This is shown in a two haplotype host-graft disparity between C57BL/6 recipients treated with either RmT1 or 30-H12 before challenging them with (BALB/c X AKR/J)F1 grafts, where the Thy-1.2 antigen concentration is approximately 50% compared to the density on BALB/c lymphocytes. Here, mAb 30-H12 loses its remarkable in vivo immunosuppressive quality, whereas RmT1 treatment protects mice against lethal GVHD. Binding sites were quantitated using a computerized approach for the analysis of data from ligand binding experiments of the respective mAb, RmT1 and 30-H12, coated to LN cells of BALB/c and F1 hybrid origin. Furthermore, the in vivo immunosuppressive activity of rat IgG2b antibodies directed against Thy-1 was found to correlate with their ability to generate stable antibody-C1q complexes on the cell surface of immunocompetent T cells.
A microplate-adapted enzyme linked immunosorbent assay (ELISA) for detection of C1q-binding immune complexes (IC) and aggregated IgG (delta IgG) is described. Purified human C1q was adsorbed to the wells of flat-bottomed microtiter plates and EDTA-treated serum samples were subsequently introduced. Bound IC was measured by use of alkaline phosphatase-labelled Protein A followed by the substrate para-nitro-phenyl-phosphate. A dose response was found for both delta IgG and BSA anti-BSA complexes, while variations in the concentration of monomer IgG did not affect the optical density. Elevated levels of IC were found in the majority of sera from patients with rheumatoid arthritis and SLE. The described C1q-PA-ELISA is a simple and inexpensive method for detection of C1q-binding immune complexes. The reproducibility is acceptable and the sensitivity is higher than for most IC-methods based on C1q-binding.
The receptor for human C1q which recognizes the "collagen like" tail of its ligand is expressed on a wide range of cells including those which are not involved in the immune response. Nevertheless, the interaction of C1q with its receptor triggers a variety of cellular responses and these responses are suspected to involve certain signalling pathways. The few examples cited in this report provide ample evidence to support the long held notion that the cellular responses induced by C1q-C1q-R interaction involve signal transduction pathways. Platelets, for instance, can be activated by aggregated C1q. This activation, which is accompanied by a significant increase in the release of inositol-1,4,5-triphosphate (IP3) results in the expression of alpha 11b/beta 3 integrins (GPIIb/IIIa) and procoagulant activity and may contribute to thrombotic and inflammatory reactions. The ability of C1q to induce proliferative or anti-proliferative responses depend upon the nature of the C1q or the state of the cell expressing the C1q-R. In any event, even these responses most likely involve activation of transmembrane signal transduction pathway(s) which are yet to be elucidated.
Although previous studies have shown that different cells and cell lines of murine origin bind human C1q, suggesting that they display cell surface receptors for C1q, no information is available to indicate whether mouse or human mast cells express C1q receptors. This paper presents the first evidence to show that murine mast cells express specific receptors for C1q. Western blot analysis of cell membrane proteins prepared from a bone marrow-derived mouse cell line using two monospecific polyclonal Abs, one directed against the 60-kDa C1q receptor (C1q-R) that binds to the collagen-like stalk of C1q (cC1q-R) and the other directed against the 33-kDa molecule that binds to the globular "heads" of C1q (gC1q-R), show that both of these receptors are present on these cells. In addition, C1q can induce mast cell migration in a specific and dose-dependent manner. Interestingly, the C1q-induced migratory response was found to be biphasic; the first response peaked at a C1q concentration of 0.1 nM, whereas the second phase peaked at approximately 40 nM. Checkerboard analysis of the mast cell migratory response to C1q showed that the first phase was primarily due to chemotaxis and the second phase was attributable to chemokinesis. Preincubation of C1q with Abs specific for the collagen-like tail of the molecule abolished both its chemotactic and chemokinetic response, whereas heat inactivation of C1q (56 degrees C, 1 h) resulted in 85% abrogation of the chemotactic phase and 42% reduction in the chemokinetic phase. The observed mast cell migratory responses were mediated by cell surface C1q-R(s), as inclusion of a mixture of anti-C1q-R and anti-gC1q-R Abs with the cells inhibited their migratory response toward C1q. However, incubation of cells with various doses of C1q did not result in histamine release. Furthermore, engagement of mast cell C1q-Rs by the ligand C1q induced an antiproliferative response, as coculturing of mast cells with C1q resulted in a specific and dose-dependent decrease in DNA synthesis. These data suggest that C1q-Rs may play a significant role in mast cell function and regulation by providing an important signal through which mast cells can be recruited to inflammatory sites of increased C1q concentration.
Serum bactericidal activity and phagocytic killing are two important mechanisms involved in the host defence against bacteria. Using some in vitro methods, serum bactericidal assay, phagocytic killing by polymorphonuclear leukocytes (PMN) and chemiluminescence, we have evaluated the significance of these mechanisms in the killing of Haemophilus ducreyi bacteria. Furthermore, induction of C3 conversion and deposition of immunoglobulins, C1q and C3, on the surface of bacteria was studied by crossed immunoelectrophoresis and ELISA, respectively. Transmission electron microscopy was employed to study internalization of bacteria by PMN. H. ducreyi and lipooligosaccharide preparations from these bacteria were able to induce conversion of complement factor C3 in normal human serum (NHS). Exposure of bacteria to NHS resulted in deposition of IgG, IgM and complement factors C1q and C3 on the surface of bacteria. H. ducreyi bacteria lost their viability when incubated with fresh but not inactivated normal serum at high concentrations, indicating that the bacteria are sensitive to the complement-dependent bactericidal activity of serum. There were some variations between different strains regarding their susceptibility to the bactericidal activity of NHS, but for eight strains tested, all of the bacteria exposed were not killed in medium containing up to 70% of fresh serum. Complement-mediated opsonophagocytic killing of H. ducreyi by PMN was more effective than complement-dependent bactericidal activity of fresh normal sera. Bacteria treated with heat inactivated immune sera, on the other hand, were as sensitive to the bactericidal effect of PMN as those treated with non-inactivated immune sera, indicating the role of antibodies in opsonophagocytosis. H. ducreyi bacteria were also killed by PMN in the absence of serum antibodies and complement. Using the chemiluminescence assay, H. ducreyi was shown to activate PMN in the absence of serum as well as after opsonization with complement and antibodies. Our results therefore indicate that both opsonic- and non-opsonic mechanisms are involved in the phagocytosis and the subsequent killing of H. ducreyi bacteria. Although both complement and antibodies enhance the ability of phagocytes to kill H. ducreyi, neither component is sufficient for effective killing of H. ducreyi.
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