Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR

Dendritic cells (DCs) are recruited to inflammatory sites where they phagocytose and process antigens for subsequent presentation to the T lymphocytes in the lymphoid tissue. Several leukocyte chemoattractants and their specific receptors have been shown to induce the migration of DC. The complement protein C1q has multiple immune functions including acting as a chemoattractant for neutrophils, eosinophils and mast cells. Therefore, the objective of this study was to determine if soluble C1q can induce chemotaxis of DC. Culturing cells in GM-CSF and IL-4 for 5 to 7 days generated human monocyte-derived DCs. In addition, LPS was added from day 5 to 7 to induce DC maturation. Cells were classified as either immature or mature DC by assessing the cell surface markers by flow cytometry, phagocytosis of dextran-FITC and T cell proliferation in an allogenic MLR. Immature DCs express the C1q receptors (C1qR), gC1qR and cC1qR/CR and, accordingly, display a vigorous migratory response to soluble C1q with maximal cell movement observed at 10-50nM. In contrast, mature DCs neither express C1qR nor do move to a gradient of soluble C1q. Varying the concentration gradient of C1q (checkerboard assay) showed that the protein largely induces a chemotactic response. Finally, blocking gC1qR and cC1qR/CR by using specific antibodies abolished the chemotactic response to C1q but had no effect on a different chemoattractant C5a. These results clearly demonstrate that C1q functions as a chemotactic factor for immature DC, and migration is mediated through ligation of both gC1qR and cC1qR/CR.     

C1q and phagocytosis: the perfect complement to a good meal

Complement component C1q is a member of a family of soluble proteins called defense collagens, which are important in host defense and apoptotic cell clearance. Failure to efficiently clear apoptotic cells in the absence of C1q is associated with autoimmunity. Here, we review the literature describing a central role for C1q in the enhancement of phagocyte function and focus specifically on C1q in apoptotic cell clearance. In addition, we highlight our recent findings demonstrating that C1q elicits a macrophage phenotype that is tailored specifically for clearance of apoptotic cells.     

Charge-based binding of complement component C1q to the Alzheimer amyloid beta-peptide.

Activation of the classical pathway in Alzheimer''s disease derives from the binding of the first protein, subcomponent C1q, to the amyloid beta-peptide (A beta). Analysis of the binding of C1q to A beta by competitive enzyme-linked immunosorbent assay shows that A beta fragments 1-16 and 1-28 but not 12-28 and 17-42 are capable of inhibiting the A beta/C1q interaction, implicating the A beta 1-11 region as the C1q binding site. Binding is also shown to be inhibited by conditions of high ionic strength, suggesting that charged side chains in the A beta 1-11 region are critical to the A beta/c1q interaction. Ultrastructural evidence of binding is provided by platinum replica electron microscopy. Along with a previous demonstration of the 14-26 region of the C1q A chain as the A beta binding site, these findings suggest that attractions between a negative charge cluster in A beta 1-11 and a positive charge cluster in C1qA14-26 mediate the binding of A beta and C1q.     

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.     

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups—one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies.We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups—ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.     

Regulation of the alternative pathway of human complement by C1q

The interaction of C1q with C3b and its effect on C3b activities in the alternative pathway of complement (APC) have been studied. Purified C1q markedly inhibited C3b deposition on and lysis of rabbit erythrocytes by the isolated cytolytic APC. It also blocked formation of the C3 convertase, C3b, Bb as well as binding of Factors B and H to sheep erythrocytes (E) bearing C3b. The direct and specific binding of C1q to C3b was clearly demonstrated using the hemagglutination technique at low ionic strength (0.1 M NaCl). C1q concns of 2 micrograms/ml and higher agglutinated, in a dose-dependent fashion, EC3b but not E, EC3bi or EC3d. Addition of C1r and C1s to C1q and formation of C1 did not affect its capacity to agglutinate EC3b. The C1q-mediated agglutination of EC3b was inhibited by EDTA, MgEGTA, C3b and Factor B but not by native C3 or collagen. Heating C1q (56 degrees C) markedly potentiated its agglutinating activity whereas collagenase-treated C1q lost most of its activity. Taken together, these results suggest that C1q binds through its "heads" and in the presence of calcium ions to a site on C3b that is adjacent to the Factor B and Factor H binding sites. This interaction may down-regulate the activity of the alternative pathway of complement on surfaces which activate both the classical and alternative pathways of complement.     

Binding of complement component C1q by rat adipocyte membranes

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.     

Interaction of plasma fibronectin with gelatin and complement C1q

A variety of techniques have been used to examine the interaction of human plasma fibronectin (Fn) with complement C1q in comparison to that with gelatin in phosphate buffered saline at pH 7.4. The precipitation of 3H-Fn by polyethylene glycol (PEG) was shifted to much lower concentrations of the polymer by addition of gelatin, and to a lesser extent, by C1q. Precipitation of 3H-Fn in the presence of C1q was close to that of C1q alone under identical conditions suggesting an affinity of Fn for solid phase C1q; a similar interaction was seen with heat-insolubilized C1q. Fibronectin bound tightly to gelatin-Sepharose and C1q-Sepharose and this binding could be inhibited by gelatin but not by C1q. The presence of gelatin retarded the anodal migration of Fn during immunoelectrophoresis under physiological conditions whereas C1q had an effect only at low ionic strength. Exclusion chromatography of Fn, alone and preincubated with gelatin or C1q, was also consistent with the formation of strong complexes with gelatin but not with C1q, whereas similar mixtures of Fn and gelatin exhibited a fast-sedimenting boundary and marked depletion of the 12S Fn peak. Titration of fluorescein-labeled alpha 2 chains of type I collagen with Fn produced an increase in fluorescence polarization which could be reversed by addition of unlabeled alpha 2 chains or gelatin but not by C1q or the pepsin-derived collagen-like domain of C1q. These observations indicate that the fluid-phase interaction of Fn with C1q is much weaker than that with gelatin but that Fn does have appreciable affinity for solid-phase C1q. Such interaction could signify a role for Fn in the clearance of immune complexes from circulation.     

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.     

Staphylococcus aureus VraX specifically inhibits the classical pathway of complement by binding to C1q

VraX is a protein secreted by Staphylococcus aureus, an important human pathogen. A dramatic over expression of VraX is observed when S. aureus is exposed to several antimicrobial agents; however, its function remains unclear. Here, we aimed to reveal the function of this protein and the mechanism by which it affects the immune system to enhance the pathogenesis of the bacterium. Our results showed that VraX specifically inhibited the classical pathway of the complement system. In particular, VraX could bind to the C1q protein and block the formation of the C1 complex. Deletion of VraX decreased the pathogenesis of S. aureus. Our findings indicate that over expression of VraX might be a protective response for S. aureus survival.     

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ~465-kD catalytic subunit, DNA-PK_cs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PK_cs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.     

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.     

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.     

Structural and functional anatomy of the globular domain of complement protein C1q

C1q is the first subcomponent of the classical pathway of the complement system and a major connecting link between innate and acquired immunity. As a versatile charge pattern recognition molecule, C1q is capable of engaging a broad range of ligands via its heterotrimeric globular domain (gC1q) which is composed of the C-terminal regions of its A (ghA), B (ghB) and C (ghC) chains. Recent studies using recombinant forms of ghA, ghB and ghC have suggested that the gC1q domain has a modular organization and each chain can have differential ligand specificity. The crystal structure of the gC1q, molecular modeling and protein engineering studies have combined to illustrate how modular organization, charge distribution and the spatial orientation of the heterotrimeric assembly offer versatility of ligand recognition to C1q. Although the biochemical and structural studies have provided novel insights into the structure-function relationships within the gC1q domain, they have also raised many unexpected issues for debate.     

A mutant human IgG molecule with only one C1q binding site can activate complement and induce lysis of target cells

There are potentially two binding sites for C1q on IgG, one on each C(H)2 domain of the gamma heavy chains, close to the lower hinge region. It is not clear whether the presence and involvement of both the C1q binding sites is necessary to induce the activation signal of human IgG. In order to clarify this issue, we made a hybrid mutant IgG1/IgG3 molecule where the IgG1 half of the molecule was made unable to activate complement through the introduction of a P329A mutation. The IgG3 half of the molecule was mutated to harbor a hinge region identical to that of IgG1, and for detection a peptide tag derived from p21ras was introduced into the FG loop of the C(H)1 domain. The hybrid IgG1P329A/IgG3h1-ras molecules were isolated by Protein A affinity chromatography and shown to activate complement and induce complement-mediated lysis at the same levels as wild-type IgG1 and IgG3h1-ras molecules. Thus, one C1q binding site per IgG is sufficient to induce activation. Wild-type human IgG molecules might also normally expose only one C1q binding site as already shown for interaction with FcgammaR, were IgG expose one binding site per molecule.