Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca(2+) ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

M-ficolin interacts with the long pentraxin PTX3: a novel case of cross-talk between soluble pattern-recognition molecules.

Ficolins and pentraxins are soluble oligomeric pattern-recognition molecules that sense danger signals from pathogens and altered self-cells and might act synergistically in innate immune defense and maintenance of immune tolerance. The interaction of M-ficolin with the long pentraxin pentraxin 3 (PTX3) has been characterized using surface plasmon resonance spectroscopy and electron microscopy. M-ficolin was shown to bind PTX3 with high affinity in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by N-acetyl-D-glucosamine, indicating involvement of the fibrinogen-like domain of M-ficolin. Removal of sialic acid from the single N-linked carbohydrate of the C-terminal domain of PTX3 abolished the interaction. Likewise, an M-ficolin mutant with impaired sialic acid-binding ability did not interact with PTX3. Interaction was also impaired when using the isolated recognition domain of M-ficolin or the monomeric C-terminal domain of PTX3, indicating requirement for oligomerization of both proteins. Electron microscopy analysis of the M-ficolin-PTX3 complexes revealed that the M-ficolin tetramer bound up to four PTX3 molecules. From a functional point of view, immobilized PTX3 was able to trigger M-ficolin-dependent activation of the lectin complement pathway. These data indicate that interaction of M-ficolin with PTX3 arises from its ability to bind sialylated ligands and thus differs from the binding to the short pentraxin C-reactive protein and from the binding of L-ficolin to PTX3. The M-ficolin-PTX3 interaction described in this study represents a novel case of cross-talk between soluble pattern-recognition molecules, lending further credit to the integrated view of humoral innate immunity that emerged recently.

Cutting edge: C1q binds deoxyribose and heparan sulfate through neighboring sites of its recognition domain.

C1q, the recognition subunit of the C1 complex of complement, is an archetypal pattern recognition molecule with the striking ability to sense a wide variety of targets, including a number of altered self-motifs. The recognition properties of its globular domain were further deciphered by means of x-ray crystallography using deoxy-D-ribose and heparan sulfate as ligands. Highly specific recognition of deoxy-D-ribose, involving interactions with Arg C98, Arg C111, and Asn C113, was observed at 1.2 A resolution. Heparin-derived tetrasaccharide interacted more loosely through Lys C129, Tyr C155, and Trp C190. These data together with previous findings define a unique binding area exhibiting both polyanion and deoxy-D-ribose recognition properties, located on the inner face of C1q. DNA and heparin compete for C1q binding but are poor C1 activators compared with immune complexes. How the location of this binding area in C1q may regulate the level of C1 activation is discussed.

Functional characterization of the recombinant human C1 inhibitor serpin domain: insights into heparin binding.

Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhDelta97), a single carbohydrate at position 330 (C1inhDelta97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhDelta97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding K(D) values of 16.7 x 10(-8) M (C1 inhibitor), 2.3 x 10(-8) M (C1inhDelta97), and 3.6 x 10(-8) M (C1inhDelta97DM). C1s also bound to heparin, with lower affinity (K(D) = 108 x 10(-8) M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a "sandwich" mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively.

Analysis of human C1q by combined bottom-up and top-down mass spectrometry: detailed mapping of post-translational modifications and insights into the C1r/C1s binding sites.

C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses. C1q was submitted to proteolytic digestion by a combination of collagenase and trypsin for bottom-up analyses. In addition to classical LC-MS/MS analyses, which provided reliable identification of hydroxylated proline and lysine residues, sugar loss-triggered MS(3) scans were acquired on an LTQ-Orbitrap (Linear Quadrupole Ion Trap-Orbitrap) instrument to strengthen the localization of glucosyl-galactosyl disaccharide moieties on hydroxylysine residues. Top-down analyses performed on the same instrument allowed high accuracy and high resolution mass measurements of the intact full-length C1q polypeptide chains and the iterative fragmentation of the proteins in the MS(n) mode. This study illustrates the usefulness of combining the two complementary analytical approaches to obtain a detailed characterization of the post-translational modification pattern of the collagen-like domain of C1q and highlights the structural heterogeneity of individual molecules. Most importantly, three lysine residues of the collagen-like domain, namely Lys(59) (A chain), Lys(61) (B chain), and Lys(58) (C chain), were unambiguously shown to be completely unmodified. These lysine residues are located about halfway along the collagen-like fibers. They are thus fully available and in an appropriate position to interact with the C1r and C1s protease partners of C1q and are therefore likely to play an essential role in C1 assembly.

Structure and properties of the Ca(2+)-binding CUB domain, a widespread ligand-recognition unit involved in major biological functions.

CUB domains are 110-residue protein motifs exhibiting a ß-sandwich fold and mediating protein-protein interactions in various extracellular proteins. Recent X-ray structural and mutagenesis studies have led to the identification of a particular CUB domain subset, cbCUB (Ca(2+)-binding CUB domain). Unlike other CUB domains, these harbour a homologous Ca(2+)-binding site that underlies a conserved binding site mediating ionic interaction between two of the three conserved acidic Ca(2+) ligands and a basic (lysine or arginine) residue of a protein ligand, similar to the interactions mediated by the low-density lipoprotein receptor family. cbCUB-mediated protein-ligand interactions usually involve multipoint attachment through several cbCUBs, resulting in high-affinity binding through avidity, despite the low affinity of individual interactions. The aim of the present review is to summarize our current knowledge about the structure and functions of cbCUBs, which represent the majority of the known CUB repertoire and are involved in a variety of major biological functions, including immunity and development, as well as in various cancer types. Examples discussed in the present review include a wide range of soluble and membrane-associated human proteins, as well as some archaeal and invertebrate proteins. The fact that these otherwise unrelated proteins share a common Ca(2+)-dependent ligand-binding ability suggests a mechanism inherited from very primitive ancestors. The information provided in the present review should stimulate further investigations on the crucial interactions mediated by cbCUB-containing proteins.

Mapping surface accessibility of the C1r/C1s tetramer by chemical modification and mass spectrometry provides new insights into assembly of the human C1 complex.

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.

Complement protein C1q forms a complex with cytotoxic prion protein oligomers.

A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8-15 PrP protomers being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes, C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation.

Carbohydrate recognition properties of human ficolins: glycan array screening reveals the sialic acid binding specificity of M-ficolin.

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr(271) in this respect.

Residue Lys57 in the collagen-like region of human L-ficolin and its counterpart Lys47 in H-ficolin play a key role in the interaction with the mannan-binding lectin-associated serine proteases and the collectin receptor calreticulin.

L- and H-ficolins are serum oligomeric defense proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. They share with mannan-binding lectin (MBL) the ability to associate with MBL-associated serine proteases (MASP)-1, -2, -3, and protein MAp19 and to trigger the lectin complement pathway through MASP-2 activation. Recent studies have revealed the essential role of Lys(55) in the collagenous region of MBL in the interaction with the MASPs and calreticulin (CRT). To test the possible involvement of the homologous residues Lys(57) of L-ficolin and Lys(47) of H-ficolin, point mutants of both proteins were produced in which these residues were mutated to Ala, Glu, or Arg. The resulting mutants exhibited oligomerization patterns and ligand binding properties similar to those of their wild-type counterparts. In contrast, all three mutations strongly inhibited the interaction of L- and H-ficolins with MAp19 and MASP-2 and impaired the ability of each ficolin to trigger the lectin pathway. In the case of MASP-1 and MASP-3, replacement of the target Lys residues by Ala or Glu abolished interaction, whereas the Lys to Arg mutations had only slight inhibitory effects. Likewise, binding of each ficolin to CRT was inhibited by mutation of Lys to Ala or Glu, but not to Arg. In conclusion, residues Lys(57) of L-ficolin and Lys(47) of H-ficolin are key components of the interaction with the MASPs and CRT, providing strong indication that MBL and the ficolins share homologous binding sites for both types of proteins.

Complement protein C1q recognizes enzymatically modified low-density lipoprotein through unesterified fatty acids generated by cholesterol esterase.

We previously reported that enzymatically modified low-density lipoprotein (E-LDL) particles obtained by LDL treatment with trypsin and then cholesterol esterase are recognized by C1q and activate the C1 complex of complement. The objective of this study was to identify the E-LDL component(s) recognized by C1q. In addition to trypsin, plasmin, thrombin, tryptase, and matrix metalloprotease-2 each yielded E-LDL particles with high C1-activating efficiency, and the C1 activation extent was strictly dependent on cholesterol esterase treatment in all cases. When incorporated into vesicles, the lipid fraction of E-LDL, but not of native LDL, triggered C1 activation, and activation correlated with the amount of unesterified cholesterol generated by cholesterol esterase. Whereas treatment of E-LDL particles with human serum albumin reduced their fatty acid content, both cholesterol and unesterified fatty acids were decreased by methyl-beta-cyclodextrin, both treatments resulting in dose-dependent inhibition of the C1-activating ability of the particles. Incorporation of linoleic acid into phosphatidylcholine-containing model vesicles enabled them to interact with the C1q globular domain and to trigger C1 activation, and cholesterol enhanced both processes by facilitating incorporation of the fatty acid into the vesicles. Direct evidence that C1q binds E-LDL through its globular domains was obtained by electron microscopy. This study demonstrates that C1 binding to E-LDL particles involves recognition by the C1q globular domain of the unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed.

Structural and functional characterization of the recombinant human mitochondrial trifunctional protein.

The α and ß subunits of the human mitochondrial trifunctional protein (TFP), the multienzyme complex involved in fatty acid ß-oxidation, were coexpressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography. The resulting α/His-ß construct was analyzed by gel filtration, sedimentation velocity, and electron microscopy, indicating a predominance of α(2)ß(2) and α(4)ß(4) complexes, with higher order oligomers. Electron microscopy indicated that the elementary species α(2)ß(2) had overall structural similarity with its bacterial homologue. As shown by cosedimentation and surface plasmon resonance analyses, recombinant TFP interacted strongly with cardiolipin and phosphatidylcholine, suggesting that the natural complex associates with the inner mitochondrial membrane through direct interactions with phospholipids. Recombinant TFP displayed 2-enoyl-CoA hydratase (ECH), l-3-hydroxyacyl-CoA dehydrogenase (HACD), and 3-ketoacyl-CoA thiolase (KACT) activities, and ECH and HACD each reached equilibrium when the downstream enzymes (HACD and KACT, respectively) were made inactive, indicating feed-back inhibition. The KACT activity was optimal at pH 9.5, sensitive to ionic strength, and inhibited at concentrations of its substrate 3-ketohexadecanoyl-CoA >5 µM. Its kinetic constants (k(cat) = 169 s(-1), K(m) = 4 µM) were consistent with those determined previously on a purified porcine TFP preparation. Using different assays, trimetazidine, an efficient antiaginal agent, had no significant inhibitory effect on any of the three enzymatic activities of the recombinant TFP preparation, in contrast with other reports. This study provides the first detailed structural and functional characterization of a recombinant human TFP preparation and opens the way to in-depth analyses through site-directed mutagenesis.

How phagocytes track down and respond to apoptotic cells.

The uptake of apoptotic cells by macrophages and dendritic cells or nonprofessional phagocytes is crucial for development and tissue homeostasis. This is of special importance because deficiencies in the recognition or removal of apoptotic cells may result in autoimmune diseases. The efficient elimination of an apoptotic cell involves contact between the altered cell and the phagocyte, specific recognition, and phagocytosis of the target. These processes are closely associated with the release of anti-inflammatory cytokines and the induction of self-tolerance. This review focuses on the different types of signals, bridging molecules and receptors involved in both steps of the uptake process. Additionally, the role of soluble pattern recognition molecules from the innate immune system, known for a long time to discriminate pathogens from self and more recently to sense altered self, is discussed. This applies to complement Clq, which appears to sense multiple ligands exposed at the apoptotic cell surface, to be involved in their engulfment by phagocytes, and to modulate dendritic cell maturation.

Trimeric reassembly of the globular domain of human C1q.

C1q is a versatile recognition protein which binds to a variety of targets and consequently triggers the classical pathway of complement. C1q is a hetero-trimer composed of three chains (A, B and C) arranged in three domains, a short N-terminal region, followed by a collagenous repeat domain that gives rise to the formation of (ABC) triple helices, each ending in a C-terminal hetero-trimeric globular domain, called gC1q, which is responsible for the recognition properties of C1q. The mechanism of the trimeric assembly of C1q and in particular the role of each domain in the process is unknown. Here, we have investigated if the gC1q domain was able to assemble into functional trimers, in vitro, in the absence of the collagenous domain, a motif known to promote obligatory trimers in other proteins. Acid-mediated gC1q protomers reassembled into functional trimers, once neutralized, indicating that it is the gC1q domain which possesses the information for trimerization. However, reassembly occurred after neutralization, only if the gC1q protomers had preserved a residual tertiary structure at the end of the acidic treatment. Thus, the collagenous domain of C1q might initialize the folding of the gC1q domain so that subsequent assembly of the entire molecule can occur.

The lectin-like activity of human C1q and its implication in DNA and apoptotic cell recognition.

C1q, the binding subunit of the C1 complex of complement, is an archetypal pattern recognition molecule known for its striking ability to recognize a wide variety of targets, ranging from pathogenic non self to altered self. DNA is one of the C1q ligands, but the precise region of C1q and the DNA motifs that support interaction have not been characterized yet. Here, we report for the first time that the peripheral globular region of the C1q molecule displays a lectin-like activity, which contributes to DNA binding through interaction with its deoxy-d-ribose moiety and may participate in apoptotic cell recognition.

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases.

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.

Molecular organization of human Ficolin-2.

Human Ficolin-2 (L-Ficolin) is an oligomeric serum protein consisting of a collagen-like stalk and fibrinogen-like recognition domains. The protein binds to arrays of sugars present on different microorganisms, enhances phagocytosis and promotes activation of the lectin complement pathway. So far the detailed oligomeric structure and composition of human Ficolin-2 has not been determined. Recombinant human Ficolin-2 was expressed in Chinese hamster ovary cells and its structure and biological functions were investigated by gel filtration, sucrose density gradient ultracentrifugation, mass spectrometry and surface plasmon resonance spectroscopy. It was revealed that Ficolin-2 has a high molecular weight due to extensive disulfide bridge formation. It was able to bind to different ligands, interact with mannose-binding lectin associated serine proteases and activate the complement system. Mass values of 807 and 403 kDa were determined corresponding to a 24-mer and a 12-mer of 34.4 kDa polypeptides. However, the 24-mer was unstable and the 12-mer is likely the major functional form of the protein. Our results are consistent with the view that Ficolin-2 is built up by a mixture of stable homodimers and homotrimers. Based on our findings we propose a model in which disulfide bridges located in the N-terminal region of the polypeptides explain the oligomerization pattern of human Ficolin-2.

Modified low density lipoproteins differentially bind and activate the C1 complex of complement.

Several studies suggest that complement plays an important role in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of low density lipoprotein (LDL) to bind and activate C1, the complex that triggers the classical pathway of complement. For this purpose, LDL was both obtained commercially and purified according to an established procedure, and oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were generated from each preparation. Whereas the unmodified LDL and oxLDL samples did not activate C1 in the presence of excess C1 inhibitor, the E-LDL derivatives obtained by sequential treatment of LDL with a protease and then with cholesterol esterase triggered efficient C1 activation under these conditions, with activation levels approximately 60% upon incubation with 1 microM E-LDL for 90 min at 37 degrees C. In agreement with these findings, as shown by surface plasmon resonance spectroscopy (SPR), the C1q recognition subunit of C1 showed no interaction with unmodified LDL but bound to both E-LDL samples with high affinity (K(D)=58-75 nM). More unexpectedly, although they did not trigger direct C1 activation, both oxLDL samples were also efficiently recognized by C1q. Whereas the oxLDL derivative of commercial LDL activated C1 to a significant extent ( approximately 30%) in the presence of C-reactive protein (CRP), much lower activation levels (<10%) were obtained using the oxLDL derivative of purified LDL. As measured by SPR, CRP bound equally well to the oxLDL and E-LDL derivatives obtained from purified LDL. These data provide the first experimental evidence that E-LDL triggers efficient C1 activation under conditions close to the physiological situation, suggesting that activation of the classical complement pathway by this derivative may be a crucial factor in the pathogenesis of atherosclerosis. In contrast, it appears unlikely that oxLDL significantly activates C1 directly or in a CRP-dependent manner in vivo.

Complement protein C1q induces maturation of human dendritic cells.

Maturation of dendritic cells (DCs) is known to be induced by several stimuli, including microbial products, inflammatory cytokines and immobilized IgG, as demonstrated recently. Since immune complexes formed in vivo also contain C1q, moreover apoptotic cells and several pathogens fix C1q in the absence of antibodies, we undertook to investigate whether this complement protein has an impact on various functions of human DCs. Maturation of monocyte-derived immature DCs (imMDCs) cultured on immobilized C1q was followed by monitoring expression of CD80, CD83, CD86, MHCII and CCR7. The functional activity of the cells was assessed by measuring cytokine secretion and their ability to activate allogeneic T lymphocytes. Cytokine production by T cells co-cultured with C1q-matured DCs was also investigated. C1q, but not the structurally related mannose-binding lectin was found to bind to imMDC in a dose-dependent manner and induced NF-kappaB translocation to the nucleus. Immobilized C1q induced maturation of MDCs and enhanced secretion of IL-12 and TNF-alpha, moreover, elevated their T-cell stimulating capacity. As IFN-gamma levels were increased in supernatants of MDC-T cell co-cultures, our data suggest that C1q-induced DC maturation generates a Th1-type response. Interestingly, IL-10 levels were elevated by C1q-treated MDCs but not in the supernatant of their co-cultures with allogeneic T cells. Taken together, these results indicate that C1q-opsonized antigens may play a role in the induction and regulation of immune response. Moreover our data are relevant in view of the role of C1q in removal of apoptotic cells and the association between C1q-deficiency and autoimmunity.