C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.     

Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells

C1q is the initiator of the classical complement pathway and opsonizes apoptotic cells to facilitate phagocytosis. Deficiency of C1q is the strongest known risk factor for development of systemic lupus erythematosus (SLE), which appears to be related to ensuing impaired clearance of apoptotic material. The objective of the current study was to investigate new ligands for C1q on the surface of apoptotic cells. We revealed that the two phospholipid-binding proteins annexin A2 and A5 are, beside DNA, significant C1q ligands. We furthermore, demonstrated that C1q binds directly to histones exposed on the surface of dying cells but we did not detect significant interaction with phosphatidylserine. The complement inhibitors C4b-binding protein and factor H also interact with dying cells, most likely to decrease complement activation beyond the level of C3 to allow noninflammatory clearance. Despite the fact that C4b-binding protein, factor H, and C1q share some ligands on dying cells, we showed that these three proteins did not compete with one another for binding to apoptotic cells. We additionally demonstrated that the way in which apoptosis is induced influenced both the degree of apoptosis and the binding of C1q. The knowledge, that annexin A2 and A5 act as ligands for C1q on apoptotic cells, sheds new light on the pathophysiology of autoimmune diseases.     

C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in apoptotic cell recognition

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute "eat me" signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (K(D) = 3.7-7 x 10(-8) M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.     

The extracellular matrix and inflammation: fibromodulin activates the classical pathway of complement by directly binding C1q

Components that propagate inflammation in joint disease may be derived from cartilage since the inflammation resolves after joint replacement. We found that the cartilage component fibromodulin has the ability to activate an inflammatory cascade, i.e. complement. Fibromodulin and immunoglobulins cause comparable deposition of C1q, C4b, and C3b from human serum. Using C1q and factor B-deficient sera in combination with varying contents of metal ions, we established that fibromodulin activates both the classical and the alternative pathways of complement. Further studies revealed that fibromodulin binds directly to the globular heads of C1q, leading to activation of C1. However, deposition of the membrane attack complex and C5a release were lower in the presence of fibromodulin as compared with IgG. This can be explained by the fact that fibromodulin also binds complement inhibitor factor H. Factor H and C1q bind to non-overlapping sites on fibromodulin, but none of the interactions is mediated by the negatively charged keratan sulfate substituents of fibromodulin. C1q but not factor H binds to an N-terminal fragment of fibromodulin previously implicated to be affected in cartilage stimulated with the inflammatory cytokine interleukin 1. Taken together our observations indicate fibromodulin as one factor involved in the sustained inflammation of the joint.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Investigations on the C1q-calreticulin-phosphatidylserine interactions yield new insights into apoptotic cell recognition

Both C1q and calreticulin (CRT) are involved in the recognition of apoptotic cells. CRT was initially characterized as a receptor for the C1q collagen-like fragment (CLF), whereas C1q was shown to bind apoptotic cells through its globular region (GR). Using purified CRT and recombinant CRT domains, we now provide unambiguous experimental evidence that, in addition to its CLF, the C1q GR also binds CRT and that both types of interactions are mediated by the CRT globular domain. Surface plasmon resonance analyses revealed that the C1q CLF and GR domains each bind individually to immobilized CRT and its globular domain with K(D) values of (2.6-8.3) × 10(-7) M. Further evidence that CRT binds to the C1q GR was obtained by electron microscopy. The role of CRT in the recognition of apoptotic HeLa cells by C1q was analyzed. The C1q GR partially colocalized with CRT on the surface of early apoptotic cells, and siRNA (small interfering RNA)-induced CRT deficiency resulted in increased apoptotic cell binding to C1q. The interaction between CRT and phosphatidylserine (PS), a known C1q ligand on apoptotic cells, was also investigated. The polar head of PS was shown to bind to CRT with a 10-fold higher affinity (K(D)=1.5 × 10(-5) M) than that determined for C1q, and, accordingly, the C1q GR-PS interaction was impaired in the presence of CRT. Together, these observations indicate that CRT, C1q, and PS are all closely involved in the uptake of apoptotic cells and strongly suggest a combinatorial role of these three molecules in the recognition step.     

Mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype

Mannose-binding lectin (MBL) is a circulating serum protein that is sequestered to sites of inflammation and infection. MBL is a member of the collectin family with structural similarities to the lung collectins and functional similarities to C1q. Both MBL and C1q activate complement; C1q activates the classical pathway and MBL the lectin pathway. Here we demonstrate that MBL binds apoptotic cells in vitro and confirm a role for MBL in clearance of apoptotic cells in vivo. Despite MBL null mice demonstrating defective apoptotic cell clearance they did not develop spontaneous autoimmunity, lymphoproliferation, or germinal center expansion although increased numbers of peritoneal B1 cells were detected. These data demonstrate an important in vivo role for MBL in clearance of dying cells and adds the MBL null animals to the few animals with demonstrable in vivo apoptotic cell clearance defects. Moreover, it demonstrates that failure of apoptotic cell clearance can be dissociated from autoimmunity.     

Adiponectin binds C1q and activates the classical pathway of complement

The adipose-specific protein adiponectin binds to a number of target molecules, including damaged endothelium and the surface of apoptotic cells. However, the significance of this binding remains unclear. This study demonstrates the binding of purified C1q to recombinant adiponectin under physiological conditions, and the dependence of this upon Ca⁺⁺ and Mg⁺⁺. Binding was enhanced by metaperiodate-mediated destruction of glucosylgalactosyl sugars on adiponectin. Adiponectin was bound by the globular domain of the A chain of collagenase-digested C1q, and C1q binding induced deposition of C4 and C3 through activation of the classical complement pathway. After Western blotting, affinity-purified adiponectin from human serum bound C1q, whereas adiponectin in whole serum did not, unless pre-treated with metaperiodate. These results suggest adiponectin is member of the pattern-recognition family of defence collagens, able to bind target molecules and activate complement. It may therefore play an important role in innate immunity and autoimmune phenomena.     

SIGN-R1, a C-type lectin,enhances apoptotic cell clearance through the complement deposition pathway by interacting with C1q in the spleen

Complements, such as C1q and C3, and macrophages in the splenic marginal zone (MZMs) play pivotal roles in the efficient uptake and processing of circulating apoptotic cells. SIGN-R1, a C-type lectin that is highly expressed in a subpopulation of MZMs, regulates the complement fixation pathway by interacting with C1q, to fight blood-borne Streptococcus pneumoniae. Therefore, we examined whether the SIGN-R1-mediated classical complement pathway plays a role in apoptotic cell clearance and immune tolerance. SIGN-R1 first-bound apoptotic cells and this binding was significantly enhanced in the presence of C1q. SIGN-R1–C1q complex then immediately mediated C3 deposition on circulating apoptotic cells in the MZ, leading to the efficient clearance of them. SIGN-R1-mediated C3 deposition was completely abolished in the spleen of SIGN-R1 knockout (KO) mice. Given that SIGN-R1 is not expressed in the liver, we were struck by the finding that C3-deposited apoptotic cells were still found in the liver of wild-type mice, and dramatically reduced in the SIGN-R1 KO liver. In particular, SIGN-R1 deficiency caused delayed clearance of apoptotic cells and aberrant secretion of cytokines, such as TNF-α, IL-6, and TGF-β in the spleen as well as in the liver. In addition, anti-double- and single-stranded DNA antibody level was significantly increased in SIGN-R1-depleted mice compared with control mice. These findings suggest a novel mechanism of apoptotic cell clearance which is initiated by SIGN-R1 in the MZ and identify an integrated role of SIGN-R1 in the systemic clearance of apoptotic cells, linking the recognition of apoptotic cells, the opsonization of complements, and the induction of immune tolerance.     

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.     

A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.     

CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells

Complement is the canonical innate immune system involved in host defense and tissue repair with the clearance of cell debris. In contrast to the robust armory mounted against microbial nonself-pathogens, complement is selectively activated on altered self (i.e. apoptotic and necrotic cells) to instruct the safe demise by poorly characterized mechanisms. Our data shed new light on the role of complement C1q in sensing nucleic acids (NA) rapidly exposed on apoptotic Jurkat T cell membranes and in driving C3 opsonization but without the lytic membrane attack complex. DNA/RNase-treated apoptotic cells failed to activate complement. We found that several other apoptotic cell models, including senescent keratinocytes, ionophore-treated sperm cells, and CMK-derived platelets, stained for cleaved caspase 3 were rapidly losing the key complement regulator CD46. CD46 from nuclear and membrane stores was found to cluster into blebs and shed into microparticles together with NA, phosphatidylserine, C1q, and factor H. Classical and alternative pathways of complement were involved in the recognition of H2O2-treated necrotic cells. Membrane attack complex was detected on necrotic cells possibly as a result of CD46 and CD59 shedding into soluble forms. Our data highlight a novel and universal paradigm whereby the complement innate immune system is using two synergistic strategies with the recognition of altered self-NA and missing self-CD46 signals to instruct and tailor the efficient removal of apoptotic and necrotic cells in immunoprivileged sites.     

Mass spectrometry analysis of the oligomeric C1q protein reveals the B chain as the target of trypsin cleavage and interaction with fucoidan

C1q is a subunit of the C1 complex that triggers activation of the complement classical pathway through recognition and binding of immune complexes. C1q also binds to nonimmune ligands such as the sulfated polysaccharide fucoidan, a potent anticomplementary agent. C1q was submitted for the first time to mass spectrometry analysis, yielding insights into its assembly and its interaction with fucoidan. The MALDI-TOF mass spectrometry technique on membrane allowed partial preservation of noncovalent interactions, allowing precise analysis of its substructure and estimation of the C1q molecular weight at 459520-461883, with an average mass of 460793 g x mol(-1). The disulfide-linked A-B and C-C dimers as well as the noncovalent structural unit (A-B:C)-(C:B-A) were detected, providing experimental support to the C1q model based on covalent and noncovalent associations of six heterotrimers. Trypsin treatment of native C1q led to proteolysis of the B chain only, at a single cleavage site (Arg(109)) located in the globular region. Unlike DNA, fucoidan protected C1q from trypsin cleavage, indicating that this polysaccharide binds to the B moiety of the globular head. Given the involvement of the C1q globular heads in the recognition of IgG, this interaction may account for the observed anticomplementary activity of fucoidan.     

Receptor-mediated binding of C1q on pulmonary endothelial cells

Normal undamaged pulmonary endothelial cells appear to be immunologically privileged in that they do not express receptors for the Fc portion of IgG nor for C3b. However, these receptors become unmasked on endothelial cells injured by viral infection or exposure to white cell lysates. We now present evidence to indicate that C1q binds to specific receptors on the surface of normal healthy endothelial cells. The binding is dose-dependent, reversible and saturable. Furthermore our data show that binding of C1q to endothelial cells is via the collagenous portion of the molecule not via the globular head regions. Thus binding of C1q to endothelium would have the effect of exposing Fc receptors that could then bind to IgG of circulating immune complexes. That Fc receptors are in fact exposed is shown by rosette formation with antibody sensitized erythrocytes. With 2C1r-2C1s-associated C1q, no binding occurred using C1 fixation and transfer assays. Our results indicate that C1q binding to endothelium provides a means for localizing immune complexes on pulmonary vessels and may be important in the initiation and progression of the inflammatory response.     

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.     

Blebs and apoptotic bodies are B cell autoantigens

Mounting evidence suggests that systemic lupus erythematosus autoantigens are derived from apoptotic cells. To characterize the potential interactions between apoptotic cells and B cells, the D56R/S76R variant of 3H9, a murine autoantibody that binds to DNA, chromatin, and anionic phospholipids, was compared with DNA4/1, a human anti-DNA autoantibody. Flow cytometry revealed that only D56R/S76R bound to Jurkat cells treated with either of three distinct proapoptotic stimuli, Ab binding was dependent on caspase activity, and immunoreactivity developed subsequent to annexin V binding. Confocal microscopy established a structural basis for the distinct kinetics of binding. D56R/S76R preferentially bound to membrane blebs of apoptotic cells, whereas annexin V binding did not require blebs. Inhibition of ROCK I kinase, an enzyme that stimulates nuclear fragmentation and fragment distribution into blebs, significantly reduced Ab binding. Because members of the collectin and pentraxin families of serum proteins bind to blebs on apoptotic cells and assist in the clearance of cellular remains, our results suggest that Abs to blebs could affect the recognition of apoptotic cells by cells of the innate immune system and thus modify tolerance to nuclear Ags.     

Contribution of the innate immune system to autoimmune diabetes: a role for the CR1/CR2 complement receptors.

B lymphocytes are required for diabetogenesis in nonobese diabetic (NOD) mice. The complement component of the innate immune system regulates B cell activation and tolerance through complement receptors CR1/CR2. Thus, it is important to assess the contribution of complement receptors to autoimmune diabetes in NOD mice. Examination of the lymphoid compartments of NOD mice revealed striking expansion of a splenic B cell subset with high cell surface expression of CR1/CR2. This subset of B cells exhibited an enhanced C3 binding ability. Importantly, long-term in vivo blockade of C3 binding to CR1/CR2 prevented the emergence of the CR1/CR2(hi) B cells and afforded resistance to autoimmune diabetes in NOD mice. These findings implicate complement as an important regulatory element in controlling the T cell-mediated attack on islet beta cells of NOD mice.     

CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.     

Modular organization of the carboxyl-terminal,globular head region of human C1q A,B, and C chains

The first step in the activation of the classical complement pathway, by immune complexes, involves the binding of the globular heads of C1q to the Fc regions of aggregated IgG or IgM. Located C-terminal to the collagen region, each globular head is composed of the C-terminal halves of one A (ghA), one B (ghB), and one C chain (ghC). To dissect their structural and functional autonomy, we have expressed ghA, ghB, and ghC in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP). The affinity-purified fusion proteins (MBP-ghA, -ghB, and -ghC) bound differentially to heat-aggregated IgG and IgM, and also to three known C1q-binding peptides, derived from HIV-1, HTLV-I, and beta-amyloid. In the ELISAs, the MBP-ghA bound to heat-aggregated IgG and IgM as well as to the HIV-1 gp41 peptide; the MBP-ghB bound preferentially to IgG rather than IgM, in addition to binding beta-amyloid peptide, whereas the MBP-ghC showed a preference for IgM and the HTLV-I gp21 peptide. Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. However, for IgM-coated erythrocytes, MBP-ghC was a better inhibitor of C1q than MBP-ghB. The recombinant forms of ghA, ghB, and ghC also bound specifically to apoptotic PBMCs. We conclude that the C1q globular head region is likely to have a modular organization, being composed of three structurally and functionally independent modules, which retains multivalency in the form of a heterotrimer. The heterotrimeric organization thus offers functional flexibility and versatility to the whole C1q molecule.     

Opsonization with C1q and mannose-binding lectin targets apoptotic cells to dendritic cells

Deficiencies of early components of the classical complement pathway, particularly C1q, are strongly associated with susceptibility to systemic lupus erythematosus. Recent data link this predisposal to autoimmunity to an inappropriate clearance of apoptotic cells, which could lead to a loss of self-tolerance. In the present study, we demonstrate that opsonization of apoptotic cells with C1q and mannose-binding lectin allows and facilitates their uptake not only by macrophages but also by human immature dendritic cells (DCs). Both C1q and mannose-binding lectin enhance the uptake of apoptotic cells by DCs in a dose-dependent way. The uptake of C1q-opsonized apoptotic cells, but not nonopsonized apoptotic cells, by DCs stimulated the production of IL-6, IL-10, and TNF-alpha, without an effect on IL-12p70. We conclude that these recognition molecules of the complement system do not sequester apoptotic cells from DCs, but rather promote their uptake by immature DCs. Therefore, we propose that early complement components support safe clearance of cellular debris by facilitating phagocytosis and possibly by immunomodulatory mechanisms, thus preventing autoimmunity.