Complement classical pathway components are all important in clearance of apoptotic and secondary necrotic cells

Inherited deficiencies in components of the classical complement pathway are strong disease susceptibility factors for the development of systemic lupus erythematosus (SLE) and there is a hierarchy among deficiency states, the strongest association being with C1q deficiency. We investigated the relative importance of the different complement pathways regarding clearance of apoptotic cells. Phagocytosis of labelled apoptotic Jurkat cells by monocyte‐derived macrophages in the presence of sera from individuals with complement deficiencies was studied, as well as C3 deposition on apoptotic cells using flow cytometry. Sera from individuals deficient in C1q, C4, C2 or C3 all showed decreased phagocytosis. Mannose binding lectin (MBL) and the alternative pathway did not influence phagocytosis. Notably, the components of the complement classical pathway, including C1q, were equally important in clearance of apoptotic cells. This indicates that deposition of C3 fragments is of major significance; we therefore studied C3 deposition on apoptotic cells. Experiments with MBL‐deficient serum depleted of C1q or factor D confirmed the predominance of the classical pathway. At low dilution, sera deficient of C1q, C4 or C2 supported C3 fragment deposition demonstrating alternative pathway activation. In conclusion, we have found that complement‐mediated opsonization and phagocytosis of apoptotic cells, particularly those undergoing secondary necrosis, are dependent mainly upon an intact classical pathway. The alternative pathway is less important, but may play a role in some conditions. C1q was not more important than other classical pathway components, suggesting a role in additional pathogenetic processes in SLE other than clearance of apoptotic cells.     

The tissue pentraxin PTX3 limits C1q-mediated complement activation and phagocytosis of apoptotic cells by dendritic cells

Pentraxins (PTX) and complement belong to the humoral arm of the innate immune system and have essential functions in immune defense to microbes and in scavenging cellular debris. The prototypic long PTX, PTX3, and the first component of the classical complement pathway, C1q, are innate opsonins involved in the disposal of dying cells by phagocytes. Whether the interaction between various innate opsonins impacts on their function is not fully understood. We show here that characterized Toll-like receptor (TLR) ligands elicit the production of C1q and PTX3 by immature dendritic cells (DC). Moreover, these molecules bind to dying cells with similar kinetics, although they recognize different domains on the cell membranes. PTX3 binds in the fluid phase to C1q, decreasing C1q deposition and subsequent complement activation on apoptotic cells. C1q increases the phagocytosis of apoptotic cells by DC and the release of interleukin-12 in the presence of TLR4 ligands and apoptotic cells; PTX3 inhibits both events. Moreover, PTX3 inhibited the cross-presentation of the MELAN-A/melanoma antigen-reactive T cell 1 (MART-1) tumor antigen expressed by dying cells, even in the presence of C1q. These results suggest that interaction of C1q and PTX3 influences the clearance of apoptotic cells by DC. The coordinated induction by primary, proinflammatory signals of C1q and PTX3 and their reciprocal regulation during inflammation influences the clearance of apoptotic cells by antigen-presenting cells and possibly plays a role in immune homeostasis.     

IGM is required for efficient complement mediated phagocytosis of apoptotic cells in vivo

A variety of complement components have been detected on apoptotic cells and proposed to facilitate recognition and/or ingestion by phagocytes. The triggers for complement activation remain uncertain. To determine the role of IgM in classical pathway activation and clearance of apoptotic cells in vitro and in vivo, we quantified these parameters in mice deficient in serum IgM (sIgM). Phagocytosis by bone marrow-derived macrophages of apoptotic cells incubated with serum deficient in sIgM was markedly reduced, similar to apoptotic cells incubated with C1q deficient serum in vitro. Similarly, intraperitoneal clearance of apoptotic cells and cellular C3 deposition were significantly reduced in mice deficient in sIgM compared to wild-type mice. Clearance and C3 deposition were reconstituted by addback of IgM. In mice deficient in both sIgM and C1q, addback of both serum factors was required for restoration of clearance. These findings indicate that, on a quantitative basis, sIgM is a potent factor required for intraperitoneal phagocytosis of apoptotic cells, and further demonstrate that IgM and C1q work in concert to activate complement, resulting in C3 deposition on the apoptotic cell surface and ultimately, efficient clearance of the apoptotic cell by macrophages.     

IGM is required for efficient complement mediated phagocytosis of apoptotic cells in vivo

A variety of complement components have been detected on apoptotic cells and proposed to facilitate recognition and/or ingestion by phagocytes. The triggers for complement activation remain uncertain. To determine the role of IgM in classical pathway activation and clearance of apoptotic cells in vitro and in vivo, we quantified these parameters in mice deficient in serum IgM (sIgM). Phagocytosis by bone marrow-derived macrophages of apoptotic cells incubated with serum deficient in sIgM was markedly reduced, similar to apoptotic cells incubated with C1q deficient serum in vitro. Similarly, intraperitoneal clearance of apoptotic cells and cellular C3 deposition were significantly reduced in mice deficient in sIgM compared to wild-type mice. Clearance and C3 deposition were reconstituted by addback of IgM. In mice deficient in both sIgM and Clq, addback of both serum factors was required for restoration of clearance. These findings indicate that, on a quantitative basis, sIgM is a potent factor required for intraperitoneal phagocytosis of apoptotic cells, and further demonstrate that IgM and C1q work in concert to activate complement, resulting in C3 deposition on the apoptotic cell surface and ultimately, efficient clearance of the apoptotic cell by macrophages.     

Factor H as a regulator of the classical pathway activation

C1q, the first subcomponent of the classical pathway, is a charge pattern recognition molecule that binds a diverse range of self, non-self and altered self ligands, leading to pro-inflammatory complement activation. Although complement is required for tissue homeostasis as well as defence against pathogens, exaggerated complement activation can be damaging to the tissue. Therefore, a fine balance between complement activation and inhibition is necessary. We have recently found that factor H, a polyanion recognition molecule and soluble regulator of alternative pathway activation in blood and on cell surfaces, can directly compete with C1q in binding to anionic phospholipids (cardiolipin), lipid A and Escherichia coli (three known activators of the classical pathway) and acts as a direct down regulator of the complement classical pathway. This ability of factor H to dampen classical pathway activation is distinct from its role as an alternative pathway down-regulator. Thus, by directly competing for specific C1q ligands (exogenous as well as endogenous), factor H is likely to be involved in fine-tuning and balancing the C1q-driven inflammatory processes in autoimmunity and infection. However, in the case of apoptotic cells, C1q-mediated enhancement of uptake/adhesion of the apoptotic cells by monocytes was reduced by factor H. Thus, factor H may be important in controlling the inflammation, which might arise from C1q deposition on apoptotic cells.     

Complement activation by apoptotic cells occurs predominantly via IgM and is limited to late apoptotic (secondary necrotic) cells

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation. Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM. We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.     

The regulatory roles of C1q

C1q binds to immune complexes to elicit complement-dependent microbial killing and enhance phagocytosis. Besides this classical role, C1q also opsonizes apoptotic cells for clearance by phagocytes. C1q deficiency increases susceptibility to microbial infections and is also associated with elevated autoimmunity as characterized by increased apoptotic bodies in tissues. Most complement proteins are of liver origin, but C1q is predominantly synthesized by peripheral tissue macrophages and dendritic cells. Besides being found in the blood, C1q has also been found deposited in extracellular tissues around these cells. In vitro, immobilized C1q inhibits monocyte, macrophage and T-cell production of inflammatory cytokines. It also regulates T-cell activation. Therefore, mounting evidence suggest a major regulatory role for C1q in inflammation and autoimmunity.     

Complement Activation by Apoptotic Cells Occurs Predominantly via IgM and is Limited to Late Apoptotic (Secondary Necrotic) Cells

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation.

Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM.

We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.     

C1q,autoimmunity and apoptosis

Deficiency of classical pathway complement components displays a hierarchical association with the development of systemic lupus erythematosus (SLE). Individuals with deficiency of C1q, the first component of the classical pathway of activation, have the highest prevalence of SLE and the most severe manifestations of the disease. However, complement is also implicated in the effector inflammatory phase of the autoimmune response that characterizes SLE. Complement proteins are deposited in inflamed tissues causing consumption of complement. In addition, autoantibodies to C1q develop as part of the autoantibody response. Understanding how C1q deficiency results in the autoimmune phenotype of SLE may provide valuable clues to the role of the complement system in the maintenance of immune tolerance. In this review firstly we discuss the relationship between C1q deficiency and/or consumption and lupus. Secondly, we consider the links between apoptosis and complement. Finally we review the lessons we have learned from a murine model of C1q deficiency discussing the experimental evidence in support of the hypothesis that C1q may critically influence the immune response to self-antigens contained within the surface blebs generated by apoptotic cells.     

MBL and C1q compete for interaction with human endothelial cells

C1q, the recognition molecule of the classical pathway of complement, binds to endothelial cells, leading to cell activation. Mannose-binding lectin (MBL), a recognition molecule of the lectin pathway, is structurally and functionally related to C1q. Therefore, we investigated the interaction of MBL with human umbilical vein endothelial cells (HUVEC). C1q and MBL were purified from normal human plasma and binding to HUVEC was evaluated by flow cytometry. Cross-competition experiments were performed using MBL and C1q labeled with digoxygenin. MBL, similar to C1q, exhibited a dose-dependent binding to HUVEC under calcium-free conditions, suggesting involvement of its collagenous domains. Pre-incubation of HUVEC with MBL inhibited the binding of digoxygenin-labeled MBL at equimolar concentrations, confirming the specificity of the interaction. Pre-incubation of HUVEC with MBL inhibited the binding of C1q and vice versa. Activation of HUVEC with LPS resulted in increased C1q and MBL binding. Stimulation of HUVEC with MBL did not result in a detectable increase in cytokine production. Based on these results, we propose that MBL and C1q bind to a shared receptor on endothelial cells. Interaction of MBL and C1q with receptors on endothelial cells may be involved in inflammatory processes, and in clearance of pathogens and apoptotic cells.     

C1q knock-out mice for the study of complement deficiency in autoimmune disease

In humans, homozygous deficiency of the first component of the classical pathway of complement, C1q, is a powerful disease susceptibility factor for the development of systemic lupus erythematosus (SLE). This strong association indicates that a functional activity of C1q protects from the development of SLE. Studies in vitro have shown that C1q can bind apoptotic keratinocytes suggesting that it may have an important role in the clearance of apoptotic cells. C1q-deficient mice, generated by gene targeting, showed an increased mortality and 25% of the mice had histological evidence of glomerulonephritis characterised by multiple apoptotic cell bodies and immune deposits, assessed by immunofluorescence and electron microscopy. These observations are compatible with the hypothesis that C1q deficiency causes autoimmunity by an impaired clearance of apoptotic cells.     

Specific binding of L-ficolin and H-ficolin to apoptotic cells leads to complement activation

The serum lectins mannose-binding lectin (MBL), L-ficolin, and H-ficolin are recognition molecules in the lectin complement pathway, which play an important role in innate immunity. To assess involvement of the lectin pathway in the clearance of apoptotic cells, we used flow cytometry to quantify binding of MBL, L-ficolin, and H-ficolin to apoptotic HL60, U937, and Jurkat cells induced by actinomycin D. When apoptotic cells were incubated with normal human serum, MBL and L-ficolin bound to all three cell lines tested; moreover, H-ficolin bound to apoptotic Jurkat cells only. Subsequently, C4 and C3 were deposited on apoptotic cells of all three cell lines. MBL, L-ficolin, and H-ficolin binding to apoptotic cells was confirmed by the use of purified proteins. Purified C4 added to apoptotic cells that had bound pure L-ficolin was deposited on the cell surfaces. In L-ficolin-depleted serum, C3 deposition on HL60 or Jurkat cells decreased to approximately 50% or 70%, respectively, in comparison to the serum before L-ficolin depletion. We conclude that L-ficolin, in addition to MBL, recognizes apoptotic cells and activates complement via the lectin pathway. We also observed in vitro binding of L-ficolin and H-ficolin to cC1q receptor (C1q receptor specific for the collagenous region of C1q)/calreticulin, a candidate receptor for the collagenous region of MBL and C1q. Thus, L-ficolin and H-ficolin as well as MBL participate in the clearance of apoptotic cells through complement activation.     

Biochemical and functional characterization of the interaction between pentraxin 3 and C1q

Pentraxin 3 (PTX3) is a recently characterized member of the pentraxin family of acute-phase proteins produced during inflammation. Classical short pentraxins, C-reactive protein, and serum amyloid P component can bind to C1q and thereby activate the classical complement pathway. Since PTX3 can also bind C1q, the present study was designed to define the interaction between PTX3 and C1q and to examine the functional consequences of this interaction. A dose-dependent binding of both C1q and the C1 complex to PTX3 was observed. Experiments with recombinant globular head domains of human C1q A, B, and C chains indicated that C1q interacts with PTX3 via its globular head region. Binding of C1q to immobilized PTX3 induced activation of the classical complement pathway as assessed by C4 deposition. Furthermore, PTX3 enhanced C1q binding and complement activation on apoptotic cells. However, in the fluid-phase, pre-incubation of PTX3 with C1q resulted in inhibition of complement activation by blocking the interaction of C1q with immunoglobulins. These results indicate that PTX3 can both inhibit and activate the classical complement pathway by binding C1q, depending on the way it is presented. PTX3 may therefore be involved in the regulation of the innate immune response.     

Complement in removal of the dead – balancing inflammation

Recognition and removal of apoptotic and necrotic cells must be efficient and highly controlled to avoid excessive inflammation and autoimmune responses to self. The complement system, a crucial part of innate immunity, plays an important role in this process. Thus, apoptotic and necrotic cells are recognized by complement initiators such as C1q, mannose binding lectin, ficolins, and properdin. This triggers complement activation and opsonization of cells with fragments of C3b, which enhances phagocytosis and thus ensures silent removal. Importantly, the process is tightly controlled by the binding of complement inhibitors C4b-binding protein and factor H, which attenuates late steps of complement activation and inflammation. Furthermore, factor H becomes actively internalized by apoptotic cells, where it catalyzes the cleavage of intracellular C3 to C3b. The intracellularly derived C3b additionally opsonizes the cell surface further supporting safe and fast clearance and thereby aids to prevent autoimmunity. Internalized factor H also binds nucleosomes and directs monocytes into production of anti-inflammatory cytokines upon phagocytosis of such complexes. Disturbances in the complement-mediated clearance of dying cells result in persistence of autoantigens and development of autoimmune diseases like systemic lupus erythematosus, and may also be involved in development of age-related macula degeneration.     

Ficolin-2 recognizes DNA and participates in the clearance of dying host cells

Ficolin-2 is a serum opsonin, which has been shown to be a pattern recognition molecule in the lectin complement activation pathway. Because innate immune mechanisms are involved in maintaining tissue homeostasis we hypothesized that Ficolin-2 also participate in the clearance of dying host cells. We found that Ficolin-2 binds to late apoptotic cells, as well as to apoptotic bodies and necrotic cells, but not to early apoptotic cells. We demonstrated that Ficolin-2 binds DNA in a calcium dependent manner and that DNA inhibits the binding to late apoptotic and necrotic cells, suggesting that DNA on permeable dying cells is a plausible ligand. Reconstituting serum deficient of Ficolin-2, C1q and mannose-binding lectin with Ficolin-2 augmented deposition of complement C4 on necrotic cells. Opsonization leads to an enhanced attachment/uptake of necrotic cells by macrophages. In conclusion dying host cells expose ligands with the capacity of binding Ficolin-2, which in turn leads to increased attachment and engulfment. Binding of Ficolin-2 to DNA points at nucleic acid exposed by permeable late apoptotic and necrotic cells as one of the ligands for Ficolin-2. Ficolin-2 may therefore be a scavenger molecule participating in the removal of host cells and maintenance of tissue homeostasis.     

Complement deficiency and systemic lupus erythematosus: consensus and dilemma

The involvement of the complement system in the pathogenesis of autoimmune diseases is a matter of debate. However, the link between complement abnormalities and systemic lupus erythematosus (SLE) is well established and widely described. Homozygous and/or heterozygous complement-component deficiencies of the classical pathway (C1q, C1r, C1s, C4A, C4B and C2) are causally associated with susceptibility to the development of SLE. Although the severity of the disease and the strength of the association are heterogeneous for deficiencies of these proteins, they commonly cause peculiar SLE syndromes with an early age of onset, a susceptibility to bacterial infections and negative anti-dsDNA antibodies. In this review, we highlight the available data on complement deficiency and SLE with a focus on deficiencies in classical complement pathway components. We also discuss the paradox of the link between complement deficiency and lupus. The complement system acts as a ‘friend’ through the clearance of immune complexes and apoptotic cells, which explains the close association between complement deficiency and lupus. It also acts as an ‘enemy’ by participating in the effector inflammatory phase of the autoimmune response. Understanding the importance of complement deficiencies should provide novel targets for therapeutic interventions in the modulation of the immune response.     

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q‐mediated complement system in the basal chordate

The origin of the classical complement pathway remains open during chordate evolution. A C1q‐like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan‐binding lectin‐associated serine protease‐2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q‐like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q‐mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway.     

Human complement Factor H modulates C1q-mediated phagocytosis of apoptotic cells

Complement is implicated in the clearance of apoptotic cells by phagocytes. Deficiencies in early complement components, particularly C1q, are associated with an increased risk of the development of systemic lupus erythematosus. C1q is considered to be important in this process through interaction with apoptotic cells and phagocytes. In the present study, we confirm that apoptotic cells are recognized not only by C1q but also by the complement regulatory protein Factor H. Both C1q and Factor H bind to apoptotic cells in a dose-dependent and saturable manner. We further examined the role of C1q and Factor H in the clearance of apoptotic cells by monocytes. C1q enhanced uptake/adhesion of apoptotic cells by monocytes whereas Factor H alone had no effect on this process. However, when both C1q and Factor H were present on the apoptotic cell surface, C1q-mediated enhancement of uptake/adhesion of the apoptotic cells by monocytes was reduced. This effect of Factor H also occurred if monocytes were pre-treated with Factor H, and then exposed to C1q-coated apoptotic cells. The results were consistent with Factor H interacting with monocytes through the integrin CD11b/CD18. We conclude that under physiological conditions, Factor H may be important in controlling the inflammation which might arise from C1q deposition on apoptotic cells.     

Mannose-binding lectin engagement with late apoptotic and necrotic cells

The serum opsonin mannose-binding lectin (MBL) has been shown to be involved in the handling of apoptotic cells. However, at what stage in the process this happens and whether this mediates activation of complement is unknown. Cells rendered apoptotic or necrotic were incubated with purified MBL/MBL-associated serine protease (MASP) complexes and assessed by flow cytometry and fluorescence microscopy. MBL bound specifically to late apoptotic cells, as well as to apoptotic blebs and to necrotic cells, but not to early apoptotic cells. Binding of MBL could be inhibited by EDTA as well as with an antibody against the CRD region. Addition of C1q, another serum opsonin involved in the handling of apoptotic cells, prior to MBL partly inhibited MBL binding to apoptotic cells and vice versa. MBL/MASP could initiate deposition of purified complement C4 on the target cells. However, addition of MBL/MASP to whole serum deficient for both C1q and MBL did not enhance deposition of C4, but MBL enhanced phagocytosis of apoptotic cells by macrophages. These results demonstrate that MBL interacts with structures exposed on cells rendered late apoptotic or necrotic and facilitates uptake by macrophages. Thus, MBL may promote non-inflammatory sequestration of dying host cells.     

Classical pathway complement activation on human endothelial cells

Endothelial cells regulate vascular integrity and express complement binding proteins including gC1qR/p33 (gC1qR), which recognize C1q, a subunit of the first component of the classical complement pathway. Experiments were performed to investigate classical complement pathway activation on resting endothelial cells and endothelial cells exposed to shear stress. C1q deposition and C4 activation (C4d) were demonstrated by solid phase ELISA and flow cytometry on human microvascular and umbilical vein endothelial cells after exposure to serum or plasma. C4d deposition was accompanied by downstream complement activation including C3b and C5b-9 deposition. C4 activation failed to occur in C1q depleted serum, but was not affected by Factor B depleted serum, confirming classical complement pathway activation. Moreover, C4 activation occurred following exposure of endothelial cells to purified C1 and C4, in the absence of other plasma proteins, and in the absence of detectable cell surface IgG and IgM. Shear stress (18 dynes/cm2) increased C1q (n=9, p<0.05) and C4d (n=9, p<0.05) deposition approximately two-fold, and enhanced endothelial cell gC1qR expression (n=7, p<0.05). Treatment of endothelial cells with anti gC1qR monoclonal antibody F(ab')2 fragments reduced C4d deposition by approximately 20% (n=5, p<0.05). These data demonstrate direct classical complement pathway activation on endothelial cells. gC1qR appears to play a minor but definable role, whereas cell surface IgG or IgM are not required.