An assay for the mannan-binding lectin pathway of complement activation.

The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs). When bound to microorganisms, the MBL complex activates the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. This C4/C2 cleaving activity is shared with the C1 complex of the classical pathway of complement activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt the C1 complex, whereas the carbohydrate-binding activity of MBL and the integrity of the MBL complex is maintained under hypertonic conditions. In the assay described here, the specific C4b-depositing capacity of the MBL pathway was determined by incubating serum diluted in buffer containing 1 M NaCl in mannan-coated microtiter wells before the addition of purified C4. The interassay coefficient of variation in the ELISA version was 7.3%. As expected no activity was found in MBL-deficient serum. When 100 normal serum samples were analysed we found that the MBL level correlated with the amount of C4b deposited on the mannan-coated surface. However, we also found a threefold variation in C4b-depositing capacity between individuals with similar MBL concentrations. The assay permits for the determination of MBL complex activity in serum and plasma samples and may thus be used to evaluate the clinical implications of complement activation via this pathway.     

Human neutrophil peptide-1 inhibits both the classical and the lectin pathway of complement activation

Human neutrophil peptide-1 (HNP-1) is a member of the alpha-defensin family. Defensins are cationic antimicrobial peptides, which play an important role in the antimicrobial response to microorganisms. In addition, recent studies have revealed the involvement of defensins in inflammation, immunity and wound repair. Defensins are present in the azurophilic granules of neutrophils and are released upon neutrophil stimulation. Previous studies showed that HNP-1 binds to C1q and inhibits the classical complement pathway. In view of the structural and functional similarity between C1q and MBL, we have now examined the interactions between HNP-1 and MBL. We observed a dose-dependent binding of HNP-1 to MBL in calcium-free buffer, indicating that HNP-1 binds to MBL most likely via the collagenous domains. To identify the binding sites in HNP-1 involved in the binding to C1q and MBL, we used a series of overlapping synthetic linear peptides that spanned the entire HNP-1 sequence. Both MBL and C1q showed a dose-dependent binding to the same set of peptides, suggesting a similar binding site in HNP-1 for both MBL and C1q. Strongest binding was observed to peptides containing the C- or N-terminal part of the HNP-1 molecule. Using an ELISA based system, we demonstrated that HNP-1 inhibits activation of both the classical pathway and lectin pathway of complement. Furthermore, we demonstrated that C1q and MBL can form complexes with HNP-1 in solution. Together, the data indicate that HNP-1 interacts with both C1q and MBL efficiently resulting in inhibition of both the classical and the lectin pathway of complement. We conclude that HNP-1 may play a role in protection against tissue injury during inflammatory conditions by inhibiting the early phase of complement activation.     

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.     

Assays for the functional activity of the mannan-binding lectin pathway of complement activation

Mannan-binding lectin (MBL) activates complement independently of the adaptive, clonal immune system and thus presents an innate anti-microbial defence mechanism. Events in the MBL pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented by the surface of micro-organisms. For the activation of complement to occur MBL must be associated with serine proteases (MBL-associated serine proteases, MASPs) in an MBL/MASP complex. When bound to micro-organisms, the MBL complex mediates the activation of C4 and C2, generating the C3 convertase, C4bC2b. The C4/C2 cleaving activity of the MBL complex is shared with the C1 complex of the classical pathway of complement activation. Different assays that allow for determination of the activity of the MBL complex in serum samples have been developed and are discussed in this report. We present data from one such assay (MBL/MASP activity test), which we have found useful for the routine evaluation of clinical samples. In this assay any influence of the classical pathway has been eliminated by using a hypertonic buffer, which inhibits the binding of C1q to immuncomplexes and disrupt the C1 complex, while leaving the function of the MBL complex intact. In parallel we determine the MBL concentration in the sample. As predicted a very high correlation is observed between the results of the two assays.     

The phylogeny of the complement system and the origins of the classical pathway

The origins of the complement system have now been traced to near to the beginnings of multi-cellular animal life. Most of the evidence points to the earliest activation mechanism having been more similar to the lectin pathway than to the alternative pathway. C1q, the immunoglobulin recognition molecule of the classical pathway of the vertebrates, has now been shown to predate the development of antibody as it has been found in the lamprey, a jawless fish that lacks an acquired immune system. In this species, C1q acts as a lectin that binds MASPs and activates the C3/C4-like thioester protein of the lamprey complement system. The classical pathway can, therefore, be regarded as a specialised arm of the lectin pathway in which the specificity of C1q for carbohydrate has been recruited to recognise the Fc region of immunoglobulin.     

Structural and functional aspects of complement activation by mannose-binding protein

Serum mannose-binding protein (MBP) is the first component of the lectin pathway of the complement cascade. It binds to sugars on the surface of pathogenic microorganisms and triggers complement fixation by activating an associated serine protease, designated MBP-associated serine protease-2 (MASP-2). Recent studies have provided insight into the interactions between MBP and MASP-2 that trigger complement activation. MBP/MASP complexes share many features with the C1 complex of the classical pathway. The relatively simple MBP/MASP complexes serve as useful models for understanding activation of the classical pathway of the complement cascade.     

The lectin-complement pathway – its role in innate immunity and evolution

Summary: Innate immunity was formerly thought to be a non‐specific immune response characterized by phagocytosis. However, innate immunity has considerable specificity and is capable of discriminating between pathogens and self. Recognition of pathogens is mediated by a set of pattern recognition receptors, which recognize conserved pathogen‐associated molecular patterns (PAMPs) shared by broad classes of microorganisms, thereby successfully defending invertebrates and vertebrates against infection. Lectins, carbohydrate‐binding proteins, play an important role in innate immunity by recognizing a wide range of pathogens. Mannose‐binding lectin (MBL) and ficolin are lectins composed of a lectin domain attached to collagenous region. However, they use a different lectin domain: a carbohydrate recognition domain (CRD) is responsible for MBL and a fibrinogen‐like domain for ficolin. These two collagenous lectins are pattern recognition receptors, and upon recognition of the infectious agent, they trigger the activation of the lectin‐complement pathway through attached serine proteases, MBL‐associated serine proteases (MASPs). A similar lectin‐based complement system, consisting of the lectin–protease complex and C3, is present in ascidians, our closest invertebrate relatives, and functions in an opsonic manner. We isolated several lectins homologous to MBLs and ficolins and several MASPs in invertebrates and lower vertebrates, and herein we discuss the molecular evolution of these molecules. Based on these findings, it seems likely that the complement system played a pivotal role in innate immunity before the evolution of an acquired immune system in jawed vertebrates.     

MASP-1 and MASP-3 Bind Directly to Aspergillus fumigatus and Promote Complement Activation and Phagocytosis

Activation of the complement system is mediated by the interaction between pathogens and pattern recognition molecules (PRMs); mannose-binding lectin (MBL), ficolins, and collectin-10/-11 from the lectin pathway and C1q from the classical pathway. Lectin pathway activation specifically depends on proteases named MBL-associated serine proteases (MASPs) that are found in complexes with PRMs. In this study, we hypothesize that MASPs can recognize selected pathogens independently of PRMs. Using different clinical strains of opportunistic fungi, we have observed that MASPs directly recognize certain fungal pathogens in a way that can facilitate complement activation. Among these were Aspergillus fumigatus − a dangerous pathogen, especially for immunocompromised patients. In flow cytometry and fluorescence microscopy, we found that MASP-1 and −3 bound to all A. fumigatus growth stages (conidia, germ tubes, and hyphae), whereas rMASP-2 and the nonproteolytic rMAP-1 did not. Bound rMASPs could recruit rMBL and rficolin-3 to A. fumigatus conidia in a nonclassical manner and activate complement via rMASP-2. In experiments using recombinant and purified components, rMASP-1 increased the neutrophilic phagocytosis of conidia. In serum where known complement activation pathways were blocked, phagocytosis could be mediated by rMASP-3. We have encountered an unknown pathway for complement activation and found that MASP-1 and MASP-3 have dual functions as enzymes and as PRMs.     

The mannan-binding lectin-associated serine proteases (MASPs) and MAp19: four components of the lectin pathway activation complex encoded by two genes

Mannan-binding lectin (MBL) and ficolins (L-ficolin and H-ficolin) initiate the lectin pathway of complement activation upon binding to microbial carbohydrates. The activation is mediated by associated serine proteases, termed MASPs, since they were discovered as MBL-associated serine proteases. The MASP family comprises three serine proteases, MASP-1, MASP-2 and MASP-3 and a non-enzymatic protein, MAp19. The MASPs show identical domain structure, shared also with C1r and C1s. MASP-1 and MASP-3 are alternative splice products of a single gene, MASP1/3, and have identical A chains, whereas they have individual B chains, encompassing the serine protease domain. MASP2 and MAp19 are alternative splice products of the MASP-2 gene, with MAp19 consisting of the first two domains of MASP-2 plus additional four amino acid residues. MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4bC2b. The biological function of the remaining three proteins has not yet been resolved.     

C3 dysregulation due to factor H deficiency is mannan‐binding lectin‐associated serine proteases (MASP)‐1 and MASP‐3 independent in vivo

Uncontrolled activation of the complement alternative pathway is associated with complement‐mediated renal disease. Factor B and factor D are essential components of this pathway, while factor H (FH) is its major regulator. In complete FH deficiency, uncontrolled C3 activation through the alternative pathway results in plasma C3 depletion and complement‐mediated renal disease. These are dependent on factor B. Mannan‐binding lectin‐associated serine proteases 1 and 3 (MASP‐1, MASP‐3) have been shown recently to contribute to alternative pathway activation by cleaving pro‐factor D to its active form, factor D. We studied the contribution of MASP‐1 and MASP‐3 to uncontrolled alternative pathway activation in experimental complete FH deficiency. Co‐deficiency of FH and MASP‐1/MASP‐3 did not ameliorate either the plasma C3 activation or glomerular C3 accumulation in FH‐deficient mice. Our data indicate that MASP‐1 and MASP‐3 are not essential for alternative pathway activation in complete FH deficiency.     

Lectin complement system and pattern recognition

Living organisms have strong defense mechanisms against invading microorganisms as survival strategies. One of the defense mechanisms is the complement system, composed of more than 30 serum and cell surface components. This system collaborates in recognition and elimination of pathogens as a part of both the innate and acquired immune systems. The two collagenous lectins, mannose-binding lectin (MBL) and ficolins, are pattern recognition proteins acting in innate immunity and, upon recognition of the pathogens, they trigger the activation of the lectin complement pathway through attached serine proteases (MASPs). A similar lectin-based complement system, consisting of the lectin-protease complex and C3, is present in ascidians, our closest invertebrate relatives and in lamprey, the most primitive vertebrate. Furthermore, a lamprey N-acetylglucosamine (GlcNAc)-binding lectin was identified as the orthlogue of mammalian C1q, and lamprey MASP is suggested as the prototype of MASP-2/C1r/C1s, indicating that the classical complement pathway arose as a part of the innate immune system. Thus, the complement system is one of the most highly organized innate immune systems in invertebrates and jawless vertebrates, and this system has survived in vertebrates with its core components little changed for 600-700 million years.     

The classical and regulatory functions of C1q in immunity and autoimmunity

A classical function of Clq is to bind immune complexes and initiate complement activation producing membrane lytic complexes, opsonins and anaphylatoxins. This classical pathway of complement activation is also elicited when Clq binds some other ligands. Besides complement activation, Clq also regulates cell differentiation, adhesion, migration, activation and survival. Clq deficiency is associated with autoimmunity as well as increased susceptibility to infections. In this article, we discuss the basic properties of Clq, its expression, and classical and regulatory functions. Cellular ＆ Molecular Immunology.     

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.     

Mechanism of complement-dependent haemolysis via the lectin pathway: role of the complement regulatory proteins.

Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b.     

Short leucine-rich glycoproteins of the extracellular matrix display diverse patterns of complement interaction and activation

The extracellular matrix consists of structural macromolecules and other proteins with regulatory functions. An important family of the latter class of molecules found in most tissues is the small leucine-rich repeat proteins (SLRPs). We have previously shown that the SLRP fibromodulin binds directly to C1q and activates the classical pathway of complement. In the present study we further examine the interactions between SLRPs and complement. Osteoadherin, like fibromodulin, binds C1q and activates the classical pathway strongly while moderate activation is seen in the terminal pathway. This can be explained by the interaction of fibromodulin and osteoadherin with factor H, a major soluble inhibitor of complement. Also, chondroadherin was found to bind C1q and activate complement, albeit to a lesser extent. Chondroadherin also binds factor H. We confirm published data showing that biglycan and decorin bind C1q but do not activate complement. In this study a similar pattern is seen for lumican although its affinity for C1q is lower than for biglycan and decorin. Furthermore, using electron microscopy and radiolabeled SLRPs, we demonstrate two different classes of SLRP binding sites on C1q, to head and stalk respectively, where only binding to the head appears to be activating. We propose a role for SLRPs in the regulation of complement activation in diseases involving the extracellular matrix, particularly those characterized by chronic inflammation such as rheumatoid arthritis, atherosclerosis, osteoarthritis and chronic obstructive lung disease.     

Effect of supraphysiologic levels of C1-inhibitor on the classical, lectin and alternative pathways of complement

C1-inhibitor is increasingly used experimentally and clinically in inflammatory conditions like septicemia and ischemia-reperfusion injury. Several mechanisms may account for the anti-inflammatory effects of C1-inhibitor, including inhibition of complement. The aim of the present study was to investigate and compare the supraphysiologic effect of C1-inhibitor on the three complement pathways. Novel assays for specific evaluation of the classical, lectin and alternative pathways were employed using normal human serum supplemented with increasing concentrations of C1-inhibitor. Solid-phase classical- and lectin pathway activation was dose-dependently and significantly reduced up to 85% in the range of 2-28 times physiologic C1-inhibitor concentration. The lectin pathway was more potently inhibited than the classical at low doses. A functional lectin pathway assay demonstrated a significant reduction of C4 deposition up to 86% even at low concentration of C1-inhibitor and documented the effect to be at the level of MBL/MASPs. In contrast, C1-inhibitor had no effect on solid-phase alternative pathway activation, but significantly reduced cobra venom factor-induced fluid-phase activation up to 88%. The negative controls albumin and IgG had no effect on complement activation. The positive inhibitory controls compstatin (C3 inhibition), EDTA- or MBL-deficient sera reduced complement activation by 82-100%. We conclude that C1-inhibitor in high physiologic doses differentially inhibits all three-complement pathways. The inhibition pattern was strikingly different in the classical and lectin pathway, compared to the alternative. Previous studies interpreting the effects of C1-inhibitor as only due to classical pathway inhibition needs reconsideration. The data has implications for the therapeutic use of C1-inhibitor.     

Ficolins and the lectin complement pathway

Ficolins, found in various tissues, are a group of proteins containing both a collagen‐like and a fibrinogen‐like domain. Recently, it was shown that ficolins present in serum are lectins with a common binding specificity for N‐acetylglucosamine (GlcNAc). The fibrinogen‐like domain is responsible for the carbohydrate binding. Mannose‐binding lectin (MBL) is also a collagenous lectin in serum that is specific for GlcNAc and mannose binding. Its domain organization is similar to that of ficolins, except that MBL has a carbohydrate‐recognition domain instead of a fibrinogen‐like domain. MBL plays a role in innate immunity by acting as an opsonin and activating complement in association with MBL‐associated serine protease (MASP) via the lectin pathway. Investigations of two types of human serum ficolins, ficolin/P35 and Hakata antigen, revealed that they are associated with MASPs and sMAP, a truncated protein of MASP‐2, and that they activate complement. These findings indicate that serum ficolins are structurally and functionally similar to MBL and have the capacity to activate the lectin pathway and thus have a role in innate immunity. This work was supported by Grants‐in‐Aid for Scientific Research (12470079 and 11670328) from the Ministry of Education, Science, Sports and Culture of Japan.     

Structural and Functional Overview of the Lectin Complement Pathway: Its Molecular Basis and Physiological Implication

The complement system is an effector mechanism in immunity. It is activated in three ways, the classical, alternative and lectin pathways. The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) or ficolins to carbohydrates on the surfaces of pathogens. In humans, MBL and three types of ficolins (L-ficolin, H-ficolin, and M-ficolin) are present in plasma. Of these lectins, at least, MBL, L-ficolin, and H-ficolin are complexed with three types of MBL-associated serine proteases (MASPs), MASP-1, MASP-2, and MASP-3 and their truncated proteins (MAp44 and sMAP). In the lectin pathway, the lectin–MASP complex (i.e., a complex of lectin, MASPs and their truncated proteins) binds to pathogens, resulting in the activation of C4 and C2 to generate a C3 convertase capable of activating C3. MASP-2 is involved in the activation of C4 and C2. MASP-1 activates C2 and MASP-2. The functions of MASP-3, sMAP, and MAp44 in the lectin pathway remain unknown. MASP-1 and MASP-3 also have a role in the alternative pathway. MBL and ficolins are able to bind to a variety of pathogens depending on their carbohydrate binding specificity, resulting in the activation of the lectin pathway. Deficiencies of the components of the lectin pathway are associated to susceptibility to infection, indicating an important role of the lectin pathway in innate immunity. The lectin-MASP complex is also involved in innate immunity by activating the coagulation system. Recent findings suggest a crucial role of MASP-3 in development.     

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.