Role of mannose-binding lectin in regulating monocytes/macrophages functions during Aeromonas hydrophila infection in grass carp, Ctenopharyngodon idella.

Mannose-binding lectin (MBL) is a vital component in host's innate immune system and the initiator of the lectin pathway of complement cascade. However, its opsonic role has rarely been reported. In this study, we revealed the biological function of Ctenopharyngodon idella MBL (CiMBL) in regulating monocytes/macrophages (MO/MФ) in the grass carp (C. idella). Flow cytometry results indicated that recombinant CiMBL (rCiMBL) significantly enhanced the phagocytotic activity of MO/MФ. Recombinant CiMBL also enhanced bactericidal activity and respiratory burst capacity in Aeromonas hydrophila-infected MO/MФ, regulated A. hydrophila-induced polarization of MO/MФ including down- and up-regulated pro- and anti-inflammatory cytokines, respectively, suppressed the inducible nitric oxide synthase activity, and enhanced the arginase activity. In addition, rCiMBL suppressed the bacteria burden in tissues and blood in vivo and enhanced the survival rate of juvenile A. hydrophila-infected grass carp. We provide evidence that CiMBL was synthesized by MO/MФ, regulating the biological function of MO/MФ against A. hydrophila infection.

Pharmacological inhibition of mannose-binding lectin ameliorates neurobehavioral dysfunction following experimental traumatic brain injury.

Mannose-binding lectin is present in the contusion area of traumatic brain-injured patients and in that of traumatic brain-injured mice, where mannose-binding lectin-C exceeds mannose-binding lectin-A. The reduced susceptibility to traumatic brain injury of mannose-binding lectin double knock-out mice (mannose-binding lectin-/-) when compared to wild type mice suggests that mannose-binding lectin may be a therapeutic target following traumatic brain injury. Here, we evaluated the effects of a multivalent glycomimetic mannose-binding lectin ligand, Polyman9, following traumatic brain injury in mice. In vitro surface plasmon resonance assay indicated that Polyman9 dose-dependently inhibits the binding to immobilized mannose residues of plasma mannose-binding lectin-C selectively over that of mannose-binding lectin-A. Male C57Bl/6 mice underwent sham/controlled cortical impact traumatic brain injury and intravenous treatment with Polyman9/saline. Ex-vivo surface plasmon resonance studies confirmed that Polyman9 effectively reduces the binding of plasma mannose-binding lectin-C to immobilized mannose residues. In vivo studies up to four weeks post injury, showed that Polyman9 induces significant improvement in sensorimotor deficits (by neuroscore and beam walk), promotes neurogenesis (73% increase in doublecortin immunoreactivity), and astrogliosis (28% increase in glial fibrillary acid protein). Polyman9 administration in brain-injured mannose-binding lectin-/- mice had no effect on post-traumatic brain-injured functional deficits, suggestive of the specificity of its neuroprotective effects. The neurobehavioral efficacy of Polyman9 implicates mannose-binding lectin-C as a novel therapeutic target for traumatic brain injury.

A New Surface Plasmon Resonance Assay for In Vitro Screening of Mannose-Binding Lectin Inhibitors.

Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro screening of these compounds, which may be useful before the more expensive and time-consuming in vivo studies. The assay measures the inhibitor's ability to interfere with the binding of murine MBL-A or MBL-C, or of human recombinant MBL, to mannose residues immobilized on the sensor chip surface. We have applied the assay to measure the IC50 of synthetic glycodendrimers, two of them with neuroprotective properties in animal models of MBL-mediated injuries.

Tunicamycin-induced inhibition of protein secretion into culture medium of Arabidopsis T87 suspension cells through mRNA degradation on the endoplasmic reticulum.

The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.

Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 µg/mL in MBL2(+/+)Mbl1(-/-)Mbl2(-/-) [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 µg/mL) or 14 (9 µg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride-induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.

Functional assessment of rat complement pathway activities and quantification of soluble C5b-9 in an experimental model of renal ischemia/reperfusion injury.

There is a growing interest in the monitoring of complement activation, not only in clinical settings but also in experimental models. However, for rodents only a limited number of tools are available to assess complement activity and activation. Here we describe three ELISAs for the measurement of rat classical (CP), MB-lectin (LP) and alternative (AP) pathway activities in serum and plasma. Moreover, we optimised a soluble C5b-9 (sC5b-9) ELISA for the detection of low level complement activation in rat. We determined the conditions for correct sample handling and showed that the assays had low inter- and intra-assay variation. We applied these assays to monitor complement activation in an experimental rat model of renal ischemia/reperfusion injury. We did not observe major complement consumption following reperfusion in CP or LP, and only minor AP consumption at 24h post reperfusion. However, MBL depletion prior to ischemia/reperfusion using a monoclonal antibody, transiently and specifically inhibited 75% of LP activity and ameliorated the AP consumption at 24h. To further assess complement activation during renal IRI, we monitored serum sC5b-9 and found that it was only significantly increased 72h post-reperfusion, but not when rats were pre-treated with anti-MBL or after sham surgery. In conclusion the described assays enable sensitive, reproducible and comprehensive assessment of complement activation in experimental rat models.

Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.

Impact of mannose-binding lectin deficiency on radiocontrast-induced renal dysfunction.

Contrast-induced nephropathy (CIN) is the third leading cause of acute renal failure in hospitalized patients. Endothelial dysfunction, renal medullary ischemia, and tubular toxicity are regarded as the most important factors in the pathogenesis of CIN. Mannose-binding lectin (MBL), a pattern recognition protein of the lectin pathway of complement, has been found to aggravate and mediate tissue damage during experimental renal ischemia/reperfusion (I/R) injury which was alleviated by inhibition with C1 inhibitor, a potent MBL, and lectin pathway inhibitor. In this paper, we highlight the potential role of MBL in the pathogenesis of human CIN. In experimental I/R models, MBL was previously found to induce tubular cell death independent of the complement system. In addition, after binding to vascular endothelial cells, MBL and its associated serine proteases were able to trigger a proinflammatory reaction and contribute to endothelial dysfunction. In humans, urinary MBL was increased after administration of contrast media and in individuals with CIN. Moreover, individuals with normal/high MBL levels were at increased risk to develop radiocontrast-induced renal dysfunction. Hence, MBL and the lectin pathway seem to be a promising target given that a licensed, powerful, human recombinant inhibitor exits to be added to the scarce armamentarium currently available for prophylaxis of CIN.

The mannose-binding lectin pathway is a significant contributor to reperfusion injury in the type 2 diabetic heart.

The severity of ischaemic heart disease is markedly enhanced in type 2 diabetes. We recently reported that complement activation exacerbates I/R injury in the type 2 diabetic heart. The purpose of this study was to isolate and examine MBL pathway activation following I/R injury in the diabetic heart. ZLC and ZDF rats underwent 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Two different groups of ZDF rats were treated with either FUT-175, a broad complement inhibitor, or P2D5, a monoclonal antibody raised against rat MBL-A. ZDF rats treated with FUT175 and P2D5 had significantly decreased myocardial infarct size, C3 deposition and neutrophil accumulation compared with untreated ZDF controls. Taken together, these findings indicate that the MBL pathway plays a key role in the severity of complement-mediated I/R injury in the type 2 diabetic heart.

A novel interpretation of immune redundancy and duality in reperfusion injury with important implications for intervention in ischaemic disease.

The majority of ischaemia related injury occurs upon tissue reperfusion. Knock-out mouse models have recently shed light on the underlying molecular mechanisms, and suggest that this may be the result of an innate autoimmune response. Based on these new findings we present a novel model of immune redundancy and duality in reperfusion injury. Natural antibody, mannan-binding lectin and toll-like receptor 4 are three pre-formed innate immune receptors that recognise pathogenic molecular patterns. Removing either significantly ameliorates reperfusion injury. We propose that these three receptors serve as key parallel recognition elements that respond to the same or similar ischaemic neo-antigens, of which at least one may have a lipopolysaccharide-like motif. This would fit both with the ligand preference of the three receptors, and the observation that giving monoclonal antibody to lipopolysaccharide reduces reperfusion injury. The consequent injury caused by receptor activation appears to be mainly related to the complement anaphylatoxins, and less to phagocytes, oxidative radicals, and the membrane attack complex. C5a levels in particular are predictive of overall injury, and we suggest this anaphylatoxin causes most of reperfusion injury via both direct toxic effects and a generalised immune activation. The former is illustrated by the recent observation that excess C5a alone can cause cardiac dysfunction. As for the latter, there is evidence that adaptive immunity (especially CD4+ cells) and other serum cascades (coagulation and kallikrein) are involved, and may have been recruited by complement. Furthermore, excess C5a can cause innate immune overactivation that paralyses neutrophils, reduces complement lytic function, and leads to systemic inflammation. This is analogous to what happens in sepsis, and would explain the passive role in IRI of normal immune effectors. Finally, there is a duality complement's function in reperfusion, as some elements are conductive of damage, whilst others may help inflammatory resolution. Most important among the latter are the opsonins, like C3b and apparently C1q, which help macrophages clear apoptosing cells before they undergo secondary necrosis. This model has important implications for clinical interventions. Firstly, redundancy means that inhibiting multiple receptors may achieve a larger mortality reduction than the small and inconsistent one seen in the published monotherapy trials. Secondly, duality means that a non-specific inhibition of complement would reduce both injury and resolution. Therefore, a specific inhibition of the lectin pathway and/or an inhibition of the downstream effectors upon which the receptors converge (e.g. C5a) seem to be a better interceptive strategy.

Degalactosylated and/or denatured IgA, but not native IgA in any form, bind to mannose-binding lectin.

Mannose-binding lectin (MBL) is reported to bind to agalactosyl IgG, but not to normally galactosylated (native) IgG. It was recently reported that serum polymeric IgA in its native form reacts with MBL, whereas a more recent report has claimed that native IgD and IgE, and possibly IgM, do not. This led us to investigate whether IgA is truly reactive with MBL. To accomplish this, we collected purified human Igs, of various classes, subclasses, and allotypes, and tested their ability to bind to MBL using an ELISA method. Among these preparations, only one (monoclonal IgA2m(2):Kur) exhibited significant MBL binding. In particular, polymeric or monomeric forms of our normal serum IgA preparation lacked any ability to bind to MBL whatsoever. However, all the Ig preparations which had not bound to MBL became able to do so when they were degalactosylated with a galactosidase treatment, and the binding was further enhanced by acidic denaturation of the Igs. Among the degalactosylated and/or acid-denatured IgA, the IgA2 subclass exhibited a higher level of MBL binding than did IgA1. Our results suggest that MBL does not bind to native Igs (viewed in principle as "self" components), and that only Igs with abnormal glycosylation (degalactosylated forms) and/or denaturation would be MBL reactive.

Detection of an autologous ligand for mannan-binding lectin on human B lymphocytes.

Mannan-binding lectin (MBL) is a collectin and a major soluble pattern-recognition protein. MBL can distinguish self from nonself and altered self using its C-type carbohydrate recognition domain and may also interact via its collagen-like region with autologous cells. Recently, it was found that MBL could bind to adherent cells (monocytes) and dendritic cells in a specific and sugar-sensitive manner. We have now investigated the interaction of MBL with fresh human peripheral blood cells and report binding to B lymphocytes and natural killer cells. The binding to B lymphocytes was studied in detail and was compared with the binding of MBL to monocytes and dendritic cells. Binding of MBL to B cells was evident at physiological MBL and calcium concentrations but was optimal at supraphysiological MBL concentrations. It was readily inhibited by autologous serum, mannan, mannose, GlcNAc and (to a lesser extent) galactose but not by C1q. A similar, but not identical, inhibition profile was observed with dendritic cells, but monocytes were not sensitive to mannose or mannan. We conclude that MBL is capable of binding to differently glycosylated ligands on several autologous cell types via its carbohydrate-recognition domain. We speculate that this could have functional significance at extravascular sites, but perhaps only in individuals possessing MBL genotypes conferring MBL sufficiency.

Mannose-binding lectin recognizes peptidoglycan via the N-acetyl glucosamine moiety, and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages.

Peptidoglycan (PGN) is the major cell wall component (90%, w/w) of Gram-positive bacteria and consists of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide repeating arrays that are cross-linked by short peptides. We hypothesized that PGN is a ligand for pathogen-associated pattern-recognition proteins. Mannose-binding lectin (MBL) and serum amyloid component P are two carbohydrate-binding innate immune proteins present in the blood. In this study we show that human MBL, but not serum amyloid component P, binds significantly to PGN via its C-type lectin domains, and that the interaction can be more effectively competed by GlcNAc than by MurNAc. Surface plasmon resonance analyses show that native MBL binds immobilized PGN with high avidity. Competition experiments also show that both native MBL and MBL(n/CRD), a 48-kDa recombinant trimeric fragment of MBL containing neck and carbohydrate recognition domains, have higher affinity for GlcNAc than for MurNAc. Protein arrays and ELISA show that PGN increases the secretion of TNF-alpha, IL-8, IL-10, MCP-2, and RANTES from PMA-stimulated human monocytic U937 cells. Interestingly, the presence of MBL together with PGN increases the production of IL-8 and RANTES, but reduces that of TNF-alpha. Our results indicate that Gram-positive bacterial is a biologically relevant ligand for MBL, and that the collectin preferentially binds to the GlcNAc moiety of the PGN via its C-type lectin domains. MBL inhibits PGN-induced production of proinflammatory cytokines while enhancing the production of chemokines by macrophages, which suggests that MBL may down-regulate macrophage-mediated inflammation while enhancing phagocyte recruitment.

Mannan-binding proteins from boar seminal plasma.

The interaction of boar seminal plasma proteins and sperm with yeast mannan was investigated by the enzyme-linked binding assay (ELBA) and specific detection of proteins after SDS electrophoresis and blotting using biotinylated derivative of the polysaccharide. Heparin-binding proteins (especially AQN 1 and DQH proteins) and their aggregated forms showed affinity to yeast mannan. Besides that, these proteins were shown to bind to oviductal epithelium. The mannan-binding activity of boar proteins and sperm was inhibited most efficiently by ovomucoid, ovalbumin and N-glycans released from ovalbumin, but not with d-glucose, d-mannose and their phosphates. On the other hand, yeast mannan inhibited both the interaction of boar seminal plasma and sperm with heparin and the binding of these proteins to porcine oviductal epithelium. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. Proteins adsorbed to the immobilized polysaccharide were analyzed by RP-HPLC, SDS electrophoresis and N-terminal amino acid sequencing. AQN and AWN spermadhesins and DQH protein (names are derived from the N-terminal amino acid sequence) were identified as components of the isolated fraction. The results suggest an involvement of mannan-binding proteins in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both the complex d-mannose-containing saccharide structures and the heparin may also play an important role in sperm release from the oviductal reservoir or the capacitation process.

The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi.

The high resistance of Trypanosoma cruzi trypomastigotes, the causal agent of Chagas' disease, to complement involves several parasite strategies. In these in vitro studies, we show that T. cruzi calreticulin (TcCRT) and two subfragments thereof (TcCRT S and TcCRT R domains) bind specifically to recognition subcomponents of the classical and lectin activation pathways (i.e., to collagenous tails of C1q and to mannan-binding lectin) of the human complement system. As a consequence of this binding, specific functional inhibition of the classical pathway and impaired mannan-binding lectin to mannose were observed. By flow cytometry, TcCRT was detected on the surface of viable trypomastigotes and, by confocal microscopy, colocalization of human C1q with surface TcCRT of infective trypomastigotes was visualized. Taken together, these findings imply that TcCRT may be a critical factor contributing to the ability of trypomastigotes to interfere at the earliest stages of complement activation.

Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments.

Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only approximately 0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only approximately 1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.

A hemolytic assay for the estimation of functional mannose-binding lectin levels in human serum.

A simple assay was developed to estimate functional mannose-binding lectin (MBL) levels in serum based on the principle of yeast-induced bystander lysis of chicken erythrocytes (ChE). The assay is sensitive to inhibition by ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (which allows alternative pathway activation), ethylene diamine tetraacetic acid (EDTA), mannose, N-acetylglucosamine and C1 esterase inhibitor (C1-INH), whereas it was not inhibited by galactose. A high-titer human anti-mannan antibody-containing serum with 0.06 microg MBL/ml gave a functional signal corresponding to 0.12 microg equivalents MBL/ml, indicating that anti-mannan antibodies are poorly hemolytic in the assay. The assay is well suited for the large-scale testing of patient samples for a functional MBL pathway of complement activation.