Annette G. Hansen

Distinct Pathways of Mannan-Binding Lectin (MBL)- and C1-Complex Autoactivation Revealed by Reconstitution of MBL with Recombinant MBL-Associated Serine Protease-2

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

MAp44, a Human Protein Associated with Pattern Recognition Molecules of the Complement System and Regulating the Lectin Pathway of Complement Activation

Essential effector functions of innate immunity are mediated by complement activation initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL) and the ficolins. We present a novel, phylogenetically conserved protein, MAp44, which is found in human serum at 1.4 μg/ml in Ca2+-dependent complexes with the soluble pattern recognition molecules. The affinity for MBL is in the nanomolar range (KD = 0.6 nM) as determined by surface plasmon resonance. The first eight exons of the gene for MAp44 encode four domains shared with MBL-associated serine protease (MASP)-1 and MASP-3 (CUB1-EGF-CUB2-CCP1), and a ninth exon encodes C-terminal 17 aa unique to MAp44. mRNA profiling in human tissues shows high expression in the heart. MAp44 competes with MASP-2 for binding to MBL and ficolins, resulting in inhibition of complement activation. Our results add a novel mechanism to those known to control the innate immune system.

Mannan-Binding Lectin-Associated Serine Protease (MASP)-1 Is Crucial for Lectin Pathway Activation in Human Serum, whereas neither MASP-1 nor MASP-3 Is Required for Alternative Pathway Function

The lectin pathway of complement is an important component of innate immunity. Its activation has been thought to occur via recognition of pathogens by mannan-binding lectin (MBL) or ficolins in complex with MBL-associated serine protease (MASP)-2, followed by MASP-2 autoactivation and cleavage of C4 and C2 generating the C3 convertase. MASP-1 and MASP-3 are related proteases found in similar complexes. MASP-1 has been shown to aid MASP-2 convertase generation by auxiliary C2 cleavage. In mice, MASP-1 and MASP-3 have been reported to be central also to alternative pathway function through activation of profactor D and factor B. In this study, we present functional studies based on a patient harboring a nonsense mutation in the common part of the MASP1 gene and hence deficient in both MASP-1 and MASP-3. Surprisingly, we find that the alternative pathway in this patient functions normally, and is unaffected by reconstitution with MASP-1 and MASP-3. Conversely, we find that the patient has a nonfunctional lectin pathway, which can be restored by MASP-1, implying that this component is crucial for complement activation. We show that, although MASP-2 is able to autoactivate under artificial conditions, MASP-1 dramatically increases lectin pathway activity at physiological conditions through direct activation of MASP-2. We further demonstrate that MASP-1 and MASP-2 can associate in the same MBL complex, and that such cocomplexes are found in serum, providing a scenario for transactivation of MASP-2. Hence, in functional terms, it appears that MASP-1 and MASP-2 act in a manner analogous to that of C1r and C1s of the classical pathway.

Favourable effect of TNF-α inhibitor (infliximab) on Blau syndrome in monozygotic twins with a de novoCARD15 mutation†

Blau syndrome is a hereditary granulomatous disease caused by mutations in the CARD15 gene that is diagnosed in children of young age with exanthema/erythema, arthritis/periarthritis and/or uveitis. We report two cases of Blau syndrome in Danish Caucasian monozygotic male twins, exhibiting a heterozygous de novo R334W mutation in codon 334 of CARD15. The patients were initially diagnosed as having sarcoidosis. In both twins, symptoms (exanthema, arthritis/periarthritis) started at 1 year of age, and were followed by uveitis at 7–10 years of age. There was no involvement of the lungs or other organs. An initial course of standard antituberculous treatment had no effect on the symptoms. Hydroxychloroquine and cyclosporine A were also ineffective, and the latter caused impaired renal function. Partial symptomatic relief was obtained with prednisolone and increased benefit was observed in combination with methotrexate. Subsequent introduction of the TNF‐α inhibitor eternacept did not discernibly benefit the clinical condition, but was associated with recurrent infections. In contrast, a trial of infliximab therapy demonstrated clinical efficacy and eliminated all symptoms, restoring a high quality of life. At follow up at 20 years of age (after 2–5 years of infliximab treatment) the twins had an almost normal physical appearance and a normal psychomotoric development, indicating a favourable short‐term prognosis of the disease. Blau syndrome has pathologic, clinical and therapeutic features in common with sarcoidosis, but rarely involves the lungs or other parenchymatous organs. In children, discrimination between early onset sarcoidosis and Blau syndrome should include a CARD15 mutation analysis.

Polymorphisms in Mannan-Binding Lectin (MBL)-Associated Serine Protease 2 Affect Stability, Binding to MBL, and Enzymatic Activity

Mannan-binding lectin-associated serine protease 2 (MASP-2) is an enzyme of the innate immune system. MASP-2 forms complexes with the pattern recognition molecules mannan-binding lectin (MBL), H-ficolin, L-ficolin, or M-ficolin, and is activated when one of these proteins recognizes microorganisms and subsequently cleaves complement factors C4 and C2, thus initiating the activation of the complement system. Missense polymorphisms of MASP-2 exist in different ethnic populations. To further characterize the nature of these, we have produced and characterized rMASP-2s representing the following naturally occurring polymorphisms: R99Q, D120G, P126L, H155R, 156_159dupCHNH (CHNHdup), V377A, and R439H. Only very low levels of CHNHdup were secreted from the cells, whereas quantities similar to wild-type MASP-2 were found intracellularly, indicating that this mutation results in a misfolded protein. We found that D120G and CHNHdup could not associate with MBL, whereas R99Q, P126L, H155R, V377A, R439H, and wild-type MASP-2 bound equally well to MBL. Accordingly, when D120G and CHNHdup were mixed with MBL, no activation of complement factor C4 was observed, whereas R99Q, P126L, and V377A cleaved C4 with an activity comparable to wild-type MASP-2 and H155R slightly better. In contrast, the R439H variant was deficient in this process despite its normal binding to MBL. This variant was also not able to autoactivate in the presence of MBL and mannan. We find the R439H variant is common in Sub-Saharan Africans with a gene frequency of 10%. Our results indicate that individuals with different types of MASP-2 defects may be identified through genotyping.

Investigations on the pattern recognition molecule M‐ficolin: quantitative aspects of bacterial binding and leukocyte association

M‐ficolin is a PRM of the innate immune system, found in serum and associated with leukocytes. We used the soluble form to study specificity toward Gram‐positive bacteria and characterized and quantified cell‐associated M‐ficolin. The binding of M‐ficolin to capsulated and noncapsulated strains of Streptococcus agalactiae (GBS) and Staphylococcus aureus was investigated. We did not observe binding of M‐ficolin to any of 13 serotypes of S. aureus. Dose‐dependent binding of M‐ficolin was demonstrated for all of the capsulated GBS strains. The binding was abolished by prior treatment of the bacteria with sialidase, indicating that sialic acid is the ligand for M‐ficolin on these bacteria. GlcNAc could inhibit the binding, suggesting that M‐ficolin binds via its FBG. M‐ficolin was found associated with the complement‐activating enzyme in serum, and M‐ficolin bound to GBS mediated activation of the complement system. M‐ficolin expression on leukocytes was evaluated by flow cytometry with anti‐M‐ficolin mAb. Total M‐ficolin of different leukocytes was quantified in detergent extracts. Monocytes and granulocytes showed similar M‐ficolin surface expression, 1.1 × 105 and 0.7 × 105 M‐ficolin molecules/cell, respectively. The total M‐ficolin content of the cells was 1.5 × 106 molecules/monocyte and approximately one‐third of this for granulocytes. Lymphocytes contained <1.5% of the amount estimated for monocytes, and none was revealed on the surface of lymphocytes by flow cytometry. Immunohistochemical analysis of the distribution of M‐ficolin in 25 tissues revealed staining of only granulocytes and monocytes. Reported M‐ficolin expression by type II pneumocytes could not be verified. We demonstrate the specific binding of M‐ficolin to sialic acids in the capsule of GBS and give quantitative aspects of the cell‐associated M‐ficolin.

H-ficolin serum concentration and susceptibility to fever and neutropenia in paediatric cancer patients

H‐ficolin (Hakata antigen, ficolin‐3) activates the lectin pathway of complement similar to mannose‐binding lectin. However, its impact on susceptibility to infection is currently unknown. This study investigated whether the serum concentration of H‐ficolin at diagnosis is associated with fever and neutropenia (FN) in paediatric cancer patients. H‐ficolin was measured by time‐resolved immunofluorometric assay in serum taken at cancer diagnosis from 94 children treated with chemotherapy. The association of FN episodes with H‐ficolin serum concentration was analysed by multivariate Poisson regression. Median concentration of H‐ficolin in serum was 26 mg/l (range 6–83). Seven (7%) children had low H‐ficolin (< 14 mg/l). During a cumulative chemotherapy exposure time of 82 years, 177 FN episodes were recorded, 35 (20%) of them with bacteraemia. Children with low H‐ficolin had a significantly increased risk to develop FN [relative risk (RR) 2·24; 95% confidence interval (CI) 1·38–3·65; P = 0·004], resulting in prolonged duration of hospitalization and of intravenous anti‐microbial therapy. Bacteraemia occurred more frequently in children with low H‐ficolin (RR 2·82; CI 1·02–7·76; P = 0·045). In conclusion, low concentration of H‐ficolin was associated with an increased risk of FN, particularly FN with bacteraemia, in children treated with chemotherapy for cancer. Low H‐ficolin thus represents a novel risk factor for chemotherapy‐related infections.

Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes

Significance A salient feature of the immune system is its ability to discriminate self from nonself. We define the molecular mechanism governing activation of an ancient and central component: the lectin pathway of complement. The basis is the association of two proteases in distinct complexes with at least five pattern recognition molecules. Clustering of these complexes on ligand surfaces allows cross-activation of the proteases, which subsequently activate downstream factors to initiate a proteolytic cascade. This is conceptually similar to signaling by cellular receptors and could be viewed as cellular signaling turned inside out. Different pattern recognition complexes “talk to each other” to coordinate immune activation, which may impart differential activation based on recognition of simple vs. complex ligand patterns. Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement.

MAp19, the alternative splice product of the MASP2 gene

Abstract The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217ng/ml, i.e., 11nM, comparable to the 7nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent.

M-ficolin in the neonatal period: Associations with need for mechanical ventilation and mortality in premature infants with necrotising enterocolitis

OBJECTIVE Necrotising enterocolitis (NEC) causes significant mortality in premature infants. The involvement of the innate immune system in the pathogenesis of NEC remains unclear. M-, L- and H-ficolins recognize microorganisms and activate the complement system, but their role in host defense is largely unknown. This study investigated whether ficolin concentrations are associated with NEC. STUDY DESIGN Case-control study including 30 premature infants with NEC and 60 controls. M-, L- and H-ficolins were measured in cord blood using time-resolved immunofluorometric assays. Multivariate logistic regression was performed. RESULTS Of the 30 NEC cases (median gestational age, 29.5 weeks), 12 (40%) were operated and 4 (13%) died. No difference regarding ficolin concentration was found when comparing NEC cases versus controls (p>0.05). However, infants who died of NEC had significantly lower M-ficolin cord blood concentrations than NEC survivors (for M-ficolin <300ng/ml; multivariate OR 12.35, CI 1.03-148.59, p=0.048). In the entire study population, M-, L- and H-ficolins were positively correlated with gestational age (p<0.001) and birth weight (p<0.001). Infants with low M-ficolin required significantly more often mechanical ventilation after birth multivariate (OR 10.55, CI 2.01-55.34, p=0.005). CONCLUSIONS M-, L- and H-ficolins are already present in cord blood and increase with gestational age. Low cord blood concentration of M-ficolin was associated with higher NEC-associated fatality and with increased need for mechanical ventilation. Future studies need to assess whether M-ficolin is involved in multiorgan failure and pulmonary disease.