The Lectin Complement Pathway in Patients with Necrotizing Soft Tissue Infection.

BACKGROUND: Mannose-binding lectin (MBL) and ficolins are pattern recognition molecules (PRMs) that play an important role during infection through activation of the lectin complement pathway. We assessed whether plasma PRM levels were associated with mortality in patients with necrotizing soft tissue infection (NSTI). METHODS: We conducted a prospective, observational study over 25 months involving 135 NSTI patients with a maximum follow-up of 2.7 years. Blood samples were taken upon admission. Non-infected patients served as controls. RESULTS: PRM levels were significantly lower compared with controls. A baseline Ficolin-2 level below the median was associated with mortality at the end of follow-up (p = 0.007). No significant association was found for MBL, Ficolin-1 and Ficolin-3. A Ficolin-2 level below the median had a negative predictive value of 0.94 for 28-day mortality, and a level below the optimal cut-off was independently associated with 28-day mortality when adjusted for age, sex and chronicity [hazard ratio 6.27 (95% confidence interval 2.28-17.21), p < 0.0001], also when Simplified Acute Physiology Score II was included in the analysis [hazard ratio 3.16 (95% confidence interval 1.03-9.73), p = 0.045]. CONCLUSIONS: All PRMs were significantly lower in patients with NSTI than in controls. Only baseline Ficolin-2 was associated with short- and long-term mortality. A high baseline Ficolin-2 level indicated a 94% chance of surviving the first 28 days after admission.

The Lectin Pathway of Complement and Biocompatibility.

In modern health technologies the use of biomaterials in the form of stents, haemodialysis tubes, artificial implants, bypass circuits etc. is rapidly expanding. The exposure of synthetic, foreign surfaces to the blood and tissue of the host, calls for strict biocompatibility in respect to contact activation, the coagulation system and the complement system. The complement system is an important part of the initial immune response and consists of fluid phase molecules in the blood stream. Three different activation pathways can initiate the complement system, the lectin, the classical and the alternative pathway, all converging in an amplification loop of the cascade system and downstream reactions. Thus, when exposed to foreign substances complement components will be activated and lead to a powerful inflammatory response. Biosurface induced complement activation is a recognised issue that has been broadly documented. However, the specific role of lectin pathway and the pattern recognition molecules initiating the pathway has only been transiently investigated. Here we review the current data on the field.

Genetically engineered fusion of MAP-1 and factor H domains 1-5 generates a potent dual upstream inhibitor of both the lectin and alternative complement pathways.

Inhibition of the complement cascade has emerged as an option for treatment of a range of diseases. Mannose-binding lectin/ficolin/collectin-associated protein (MAP-1) is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway. The central regulator of the alternative pathway (AP) is complement factor H (FH). Our aim was to design a dual upstream inhibitor of both human lectin and APs by fusing MAP-1 with a part of FH. There were 2 different recombinant chimeric proteins comprising full-length human MAP-1 and the first 5 N-terminal domains of human FH designed. The FH domains were orientated either in the N- or C-terminal part of MAP-1. The complement inhibition potential in human serum was assessed. Both chimeric constructs displayed the characteristics of the native molecules and bound to the PRMs with an EC50 of ∼ 2 nM. However, when added to serum diluted 1:4 in a solid-phase functional assay, only the first 5 N-terminal domains of complement FH fused to the C-terminal part of full-length MAP-1 chimeric construct were able to combine inhibition of lectin and AP activation with an half maximal inhibitory concentration of ∼ 100 and 20 nM, respectively. No effect was seen on the classical pathway. Fusion of MAP-1 with FH domains represents a novel therapeutic approach for selective targeting upstream and central complement activation at sites of inflammation.

Soluble Collectin-12 (CL-12) Is a Pattern Recognition Molecule Initiating Complement Activation via the Alternative Pathway.

Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to ∼ 120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca(2+)-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.

Ficolins and the lectin pathway of complement in patients with systemic lupus erythematosus.

The complement system plays a pathophysiological role in systemic lupus erythematosus (SLE). This study aims to investigate whether an association exists between the ficolins that are part of the lectin complement pathway and SLE. EDTA plasma samples from 68 Danish SLE patients and 29 healthy donors were included in the study. Plasma concentrations of Ficolin-1, -2, and -3 were determined in specific sandwich ELISAs. Lectin pathway activity via Ficolin-3 was measured in ELISA on acetylated bovine serum albumin (acBSA) and measured as Ficolin-3 binding and deposition of C4, C3 and the terminal complement complex (TCC). SLE patients had increased levels of Ficolin-3, 21.6µg/ml as compared to 17.0µg/ml in healthy controls (P=0.0098). The Ficolin-1 plasma concentration was negatively correlated with SLE Disease Activity Index (SLEDAI) (Rho=-0.29, P=0.015) and positively correlated to the [Systemic Lupus International Collaborating Clinics (SLICC)/American College of Rheumatology (ACR) Damage Index] (SDI) (Rho=0.27, P=0.026). The Ficolin-1 concentration was also associated with the occurrence of arterial (P=0.0053) but not venous thrombosis (P=0.42). Finally, deposition of C4, C3 and TCC in the Ficolin-3 pathway were all correlated to SLEDAI, respectively (P<0.0076). The Ficolin-1 association to SLEDAI and SDI as well as arterial thrombosis shown in this study suggests that Ficolin-1 may be a potential new biomarker for patients with SLE. Furthermore, Ficolin-3 mediated complement activation may be valuable in monitoring disease activity in SLE patients due to the high sensitivity for complement consumption in the assay independent of the Ficolin-3 concentration.

Activation of the ficolin-lectin pathway during attacks of hereditary angioedema.

BACKGROUND: The activation of plasma enzyme systems is insufficiently controlled in hereditary angioedema due to the deficiency of C1-inhibitor (C1-INH) (HAE-C1-INH). Recently, it was suggested that the ficolin-lectin pathway (ficolin-LP) might play a more dominant role than the mannose-binding lectin-lectin pathway in the pathomechanism of HAE-C1-INH. OBJECTIVE: Because the role of the ficolin-LP during edematous attacks is still enigmatic, we analyzed its activity during such episodes. METHODS: Thirty-five patients with HAE-C1-INH, who have experienced severe attacks on 106 occasions, were enrolled. We analyzed blood samples drawn during attacks, and obtained 35 samples from the same patients during symptom-free periods. The serum levels of ficolin-2, ficolin-3, MASP-2, ficolin-3/MASP-2 complex, C1-INH, and C4, as well as the extent of ficolin-3-mediated terminal complement complex (FCN3-TCC) deposition, were measured using ELISA-based methods. RESULTS: Levels of MASP-2 and of the ficolin-3/MASP-2 complex were elevated (P < .0001 and .033, respectively), whereas that of FCN3-TCC was lower (P < .0001) during attacks than during the symptom-free period. During symptom-free periods, FCN3-TCC deposition was significantly related to concentrations of ficolin-3 (R = 0.2778; P = .0022), antigenic C1-INH (R = 0.3152; P = .0006), and C4 (R = 0.5307; P < .0001). Both ficolin-3 and MASP-2 levels correlated inversely with the time from the onset of the attack until blood sampling. CONCLUSIONS: There is a marked heterogeneity of the pathomechanism and development of hereditary angioedema attacks in different patients. Our results suggest that the activation of the ficolin-LP may deplete the innately low level of C1-INH and thus, it may contribute to the uncontrolled activation of plasma cascade systems, and thereby to edema formation.

Ficolin-3-mediated lectin complement pathway activation in patients with subarachnoid hemorrhage.

OBJECTIVES: To assess the involvement of ficolin-3, the main initiator of the lectin complement pathway (LCP), in subarachnoid hemorrhage (SAH) pathology and outcome. METHODS: In this preliminary exploratory study, plasma concentration of ficolin-3 and of ficolin-3-mediated functional LCP activity was measured, along with that of other LCP initiators (mannose-binding lectin, ficolin-2, and ficolin-1), C3 activation products, and soluble C5b-9 terminal complex, in a prospective cohort of 39 patients with SAH and 20 healthy controls. The following parameters were recorded: SAH severity, assessed using the World Federation of Neurosurgical Societies grading scale; vasospasm, defined as neuro-worsening with angiographic confirmation of vessel narrowing; cerebral ischemia, defined as hypodense lesion on CT scan performed before discharge; and 6-month outcome, assessed using the Glasgow Outcome Scale. RESULTS: In patients, no changes were detected for ficolin-3 compared with controls. Notably, however, ficolin-3-mediated functional LCP activity was reduced. Low levels of plasma ficolin-3 and ficolin-3-mediated functional LCP activity were related to SAH severity, vasospasm, and cerebral ischemia. Moreover, ficolin-3 functional LCP activity was decreased in patients with unfavorable outcome. CONCLUSION: Our data provide evidence that LCP is activated after SAH and that the actual plasma concentrations of ficolin-3 reflect the severity of brain injury as evaluated by clinical and structural parameters. These results support the idea that ficolin-3-mediated functional LCP activity may be targeted to control injury progression in SAH.

Ficolin-2 reveals different analytical and biological properties dependent on different sample handling procedures.

Ficolin-2 (L-ficolin) is a germ line encoded pattern recognition molecule circulating in the blood, and functions as a recognition molecule in the lectin complement pathway. However, consistent and reliable measurements of Ficolin-2 concentration and activity have been difficult to achieve. After recurrent observations of deviations in Ficolin-2 properties between different blood sample procedures, we decided to investigate this closer. Blood samples from ten healthy donors were collected in various serum and plasma tubes and Ficolin-2 properties were evaluated by different ELISA setups. We found that serum prepared from tubes containing the clot activator silica used as a standard technique in many routine laboratories held a significantly lower concentration of Ficolin-2 as compared to the other sample types. Furthermore, Ficolin-2 binding and complement activation potential in this type of serum was impaired when using an acetylated compound as matrix. On the other hand, Ficolin-2 in serum made without clot activator and in plasma irrespective of additive used, had the same concentration and was capable of initiating the lectin pathway measured as C4 and C3 deposition on the ligand. No Ficolin-2 mediated formation of the terminal complement complex was observed under the applied assay conditions. In conclusion, our results show that Ficolin-2 is a promiscuous molecule and that care should be taken during sampling, handling and matrix chosen for measurement of Ficolin-2 levels and activity.

Pre-transplant levels of ficolin-3 are associated with kidney graft survival.

Ficolin-3 is an initiator of the lectin complement pathway. The complement system is a mediator of the pathophysiology of graft rejection in kidney transplantation, but the role of ficolin-3 in this process is unknown. Using a prospective study design, 527 kidney transplanted patients were included. 97 blood donors served as controls. Ficolin-3, C4 and C3 were measured in pre-transplant as well as in control serum samples. In controls, deposition of ficolin-3, C4, C3 and the terminal complement complex (TCC) was measured in an assay based on acetylated albumin as matrix. The ficolin-3 levels correlated with the serum levels of C4 and C3. The serum levels of ficolin-3 correlated with the deposition of ficolin-3, C4, C3 and TCC. Survival analyses showed that high pre-transplant serum levels of ficolin-3 were associated with decreased graft survival. These results suggest an important role of ficolin-3 in the pathophysiology of kidney graft rejection.

A novel assay to quantitate MASP-2/ficolin-3 complexes in serum.

Ficolin-1, -2 and -3 are recognition molecules in the lectin complement pathway and form complexes with serine proteases named MASP-1, -2 and -3 and two nonenzymatic proteins. MASP-2 is the main initiator of lectin pathway activation, while ficolin-3 is the most abundant ficolin molecule in the circulation. The significance of lectin pathway complexes in the circulation is unknown. Thus, we established an assay for the measurement of circulating MASP-2/ficolin-3 complexes. A quantitative sandwich ELISA was developed for the measurement of the MASP-2/ficolin-3 complexes in serum based on monoclonal antibodies against MASP-2 for coating and anti-ficolin-3 for detection. In addition, we assessed the serum concentrations of ficolin-3 and MASP-2 and the extent of ficolin-3 mediated C4 deposition on acetylated BSA in samples from 97 healthy donors. The median concentration of MASP-2/ficolin-3 complexes was found to be 119.7 AU/ml (range: 2.9-615.5 AU/ml). Significant correlations were found between the level of MASP-2/ficolin-3 complexes and the concentration of ficolin-3 (Spearman r=0.2532, p=0.0124), and MASP-2 (Spearman r=0.4505, p<0.0001), as well as the degree of C4 deposition (Spearman r=0.671, p<0.0001). When ficolin-3 deficient (homozygous for the rs28357092 polymorphism) and MASP-2 deficient (homozygous for the rs72550870 polymorphism) sera were incubated together, complex formation was induced between MASP-2 and ficolin-3. The complex formation disappeared in the presence of EDTA. An assay allowing quantitative measurement exclusively of MASP-2/ficolin-3 complexes in serum is described. This method may add further insight into the pathophysiology of disorders associated with the deficiency or abnormal activities of MASP-2 and ficolin-3.

Serum concentration and interaction properties of MBL/ficolin associated protein-1.

Recently, a novel protein named MBL/ficolin associated protein-1 (MAP-1) derived from the MASP1 gene through differential splicing was identified. In the present study, we established biochemical characteristics, determined the serum level and assessed the interactions between the lectin complement pathway (LCP) recognition molecules and MAP-1. We expressed recombinant MAP-1 in CHO DG44 cells, developed a quantitative ELISA assay based on a MAP-1 specific monoclonal capture antibody and measured the serum levels in 100 Danish blood donors. In addition we assessed the association properties between MAP-1 and Ficolin-2, -3 and MBL in serum using ELISA and density gradient ultra centrifugation. When recombinant MAP-1 was subjected to N-glycosidase F treatment the molecular mass decreased from ∼45 kDa to ∼40 kDa equivalent with the calculated molecular mass from the deduced amino acid sequence without the signal peptide. We found that serum MAP-1 was very stable when subjected to repeated freeze and thaw cycles. The mean serum concentration of MAP-1 was found to be 240 ng/ml (range: 115-466 ng/ml). MAP-1 was predominantly found in complex with Ficolin-3 and to a lesser degree with Ficolin-2 and MBL and by use of density gradient ultra centrifugation we could show that the major part of serum MAP-1 circulates in complex with the LCP molecules. In conclusion, these results show that MAP-1 is a highly stable glycosylated human serum protein found in complex with Ficolin-3, Ficolin-2 and MBL.

Functional analysis of Ficolin-3 mediated complement activation.

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.