Estrid Hein

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.

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.

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.

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 Ca2+-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.

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.

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.—Nordmaj, M. A., Munthe‐Fog, L., Hein, E., Skjoedt, M.‐O., Garred, P. 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. FASEB J. 29, 4945–4955 (2015). www.fasebj.org

Complement Activation and its Prognostic role in Post‐cardiac Arrest Patients

Cardiac arrest causes generalized ischaemia/hypoxia, and subsequent resuscitation inflicts reperfusion injury, the pathology of which is not fully understood. Moreover, predicting the prognosis of comatose, post‐cardiac arrest patients is a complex clinical challenge. We hypothesized that the extent of complement activation might be a reliable predictor of mortality in this population. Forty‐six comatose cardiac arrest patients were enrolled into our prospective cohort study, conducted in a tertiary care university clinic. All subjects were cooled to 32–34 °C body temperature for 24 h and then allowed to rewarm to normothermia. All patients underwent diagnostic coronary angiography. On admission, at 6 and 24 h, blood samples were taken from the arterial catheter. In these, complement products (C3a, C3, C4d, C4, SC5b9 and Bb) were measured by ELISA in blood samples. Patients were followed up for 30 days; 22 patients (47.8%) died by the end of this period. We observed that complement activation (determined as the C3a to C3 ratio) was higher in non‐survivors than in survivors at each time point. In the multivariate Cox regression analysis, the C3a/C3 ratio determined 24 h after the initiation of therapeutic hypothermia predicted 30‐day mortality regardless of age, sex and the APACHE II score. Complement activation occurs in post‐cardiac arrest patients, and its extent correlates with 30‐day survival. The C3a/C3 ratio might prove useful for estimating the prognosis of comatose post‐cardiac arrest patients.