Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel.

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

Quantification of the pro-form of human complement component factor D (adipsin).

Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1-2 µg/ml among healthy individuals. The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D. In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82-25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8-15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.

The Effect of Induced Antibodies with Respect to Neutralization, Clearance Rate and Functional Activity in a Rabbit/Infliximab Model.

BACKGROUND: Therapeutic antibodies are a developing field for treatment of an expanding number of inflammatory diseases, including Crohn's disease. Treatment with monoclonal antibodies is frequently hampered by development of anti-drug antibodies (ADAs) that may compromise the treatment. MATERIALS AND METHODS: We addressed this issue in a rabbit model of treatment with the anti-tumor-necrosis factor alpha (TNFα) antibody, infliximab (IFX). We developed an inhibition ELISA to selectively measure absolute concentrations of neutralizing antibodies and another ELISA for measuring the concentration of functional IFX in the circulation. RESULTS: We found that the concentration of functional IFX was inversely proportional to the concentration of neutralizing antibodies. CONCLUSION: Administration of IFX to rabbits showed diversity in immune responses/tolerance toward IFX, corresponding to responses observed in patients. The applied assay technology is easily adapted to human plasma samples and/or other therapeutic antibodies, including fully humanized antibodies, for which immunogenicity also is observed.

Collectin CL-LK Is a Novel Soluble Pattern Recognition Receptor for Mycobacterium tuberculosis.

Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.

Genetic variation of COLEC10 and COLEC11 and association with serum levels of collectin liver 1 (CL-L1) and collectin kidney 1 (CL-K1).

Collectin liver 1 (CL-L1, alias CL-10) and collectin kidney 1 (CL-K1, alias CL-11), encoded by the COLEC10 and COLEC11 genes, respectively, are highly homologous soluble pattern recognition molecules in the lectin pathway of complement. These proteins may be involved in anti-microbial activity and in tissue development as mutations in COLEC11 are one of the causes of the developmental defect syndrome 3MC. We studied variations in COLEC10 and COLEC11, the impact on serum concentration and to what extent CL-L1 and CL-K1 serum concentrations are correlated. We sequenced the promoter regions, exons and exon-intron boundaries of COLEC10 and COLEC11 in samples from Danish Caucasians and measured the corresponding serum levels of CL-L1 and CL-K1. The median concentration of CL-L1 and CL-K1 was 1.87 µg/ml (1.00-4.14 µg/ml) and 0.32 µg/ml (0.11-0.69 µg/ml), respectively. The level of CL-L1 strongly correlated with CL-K1 (ρ = 0.7405, P <0.0001). Both genes were highly conserved with the majority of variations in the non-coding regions. Three non-synonymous variations were tested: COLEC10 Glu78Asp (rs150828850, minor allele frequency (MAF): 0.003), COLEC10 Arg125Trp (rs149331285, MAF: 0.007) and COLEC11 His219Arg (rs7567833, MAF: 0.033). Carriers of COLEC10 Arg125Trp had increased CL-L1 serum levels (P = 0.0478), whereas promoter polymorphism COLEC11-9570C>T (rs3820897) was associated with decreased levels of CL-K1 (P = 0.044). In conclusion, COLEC10 and COLEC11 are highly conserved, which may reflect biological importance of CL-L1 and CL-K1. Moreover, the strong inter individual correlation between the two proteins suggests that a major proportion are found as heterooligomers or subjected to the same regulatory mechanisms.