S. Thiel

An assay for the mannan-binding lectin pathway of complement activation.

The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs). When bound to microorganisms, the MBL complex activates the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. This C4/C2 cleaving activity is shared with the C1 complex of the classical pathway of complement activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt the C1 complex, whereas the carbohydrate-binding activity of MBL and the integrity of the MBL complex is maintained under hypertonic conditions. In the assay described here, the specific C4b-depositing capacity of the MBL pathway was determined by incubating serum diluted in buffer containing 1 M NaCl in mannan-coated microtiter wells before the addition of purified C4. The interassay coefficient of variation in the ELISA version was 7.3%. As expected no activity was found in MBL-deficient serum. When 100 normal serum samples were analysed we found that the MBL level correlated with the amount of C4b deposited on the mannan-coated surface. However, we also found a threefold variation in C4b-depositing capacity between individuals with similar MBL concentrations. The assay permits for the determination of MBL complex activity in serum and plasma samples and may thus be used to evaluate the clinical implications of complement activation via this pathway.

Clinical manifestations of mannan-binding lectin deficiency.

n Abstractn n Mannan-binding lectin (MBL) is a plasma protein of the innate immune system with the ability to initiate antimicrobial and inflammatory actions. MBL deficiency is common. More than 10% of the general population may, depending on definition, be classified as MBL deficient, underlining the redundancy of the immune system. Ongoing research attempt to illuminate at which conditions MBL deficiency may lead to disease. With examples, this review illustrates the diversity of results obtained so far.n n

Gene frequency and partial protein characterization of an allelic variant of mannan binding protein associated with low serum concentrations

Low plasma concentration of mannan binding protein (MBP) has been shown to be the basis for a common opsonic deficiency and suggested to be caused by a single nucleotide substitution at base 230 of exon 1 in the MBP gene. This substitution causes a replacement of glycine (codon GGC) with aspartic acid (codon GAC). Of 123 healthy Danish individuals investigated by polymerase chain reaction performed on exon 1, followed by restriction fragment length polymorphism or allospecific probing, 93 were homozygous (75.6%) for GGC, 28 heterozygous (22.8%), and two homozygous for GAC (1.6%). The gene frequency of the GAC allele was found to be 013. DNA sequencing of the cloned exon 1 from one GAC homozygous individual revealed no other substitution. The median MBP concentration in the group containing the GAC allele was 6.4 times lower than in the GGC homozygous group (195 and 1234 μ/l respectively). However, the range in plasma concentrations of MBP was wide and overlapping between the groups. MBP protein was detected in both the GAC homozygotes (9 and 387 μ/l. Furthermore, no difference in relative mass and biological activity (mannan binding) was found when sera containing the two forms of MBP were investigated. Accordingly, it can be concluded that the GAC allele is able to produce a functional MBP protein which may be detected in serum at low concentrations.

M-ficolin, an innate immune defence molecule, binds patterns of acetyl groups and activates complement.

Ficolins play a role in the innate immune defence as pathogen‐associated molecular pattern recognition molecules. Three ficolins are found in humans: H‐ficolin, L‐ficolin and M‐ficolin. L‐ficolin and H‐ficolin circulate in blood in complexes with mannan‐binding lectin‐associated serine proteases (MASPs) and are capable of activating the complement system. L‐ficolin shows affinity for acetylated compounds and binds to various capsulated strains of bacteria. H‐ficolin has been shown to bind Aerococcus viridans. Less is known about M‐ficolin, but it is thought to be present only on monocytes. We have synthesized recombinant M‐ficolin and find that it, in a manner similar to L‐ficolin, is able to bind to acetylated compounds and to associate with recombinant MASP‐2. Upon binding to M‐ficolin ligands, the associated MASP‐2 zymogen is activated and cleaves C4, thus triggering the complement system. We developed a monoclonal rat anti‐human‐M/L‐ficolin antibody and verified by flow cytometric analysis the presence of ficolin on the surface of peripheral blood monocytes.

Humoral pattern recognition and the complement system.

In the context of immunity, pattern recognition is the art of discriminating friend from foe and innocuous from noxious. The basis of discrimination is the existence of evolutionarily conserved patterns on microorganisms, which are intrinsic to these microorganisms and necessary for their function and existence. Such immutable or slowly evolving patterns are ideal handles for recognition and have been targeted by early cellular immune defence mechanisms such as Toll‐like receptors, NOD‐like receptors, RIG‐I‐like receptors, C‐type lectin receptors and by humoral defence mechanisms such as the complement system. Complement is a proteolytic cascade system comprising around 35 different soluble and membrane‐bound proteins. It constitutes a central part of the innate immune system, mediating several major innate effector functions and modulating adaptive immune responses. The complement cascade proceeds via controlled, limited proteolysis and conformational changes of constituent proteins through three activation pathways: the classical pathway, the alternative pathway and the lectin pathway, which converge in common effector functions. Here, we review the nature of the pattern recognition molecules involved in complement activation, as well as their close relatives with no or unknown capacity for activating complement. We proceed to examine the composition of the pattern recognition complexes involved in complement activation, focusing on those of the lectin pathway, and arrive at a new model for their mechanism of operation, supported by recently emerging evidence.

The level of the serum opsonin, mannan-binding protein in HIV-1 antibody-positive patients.

The concentrations of mannan‐binding protein (MBP) in consecutive samples from 10 HIV+ persons were estimated using an ELISA based on polyclonal rabbit anti‐MBP. The changes in MBP with time were similar in HIV+ and HIV− persons, and did not appear to be of clinical significance. MBP was determined in a further 70 persons found HIV‐1+ during a period af 2·5 years (1984–1986). Out of the total of 80 patients, 32 have by now died from AIDS. According to the serum level of MBP the HIV‐infected persons were grouped into high (> 650 ng MBP/ml). intermediate (101–650 ng/ml), and low MBP (< 101 ng/ml). At the termination of the study the frequency of deaths/total in each of the groups were: high MBP, 14/39 (36%); intermediate MBP, 12/26 (46%); and low MBP, 6/14 (43%). There was no association between the MBP level of the individual and the progressive loss of CD4+ T cells, and the level of MBP was not predictive for the length of time between the detection of HIV antibodies and development of AIDS, nor for the duration of AIDS before death occurred. The number of HIV+ persons without detectable MBP (10%) was significantly higher than previously reported for healthy persons (2·4%, P= 0·027). The course of HIV infection does not seem to be influenced by the level of MBP, nor does the antimicrobial activity of MBP appear to affect the progression of AIDS. Further studies are required to substantiate the significance of absence of MBP in the susceptibility to HIV.

Biological Variation in Circulating Levels of Mannan-Binding Lectin (MBL) and MBL-Associated Serine Protease-2 and the Influence of Age, Gender and Physical Exercise

Mannan‐binding lectin (MBL) and MBL‐associated serine protease 2 (MASP‐2) are central components of the MBL pathway of complement activation, and may have potential as clinical biomarkers in colorectal cancer (CRC). Prior to clinical usage, knowledge of the biological variations of the molecules is needed. We here investigate variations of MBL and MASP‐2 in healthy persons over time and in relation to gender, age and physical activity. MBL and MASP‐2 concentrations were determined in serum from healthy adults over a 3‐week period and this was repeated 6u2003months later (nu2003=u200332); during a 24‐h period (nu2003=u200316); and in relation to physical exercise (nu2003=u200314). Concentrations in serum and plasma were compared (nu2003=u2003198). No significant variation over 6u2003months and no circadian variation was found for MBL (Pu2003=u20030.39 and Pu2003=u20030.34 respectively) or MASP‐2 (Pu2003=u20030.54 and Pu2003=u20030.55). Physical exercise did not affect the levels (Pu2003>u20030.8). Serum and plasma levels were only marginally different, and were independent of age and gender. Circulating levels of MBL and MASP‐2 are stable over time in healthy individuals, which is advantageous for their potential application as biomarkers.

Mannan‐binding protein—levels in plasma and upper‐airways secretions and frequency of genotypes in children with recurrence of otitis media

We have investigated a possible association between recurrence of olitis media and low concentrations of mannan‐binding protein (MBP) in plasma and upper‐airway secretions. The protein concentration was measured in plasma (n= 76), nasopharyngeal secretions (n= 83) and middle ear effusions (n= 73) from otitis‐prone children, children with less recurrence of acute otitis media, children with no previous history of acute otitis media, but suffering from secretory olitis media, and healthy children. Moreover, genetic polymorphisms associated with low MBP plasma levels were investigated in DNA from nasopharyngeal tonsils of 89 children with recurrence of otitis media. A wide range of M BP plasma concentrations was found. No statistically significant differences in M BP plasma concentration were observed between patients and controls. Nor was there any increased frequency of the genotypes associated with low MBP plasma concentrations. Thus, our results do not support the assumption that low concentration and/or MBP deficiency alone predispose to recurrence of otitis media in Caucasian children. MBP was detected in both nasopharyngeal secretions (1 /175 of plasma level) and middle ear effusions (1/4 of plasma level), suggesting a role for the protein in the local mucosal immune defence system at these locations. In contrast. MBP was undetectable in 53 samples of mixed‐saliva.

Hormonal regulation of mannan-binding lectin synthesis in hepatocytes

Activation of the complement system via the plasma protein mannan‐binding lectin (MBL) provides a first line of defence against infections. The plasma level of MBL is, in part, determined genetically, but may also be influenced by different hormones in vivo. Here we study the hormonal regulation of MBL synthesis from the human hepatocyte cell line HuH‐7. Cells were exposed to medium with growth hormone (GH), hydrocortisone, insulin‐like growth factor (IGF)‐1, insulin, interleukin (IL)‐6 or thyroid hormones (T3 or T4). After 3u2003days the concentration of MBL in the culture supernatants was determined and the amount of mRNA for MBL was measured, relative to mRNA for β2 microglobulin. GH, IL‐6, T3 and T4 significantly increased MBL synthesis in a dose‐dependent manner, while hydrocortisone, insulin and IGF‐1 had no effect. T3 caused a fourfold increase at 1u2003nM of T3 (Pu2003<u20030·001) and at 100u2003nM of T3 the production was increased more than eightfold. The effect of T4 was less potent, reaching an eightfold increase at 1u2003µM of T4 (Pu2003<u20030·001). GH augmented the production of MBL threefold at a concentration of 100u2003ng/ml (Pu2003=u20030·018) with no further effect up to 10u2003µg/ml, whereas IL‐6 caused only a very weak increase in MBL production. MBL mRNA levels were stable during the first 24u2003h of T3 stimulation but increased significantly between 24 and 48u2003h. The results suggest that MBL synthesis in humans may be increased by thyroid hormone and GH, whereas it does not exhibit a classical IL‐6‐dependent response.

Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules: normal and acute-phase levels in serum and stoichiometry of lectin pathway components.

The pattern‐recognition molecules mannan‐binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL‐associated serine proteases (MASPs). When MBL or ficolin recognizes a microorganism, activation of the MASPs occurs leading to activation of the complement system, an important component of the innate immune system. Three proteins are produced from the MASP1 gene: MASP‐1 and MASP‐3 and MAp44. We present an assay specific for MASP‐1, which is based on inhibition of the binding of anti‐MASP‐1‐specific antibody to MASP‐1 domains coated onto microtitre wells. MASP‐1 was found in serum in large complexes eluting in a position corresponding to ∼600u2003kDa after gel permeation chromatography in calcium‐containing buffer and as monomers of ∼75u2003kDa in dissociating buffer. The concentration of MASP‐1 in donor sera (nu2003=u2003105) was distributed log‐normally with a median value of 11u2003µg/ml (range 4–30u2003µg/ml). Serum and citrate plasma levels were similar, while the values in ethylenediamine tetraacetic acid plasma were slightly lower and in heparin plasma were 1·5 times higher than in serum. MASP‐1 was present at adult level at 1 year of age, while it was 60% at birth. In normal healthy individuals the level of MASP‐1 was stable throughout a 2‐month period. After induction of an acute‐phase reaction by operation we found an initial short decrease, concomitant with an increase in C‐reactive protein levels, followed by an increase, doubling the MASP‐1 concentration after 2 days. The present data prepare the ground for studies on the associations of MASP‐1 levels with disease.