A role for mannose-binding lectin dysfunction in generation of autoantibodies in systemic lupus erythematosus

M. A. Seelen, E. A. van der Bijl1, L. A. Trouw, T. C. M. Zuiverloon, J. R. Munoz, F. C. Fallaux-van den Houten, N. Schlagwein, M. R. Daha, T. W. J. Huizinga1 and A. Roos

epartments of Nephrology and 1 Rheumatology, Leiden University Medical Center, Leiden, The Netherlands.

Correspondence to: M. A. Seelen, Department of Nephrology, C3P-29, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail: majseelen{at}lumc.nl


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To investigate the possible association of the mannose binding lectin (MBL) pathway of complement activation with different disease parameters and disease activity in patients with systemic lupus erythematosus (SLE).

Methods. MBL genotype, MBL serum concentration, MBL complex activity and MBL pathway activity were assessed in 53 patients. The activity of the MBL–MASP complex was assessed on the basis of its ability to activate exogenous C4. For MBL pathway activity the formation of the terminal complex of complement activation (C5b-9) was measured. Results were analysed in relation to clinical variables and autoantibody profiles in these patients.

Results. MBL complex activity and MBL pathway activity were both reduced in patients carrying MBL variant alleles. Anticardiolipin and anti-C1q autoantibodies were observed significantly more frequently in patients with MBL variant alleles. Furthermore, the presence of these autoantibodies was associated with a decreased MBL concentration and function. In contrast, anti-MBL autoantibodies were not found in patients with MBL variant alleles, possibly related to impaired binding of variant MBL to apoptotic material.

Conclusion. In patients with SLE, a reduced functional activity of the MBL pathway of complement, in relation to expression of MBL variant alleles, is associated with increased levels of autoantibodies against cardiolipin and C1q, but not against MBL. We hypothesize that an enhanced production of autoantibodies may be related to disturbed clearance of apoptotic material due to impaired MBL function.

KEY WORDS: Mannose binding lectin, Lectin pathway activity, SLE, Autoantibodies


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Susceptibility to systemic lupus erythematosus (SLE) is related to genetic as well as environmental factors. It is thought that a combination of genes, rather than a single gene, predisposes to the development of SLE. Many genes have been reported to be linked with the development of spontaneous SLE, including several loci encoding different complement components and complement receptors [1]. Genetic deficiencies of complement components, especially of the early components of the classical pathway, are strongly associated with the occurrence of SLE [2]. In this respect, the genetic association between deficiency of C1q, the recognition factor of the classical complement activation pathway, with SLE is nearly 100%. This association between C1q deficiency and systemic autoimmunity has been linked to a role for C1q in recognition and clearance of apoptotic material [3, 4]. Defective clearance and increased generation of apoptotic material has been demonstrated in patients with SLE [5]. Furthermore, autoantibodies found in patients with SLE are mostly directed against antigens present on apoptotic material [6].

Recent studies also indicate a possible role for the lectin pathway of complement activation in the pathogenesis of SLE [7]. Variant alleles leading to reduced serum concentrations of mannose-binding lectin (MBL) are associated with a predisposition to SLE [8–11]. MBL is a major recognition factor of the lectin pathway of complement. The molecule is composed of trimeric subunits assembled to higher-order structures consisting of up to six trimers. This structure resembles the bouquet-like structure of C1q [12]. Also the MBL-associated serine proteases MASP-1, MASP-2 and MASP-3 are functionally related to the serine proteases of the classical pathway, C1r and C1s. After binding of the calcium-dependent carbohydrate recognition domain of MBL to different carbohydrates, activation of the complement cascade can take place [13]. MBL is able to bind to apoptotic cell debris via its lectin domain, which is involved in the phagocytosis of apoptotic cells by macrophages [14, 15]. Since MBL has been postulated to play a role in the clearance of apoptotic material [3], this mechanism could provide a possible explanation for the association between MBL deficiency and the occurrence of SLE.

MBL deficiency or low serum MBL levels are frequently found in the general population due to single nucleotide polymorphisms (SNPs) in the structural portion or promoter region of the MBL2 gene [7, 12, 16]. Three SNPs have been identified in the structural domain of the MBL gene, located in codon 52 [17], 54 [18] and 57 [19] of exon 1, whereas additional SNPs exist upstream of the MBL gene at position –550 (H/L alleles), –221 (X/Y alleles) and +4 (P/Q alleles) [20, 21]. The occurrence of these SNPs in the MBL gene is associated with predisposition to the development of SLE and with complications caused by bacterial infections in patients with SLE [8, 10, 22]. Furthermore, a tendency towards more renal disorders in SLE patients with MBL variant alleles has been reported [8].

Studies on the association of MBL and SLE have until now have focused on MBL genotype and MBL concentration. In the present study, we examined whether functional impairment of the MBL pathway is associated with parameters of disease activity. The results indicate that MBL gene polymorphisms, low MBL concentration and impaired functional activity of the MBL pathway are associated with the presence of anticardiolipin and anti-C1q antibodies in patients diagnosed with SLE.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Fifty-three patients with a mean age of 39 yr made up of 92% females all fulfilling at least four of the American College of Rheumatology criteria for SLE were included in this study. The patients visited the out-patient clinic of the rheumatology department at the Leiden University Medical Center from 1997 to 1998. The median disease activity in these patients was 4.3 (0–18) as measured with the SLE Disease Activity Index (SLEDAI), and organ damage measured with the Systemic Lupus International Collaborating Clinics/ACR Damage Index found in the patient group was 2 (1–4.5). Informed consent was obtained from all patients. The clinical and laboratory variables tested were the following: the ACR criteria for SLE (skin rash, photosensitivity, oral ulcers, renal involvement, joint involvement, serositis, neurological disorders, haematological disorders and autoantibody profile), age at disease onset, SLEDAI, and SLE damage index adjusted for years of disease. Paired serum and DNA samples were available.

Polymerase chain reaction amplification of exon 1 of the MBL gene and oligonucleotide ligation assay for MBL genotyping
MBL genotyping was performed using genomic DNA from SLE patients (n = 53) and from a population of healthy Caucasian controls (n = 59). Exon 1 of the MBL gene was amplified from genomic DNA by PCR. Detection of MBL mutant alleles at codons 52, 54 and 57 was carried out with three different oligonucleotide ligation assays (OLA). The polymerase chain reaction (PCR) amplification and OLA were performed exactly following the procedure as described before [23].

Measurement of MBL serum concentrations
MBL concentrations in serum were assessed precisely as described previously [24].

Functional activity of the MBL complex
The assessment of activity of MBL and its associated serine proteases (MBL complex activity) was performed using a slightly modified method as described by Petersen et al. [25]. In brief, mannan-coated plates were incubated for 16 h at 4°C with serum, diluted in GVB++ containing 1 M NaCl. Plates were washed with phosphate-buffered saline (PBS)/Tween containing 5 mM CaCl2, followed by incubation with purified C4 (1 µg/ml), diluted in Veronal-buffered saline (VBS) containing 1 mM MgCl2, 2 mM CaCl2, 0.05% Tween-20, and 1% bovine serum albumin (BSA), pH 7.5, for 1 h at 37°C. Binding of activated C4 was assessed using mAb C4-4a (anti-human C4d, from Dr C. E. Hack, Amsterdam, The Netherlands) conjugated to dig (digoxigenin-3-O-methylcarbonyl-rounded {epsilon}-aminocaproic acid-N-hydroxysuccinimide ester), followed by HRP-conjugated sheep anti-dig antibodies as described above. MBL complex activity was expressed in arbitrary units per ml, based on serial dilutions of a human pool serum that was used as a standard on each plate. Activity of this standard was set at 1000 U/ml.

Functional activity of the whole MBL pathway
The functional activity of the MBL pathway in whole serum, from MBL binding to mannan up to the deposition of C5-9, was assessed by enzyme-linked immunosorbent assay (ELISA) [23]. Plates (Nunc Maxisorb, Nunc, Roskilde, Denmark) were coated with mannan (100 µg/ml, from Saccharomyces cerevisiae; Sigma, St Louis, MO) in coating buffer (100 mM Na2CO3/NaHCO3, pH 9.6), for 2 h at 37°C. After each step, plates were washed three times with PBS containing 0.05% Tween-20. Residual binding sites were blocked by incubation with PBS containing 1% BSA for 1 h at 37°C. Next, serum samples were diluted in GVB++ [VBS (1.8 mM Na-5,5-diethylbarbital, 0.2 mM 5,5-diethylbarbituric acid, 145 mM NaCl) containing 0.5 mM MgCl2, 2 mM CaCl2, 0.05% Tween-20 and 0.1% gelatin; pH 7.5] in the presence of mAb 2204 (20 µg/ml) as an inhibitor of C1q, to exclude any contribution of the classical pathway via antibodies reactive with mannan. This mixture was pre-incubated for 15 min on ice, before addition to the plates. The plates were then sequentially incubated for 1 h at 4°C and for 1 h at 37°C. The amount of C5b-9 generated, as a measure of MBL pathway activation, was detected with mouse mAb AE11 (anti-human C5b-9, kindly provided by Dr T. E. Mollnes, Oslo, Norway) conjugated to dig (Boehringer Mannheim, Mannheim, Germany). The degree of binding of mAb was subsequently detected using dig-conjugated sheep anti-mouse antibodies (Fab fragments) followed by HRP-conjugated sheep anti-dig antibodies (Fab fragments, both from Boehringer Mannheim, Germany). Enzyme activity of HRP was detected following incubation with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (from Sigma; 2.5 mg/ml in 0.1 M citrate/Na2HPO4 buffer, pH 4.2) in the presence of 0.01% H2O2, for 30–60 min at room temperature. The optical density (OD) at 415 nm was measured using a microplate biokinetics reader (EL312e, from Biotek Instruments, Winooski, VT, USA). Functional activity of the MBL pathway was expressed in units per ml, relative to a positive standard consisting of pooled human serum arbitrarily set at 1000 U/ml.

Functional activity of the classical pathway
Activity of the classical pathway was assessed by determining C4 activation on immobilized IgM using an ELISA-based procedure as described by Roos et al. [23].

Detection of antibodies against C1q, MBL, DNA and anticardiolipin
Anti-C1q autoantibodies were assessed as described [26]. A recently described method to detect anti-MBL antibodies was used to determine MBL antibodies [27].

Antibodies against DNA were measured using an immunofluorescent method as described by Aarden et al. [28]. Antibodies against cardiolipin were measured using a commercially available Varelisa cardiolipin IgG antibodies kit (Pharmacia Diagnostics, Germany).

Statistics
The Mann–Whitney test and the {chi}2 test were used for statistical analysis. Correlation was evaluated using the Spearman rank correlation coefficient. P values less than 0.05 were considered statistically significant.

Approval of the Leiden University Medical Center research ethics committee (CME) was obtained prior to starting this study.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
MBL genotype from patients in relation to disease expression
All 53 patients were genotyped for MBL variant alleles at codon 52 (named D), at codon 54 (named B) and at codon 57 (named C) of exon 1 of the MBL2 gene. The wildtype genotype (A/A) was observed in 37 out of the 53 patients (70%), the A/B genotype was found in four patients, five patients carried the A/C genotype and five patients the A/D genotype, whereas the B/B and D/C genotypes were each found in one patient. This genotype distribution was compared with a population of healthy Caucasian controls. In both the Caucasian SLE patients (n = 44) and in the healthy controls (n = 59), 66% expressed the wildtype genotype (A/A).

We divided the patients with SLE in two groups: one group with the wildtype genotype (A/A) and one group of carriers of variant alleles (A/0 or 0/0 genotype). Because of the limited number of patients with the 0/0 allele it was not possible to study these patients as an independent group. The frequencies of the different clinical variables and MBL genotype distribution in the two patient groups are shown in Table 1. No difference was observed between carriers of wildtype alleles (A/A) and variant alleles (non-A/A) in clinical presentation of the disease variables. In contrast, significant differences were noted between carriers of MBL wildtype alleles and MBL variant alleles with respect to the autoantibody profile. A significant difference was observed concerning the presence of anticardiolipin antibodies and anti-C1q antibodies: autoantibodies against cardiolipin and against C1q were significantly associated with the occurrence of MBL gene polymorphisms (Table 2). In contrast, there was a trend towards a lower presence of anti-MBL autoantibodies in SLE patients with variant alleles.


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TABLE 1. Clinical variables and MBL genotype distribution in patients with SLE

 

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TABLE 2. The presence or absence of different autoantibodies in SLE patients with wild type MBL genotype and variant MBL genotype

 
Functional characterization of the MBL pathway in SLE
The impact of MBL gene polymorphisms on MBL protein expression and function in SLE patients was examined using three different parameters, namely MBL serum concentration, MBL complex activity and MBL pathway activity.

The MBL concentrations in all sera were assessed by ELISA (Fig. 1A). The MBL concentration in the sera from SLE patients with the A/A genotype varied from 0.3 ng/ml to 3.8 µg/ml (n = 34, median 0.829 µg/ml). The MBL concentrations in sera from patients with variant alleles were significantly decreased, as expected (median 0.279 µg/ml, n = 14, P = 0.0002). To demonstrate the functional activity of the complex of MBL with the associated MASPs, activation of exogenous C4 by MBL complexes bound to mannan was assessed in an MBL complex activity assay (Fig. 1B). The activity of the MBL complex varied from 425 to 3930 U/ml (median 1497 U/ml) in the patients with a wildtype MBL genotype and a significantly decreased activity was found in sera from patients with a variant allele (median 371 U/ml, P = 0.0003). For the activity of the MBL pathway in full serum an ELISA was used measuring the formation of the terminal complex (C5b-9) via autologous complement components on solid phase mannan. The activity of the complete MBL pathway in SLE patients with a wildtype MBL genotype varied from undetectable (below 350) up to 2126 U/ml (median 808 U/ml), which is significantly different from the decreased activity found in carriers of variant alleles (median 467 U/ml, P = 0.0036) (Fig. 1C). The classical pathway activity was also assessed in the same sera. No difference was found in classical pathway activity between patients with wildtype MBL genotype and patients with a variant allele (Fig. 1D). Both the MBL complex activity and the MBL pathway activity showed a significant correlation with the MBL concentration (R = 0.82; P<0.001, and R = 0.65; P<0.001, respectively) (Fig. 2). However, several serum samples showed a low or undetectable MBL pathway activity whereas the MBL concentration and the MBL complex activity were in the normal range (Fig. 2B). MBL pathway activity showed a significant correlation with the classical pathway activity (R = 0.41; P = 0.004) (Fig. 2C), whereas MBL concentration was not correlated with classical pathway activity (R = 0.02, P = 0.91).



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FIG. 1. MBL function and classical pathway activity in relation to MBL genotype. The MBL genotype from 53 patients with SLE was determined. MBL concentration (A), MBL complex activity (B), MBL pathway activity (C) and classical pathway activity (D) were assessed in sera from patients with the different MBL genotypes. A significant difference was found when MBL concentration (P = 0.0002), MBL complex activity (P = 0.0003) and MBL pathway activity (P = 0.0036) in patients with wildtype MBL were compared with patients carrying variant alleles (Mann–Whitney test). The dotted lines indicate the detection limit of the assay.

 


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FIG. 2. MBL function in relation to MBL concentration and classical pathway activity. The correlation of MBL concentration and MBL complex activity (A) as well as the correlation between MBL concentration and MBL pathway activity (B) is shown for different sera from SLE patients carrying wildtype or variant alleles as indicated. The correlation between MBL pathway activity and classical pathway activity is shown for the same sera (C). The dotted lines indicate the detection limit of the assay.

 
Functional activity of the MBL pathway in relation to disease
We analysed the MBL concentration, MBL complex activity and MBL pathway activity for possible association with the different clinical variables. No significant association between the different clinical variables and MBL concentration or functional activity was found. However, significantly lower MBL concentrations (Fig. 3A, P = 0.03), MBL complex activity (Fig. 3B, P = 0.03) and MBL pathway activity (Fig. 3C, P = 0.02) were found in patients with anti-C1q antibodies as compared with patients without these autoantibodies. Similarly, the presence of anticardiolipin autoantibodies was associated with low serum levels of MBL (Fig. 3D, P = 0.01), low MBL complex activity (Fig. 3E, P = 0.02) and low MBL pathway activity (Fig. 3F, P = 0.03). In sharp contrast to these findings, patients with autoantibodies against MBL in their serum had significantly higher MBL concentrations (P = 0.025) (Fig. 3G). A similar but non-significant trend was found for MBL complex activity (P = 0.07), whereas MBL pathway activity was not significantly different between patients with or without anti-MBL autoantibodies (Figs 3H and I). No significant association was found between the presences of autoantibodies and classical pathway activity (P>0.28; data not shown). Together, these data demonstrate an association of autoantibodies against cardiolipin and C1q with MBL dysfunction.



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FIG. 3. The presence of autoantibodies is associated with MBL concentration and function. Sera from SLE patients were divided into two groups on the basis of the absence or presence of autoantibodies against C1q (A–C), cardiolipin (D–F) and MBL (G–I). MBL concentration, MBL complex activity and MBL pathway activity in the different sera are shown for each group. Differences between the two groups were evaluated by the Mann–Whitney test.

 
MBL genotype and function in relation to SLE disease activity
In order to examine whether MBL pathway dysfunction is also related to disease activity we analysed the SLEDAI in SLE patients divided in two groups on the basis of their MBL genotype, MBL concentration, MBL complex activity and MBL pathway activity.

Disease activity was not significantly different between patients with MBL wildtype or variant alleles, between patients with high or low MBL concentration or between patients with high or low MBL complex activity (Figs 4A–C). However, patients with low MBL pathway activity (<500 U/ml) had significantly more disease activity, as measured with the SLEDAI, compared with patients with high (>500 U/ml) MBL pathway activity (Fig. 4D). No significant difference was found between the SLEDAI of patients with high or low classical pathway activity (P = 0.25; data not shown). The MBL genotype, MBL concentration, MBL complex activity or MBL pathway activity were not significantly associated with organ damage measured with the Systemic Lupus International Collaborating Clinics/ACR Damage Index.



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FIG. 4. SLE disease activity in relation to MBL genotype and function. SLE disease activity indices (SLEDAI) are shown for SLE patients carrying wildtype MBL genotype or variant alleles (A), and low or high levels of MBL concentration, MBL complex activity and MBL pathway activity (B–D), respectively, as indicated.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
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The present study demonstrates a significant association, in patients with SLE, of the presence of anticardiolipin and anti-C1q autoantibodies with the presence of MBL variant alleles, with low serum MBL concentrations, and with impaired MBL function. The results further indicate that expression of MBL variant alleles in SLE patients is not only associated with reduced MBL concentrations but also with impaired MBL complex activity and impaired MBL pathway activity.

The presence of a wide variety of autoantibodies is a characteristic finding in SLE. Most of the autoantibodies are directed against membranous, intracellular or nuclear target antigens. Some antibodies are directed against fluid phase proteins such as C1q [29] and MBL [27]. Autoantibodies against nuclear proteins, such as antinuclear (ANA) and anti-double-stranded DNA (dsDNA) antibodies, are used as diagnostic markers in SLE [30], whereas anti-C1q and anti-dsDNA antibodies are also used to monitor lupus activity. A rise in the titre of anti-C1q antibodies is reported to be predictive for renal flares [31]. In patients with SLE, impaired normal mechanisms of waste disposal due to complement deficiency have been hypothesized, and therefore this material can be a source of autoantigens.

Recently it has been shown that MBL binds to apoptotic material and mediates its clearance by macrophages [3, 14, 15]. Therefore, patients with low MBL serum concentrations and impaired MBL function could have a decreased rate of clearance of apoptotic material, which may result in higher autoantibody levels directed against target antigens, such as phospholipids and C1q, present on apoptotic material. In contrast to previously published results by Garcia-Laorden et al. [32], in our study anticardiolipin antibodies were more frequently found in patients with MBL variant alleles compared with patients with the MBL wildtype genotype. In addition, we demonstrated that C1q autoantibodies are more frequently present in patients with MBL variant alleles, and decreased MBL concentrations and impaired MBL activity were found in patients with autoantibodies against C1q and cardiolipin. These findings support our hypothesis of increased generation of autoantibodies to antigens present on apoptotic material in SLE patients with MBL dysfunction. Furthermore, increased MBL concentrations were found in SLE patients with anti-MBL autoantibodies and anti-MBL autoantibodies were not found in patients with variant alleles. These results could indicate that only intact wildtype MBL can bind efficiently to apoptotic material and therefore be presented by antigen-presenting cells. However, the clinical relevance of anti-MBL antibodies in patients with SLE has yet to be demonstrated.

A genetic deficiency for C1q is almost always associated with SLE in humans. However, C1q deficiency is very rare and therefore it is not a major cause for SLE in the human population. In a recent meta-analysis by Garred et al. [33], it was observed that the presence of MBL variant alleles (A/0 and 0/0) was associated with a significantly increased risk of developing SLE when the diagnosis of SLE was based on the fulfilling of at least four ACR criteria. In our study we did not find such an association, although our patients were similarly defined. However, this is probably due to the size of the patient cohort examined in the present study. It was proposed by Garred et al. [33] that the MBL2 gene is a disease modifier locus rather than a true disease susceptibility locus, being involved in accelerated progression of the disease. In this respect, our study supports this idea by showing that MBL2 variant alleles are associated with increased production of autoantibodies that are known to be associated with disease manifestations in SLE. Furthermore, our study indicates for the first time an association between the presence of autoantibodies in SLE patients and functional impairment of the MBL pathway of complement, which supports the genetic studies indicating a role for MBL in SLE.

MBL serum concentrations are known to be dependent on MBL genotype. Individuals with variant alleles have decreased MBL serum concentrations compared with serum concentrations of individuals with wildtype MBL genotype. We show that SLE patients with MBL variant alleles have less functionally active MBL as compared with patients with wildtype MBL. This was demonstrated by an impaired ability to activate exogenous C4 by MBL–MASP complexes bound to mannan, as well as by an impaired capacity to activate the whole complement cascade upon binding of MBL to mannan. Within the population carrying the wildtype MBL2 gene the concentration of MBL also varies from very low to normal levels, which could be at least partially explained by differences in promoter genotypes. Differences in serum concentrations for MASPs could also play a role in the variable functional activity of MBL in carriers of the MBL wildtype genotype [34]. Differences in functional activity of the whole MBL pathway within the population carrying the wildtype MBL genotype could be due to differences in MBL and MASP concentrations as well as differences in concentrations of the other complement components.

Therefore, we assessed MBL complex activity as well as whole MBL pathway activity and classical pathway activity to examine the role of the MBL pathway in more detail in patients with SLE. The MBL complex activity and the MBL pathway activity were both dependent on MBL genotype. Furthermore, a highly significant correlation was found between MBL concentration and MBL complex activity without evidence of MASP-2 deficiency [35]. A statistically significant correlation was also found between MBL concentrations and MBL pathway activity. However, this relation was less profound than the relation between MBL concentrations and MBL complex activity, and a few discordant samples were noticed. A possible explanation for this finding is complement consumption in patients with SLE. For a fully active MBL pathway appropriate serum complement concentrations are required. To further investigate this possibility classical pathway activity was assessed in the same samples. Classical pathway activity was significantly correlated with MBL pathway activity but not associated with MBL genotype. Since this correlation was shown to be independent of MBL, this is most likely due to differences in the levels of complement consumption shared by the MBL pathway and the classical pathway. Furthermore, in serum samples from patients with the wildtype MBL genotype with a remarkable discrepancy between MBL complex activity and MBL pathway activity, low classical pathway activity was found. Therefore one could speculate that some of the patients with lower levels of MBL pathway activity are those patients with more active disease. Indeed, for the SLE patients examined in the present study an association was found between disease activity and MBL pathway activity, as a functional parameter of the whole complement cascade starting from MBL binding to mannan. However, MBL complex activity and MBL serum concentrations were not significantly associated with the disease activity index, indicating that the association between MBL pathway activity and SLEDAI cannot be solely explained by MBL deficiency. Therefore, these results suggest that a combination of factors, such as possible defects at the level of MBL as well as acquired defects at other levels of the cascade, could be critical in this respect. These acquired defects may include decreased levels of C4 and/or C3, which are well know to be associated with severe disease in patients with SLE [36].

The results of our study could implicate that patients with less functional MBL may present with more severe disease because of impaired clearance of apoptotic material and therefore enhanced antibody production. The combined presence of anticardiolipin and anti-C1q autoantibodies was strongly associated with increased disease activity in the present study (P = 0.006, not shown). In addition, it has been shown that SLE patients with MBL variant alleles show higher incidence of infections [33]. Therefore, the increased production of antibodies in SLE patients with MBL dysfunction could possibly also be related to impaired MBL-dependent clearance of non-self antigens. Active disease and therefore more complement consumption in patients with lower MBL concentrations could further deteriorate MBL pathway activity as well as classical pathway activity, thus enhancing the susceptibility to infections, and possibly also the disturbed clearance of apoptotic material. In a recent study on the binding of MBL to apoptotic cells, it was observed that complement activation by apoptotic cells is predominantly due to activation of the classical pathway and not the MBL pathway of complement [5].

The present study supports a role for MBL as a disease modifier in patients with SLE. Functional impairment of the MBL pathway of complement, which is common in the human population, appears to be associated with enhanced production of autoantibodies against C1q and cardiolipin. In view of the role of MBL and C1q in the recognition of self debris, MBL deficiency may lead to disturbed in vivo clearance of apoptotic material, resulting in enhanced production of autoantibodies against antigens associated with apoptotic material, including C1q. Binding of autoantibodies to apoptotic cells could subsequently further modulate the response of phagocytic cells towards phagocytosis of apoptotic cells [3], thus leading to an amplification of systemic autoimmunity.


    Acknowledgments
 
Part of this work was supported by the Dutch Kidney Foundation (PC 95, C98-1763) and by a grant of the European Union (QLGT-CT2001–01039).

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 18 May 2004; revised version accepted 27 August 2004.



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