Interleukin-10 promoter microsatellite polymorphisms in systemic lupus erythematosus: association with the anti-Sm immune response

H. Schotte, M. Gaubitz, P. Willeke, N. Tidow1, G. Assmann1, W. Domschke and B. Schlüter1

Medizinische Klinik und Poliklinik B and 1 Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Münster, Germany.

Correspondence to: H. Schotte, Department of Medicine B, Münster University Hospital, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. E-mail: h.schotte{at}uni-muenster.de


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. Overproduction of interleukin-10 (IL-10) is a pivotal feature in the pathophysiology of systemic lupus erythematosus (SLE). In vitro IL-10 secretion has previously been related to haplotypes of the IL-10 promoter microsatellite polymorphisms IL10.R and IL10.G. Published data concerning the association of IL10.G alleles with susceptibility to SLE are inconsistent in different ethnic populations. We analysed the association of IL-10 promoter microsatellite polymorphisms with disease susceptibility and manifestations in German Caucasian patients with SLE.

Methods. Two hundred and ten (210) SLE patients fulfilling the 1997 revised ACR criteria and 158 ethnically, age- and sex-matched healthy controls were genotyped for the IL-10 promoter microsatellite polymorphisms by fragment length analysis. Haplotypes were reconstructed using a Bayesian coalescent theory-based method with PHASE software. Allele and haplotype distributions were compared between patients and controls and between subgroups of patients with different clinical and immunopathological findings.

Results. In the study population no significant associations of individual IL10.R and G alleles or their haplotypes with susceptibility to SLE or major clinical manifestations were observed. By contrast, alleles G14 and G15 and haplotypes R2-G14 and R2-G15 were significantly over-represented in anti-Sm antibody-positive patients.

Conclusions. The IL-10 promoter microsatellite polymorphisms and their haplotypes do not constitute a major risk factor for SLE in German Caucasians. However, the identification of genetic markers such as the IL-10 high-response haplotype R2-G14 predisposing for the production of anti-Sm antibodies may help to elucidate the conditions that lead to the development of SLE.

KEY WORDS: Systemic lupus erythematosus, Interleukin-10, Promoter microsatellite polymorphisms, Anti-Sm antibodies, German Caucasian patients


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects virtually any organ in the body. The immunological dysregulation typical of SLE is characterized by polyclonal B-cell activation, production of pathogenic autoantibodies, and an impaired cell-mediated immunity resulting from T-lymphocyte and antigen-presenting cell abnormalities [1]. Though certain susceptibility factors and triggering events like the hormonal milieu or exposure to ultraviolet radiation have been identified, the exact aetiology of SLE remains elusive. A strong genetic basis has to be assumed, and several genetic polymorphisms have been suspected to contribute to SLE susceptibility [2].

Interleukin-10 (IL-10) was originally defined as a cytokine with properties to alter the balance of murine Th1/Th2 activity in favour of the Th2-type response [3]. It is a negative autocrine regulator of tumour necrosis factor production and a potent stimulator of B lymphocytes [4, 5]. Moreover, IL-10 suppresses macrophage activation and antigen presentation, thereby directly and indirectly inhibiting T-cell function [6]. Several studies have demonstrated that IL-10 is overproduced in SLE patients [7–10]. IL-10 serum concentrations correlate with disease activity, and the ratio of the IL-10 to interferon-{gamma} secreting cells has been attributed to be an indicator of disease severity [11–14]. However, patients’ B lymphocytes and monocytes also spontaneously produce increased quantities of IL-10 independently of disease status. Treatment of SLE patients with an anti-IL-10 monoclonal antibody resulted in a rapid amelioration of the clinical status, in particular the cutaneous and the articular symptoms [15]. Additionally, dysregulation of IL-10 production has been described in healthy relatives of SLE patients [16, 17]. A larger proportion of them displayed impaired cell-mediated immune responses, diminished IL-2 production and polyclonal B-cell activation. Thus a constitutionally altered production of IL-10 could explain some of the principal features of immunological dysregulation occurring in SLE [18].

The genetic background accounts for about 75% of the interindividual variability in IL-10 secretion [19]. The IL-10 gene maps to chromosome 1q31–q32 [20]. Its promoter region contains several polymorphic elements. Approximately 1.1 kilobase pairs (kb) upstream of the transcription initiation site the microsatellite IL10.G, a CA dinucleotide repeat with at least 11 alleles, is found [21]. A further CA repeat with at least four alleles, IL10.R, has been identified approximately 4.0 kb upstream of the IL-10 gene [22]. In Dutch individuals, the lipopolysaccharide-induced IL-10 secretion in vitro varied according to the haplotypic arrangement of these microsatellite alleles [23]. Haplotypes containing the allele IL10.R3 were associated with lower IL-10 secretion than haplotypes containing any other IL10.R allele, and the haplotype IL10.R2-G14 was associated with the highest IL-10 secretion overall. Although these functional data await confirmation in other ethnic groups, the IL10.G and IL10.R microsatellite loci may represent candidate polymorphisms associated with susceptibility to SLE. However, previous studies in SLE patients of different ethnic origins have shown conflicting results [24–27]. In addition, none of these studies considered the haplotypic architecture of these polymorphic sites within the IL-10 promoter. We therefore analysed the IL-10 microsatellite polymorphisms in a series of German Caucasian SLE patients and reconstructed haplotypes by means of a mathematical model. We compared allele and haplotype distributions between patients and healthy controls and analysed subgroups of patients defined by major clinical or immunopathological findings.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Study subjects
The study protocol was approved by the local independent ethics committee (Ethikkommission der Ärztekammer Westfalen-Lippe und der Medizinischen Fakultät der Westfälischen Wilhelms-Universität Münster, Germany). After receipt of informed consent according to the Declaration of Helsinki, blood samples were collected from 210 German Caucasian SLE patients at the out-patient clinic for rheumatology, Department of Medicine B, Münster University Hospital, Germany. The patients satisfied at least four of the 1997 updated American College of Rheumatology (ACR) criteria for SLE [28]. Medical history was reviewed from the onset of disease until admission to the study. Anti-dsDNA antibodies were assessed by radioimmunoassay (Biermann, Bad Nauheim, Germany). Anti-Sm antibodies were tested by enzyme-linked immunoassay (Pharmacia, Freiburg, Germany). Clinical manifestations of the disease as defined by the ACR criteria and immunopathological findings were recorded in standardized questionnaires. Thereby we surveyed a median disease duration of 11 yr (range 1–35). German Caucasian age- and sex-matched controls (n = 158) were taken from the Prospective Cardiovascular Münster (PROCAM) study [29]. Participants in this study were employees of Westphalian companies from the same geographical area as the patients. Significant cardiovascular, pulmonary, metabolic, rheumatic and renal disease was excluded by review of medical history and physical examination.

Genotyping
DNA was extracted from EDTA-anticoagulated blood according to standard protocols [30]. The multiallelic IL-10 microsatellites IL10.R and IL10.G were genotyped by fragment length analysis. Flanking primers were constructed as follows: IL10.1 5'-GTC.CTT.CCC.CAG.GTA.GAG.CAA.CAC.TCC-3' (5'-labelled with 6-FAM fluorescent dye, PE Applied Biosystems, Weiterstadt, Germany), IL10.2 5'-CTC.CCA.AAG.AAG.CCT.TAG.TAG.TGT.TG-3', IL10.3 5'-CCC.TCC.AAA.ATC.TAT.TTG.CAT.AAG-3' (5'-labelled with HEX fluorescent dye, PE Applied Biosystems) and IL10.4 5'-CTC.CGC.CCA.GTA.AGT.TTC.ATC.AC-3'. IL10.1 and IL10.2 amplified the IL10.G microsatellite, IL10.3 and IL10.4 the IL10.R microsatellite. A multiplex polymerase chain reaction (PCR) was carried out in a thermal cycler (GeneAmp® PCR System 9700, PE Applied Biosystems) under the following conditions: hot start at 94°C for 10 min, 30 cycles of 30 s at 95°C, 45 s at 65°C, 45 s at 72°C, final extension for 7 min at 72°C. The PCR reaction mixture contained 0.6 U AmpliTaq GoldTM, primers IL10.1, IL10.2, IL10.3 and IL10.4 at 0.2 µM, dNTP at 50 µM, MgCl2 at 1 mM, and approximately 50 ng DNA in a total volume of 20 µl. A DNA size standard (20 µl of a 1:40 dilution of Genescan-ROX 500, PE Applied Biosystems) was added to 1 µl of PCR product in a 96-well MicroAmp optical reaction plate (PE Applied Biosystems). The samples were denatured after heating at 90°C for 2 min and then subjected to fragment length analysis on a four-colour laser-induced fluorescence capillary electrophoresis system (ABI Prism 3700 Genetic Analyser, PE Applied Biosystems) using POP6 as polymer. The size of microsatellite-containing DNA fragments was measured by comparison with the DNA size standard using GeneScanTM software. The analysis of the IL10.R microsatellite revealed four alleles which ranged in size between 111 base pairs (bp) (IL10.R2) and 117 bp (IL10.R5). The 11 alleles of the IL10.G microsatellite ranged in size between 128 bp (IL10.G6) and 148 bp (IL10.G16). Each electrophoresis run included a negative control and a DNA sample with known IL10.R and IL10.G genotypes as positive control. Specificity of genotyping was confirmed in selected cases (n = 20 chromosomes) by direct sequencing of PCR products (data not shown).

Haplotype reconstruction and statistical analysis
Haplotypes were reconstructed using a Bayesian, coalescent theory-based method with the PHASE software (Version 2.0.2 for DOS) [31, 32]. The software incorporates a model that allows for recombination and decay of linkage disequilibrium with physical distance of alleles. Moreover, the type of polymorphism (SNP or multiallelic with/without stepwise mutation mechanism, respectively) is taken into account. Allele and haplotype frequencies in SLE patients and healthy controls as well as in patient subgroups with different clinical or immunopathological findings were compared by Monte Carlo simulation [33]. The T4 statistic was applied to test for significance which was assumed in case of a P value of <0.05. The strength of association was calculated as the odds ratio (OR) and is presented with 95% confidence intervals (CI). For calculation of linkage disequilibria {Delta} expected haplotype frequencies were estimated by multiplication of allele frequencies and subtracted from observed haplotype frequencies. {chi}2 analysis was used to determine the significance of the deviation from 0 to the {Delta} value. All statistical calculations were performed on a personal computer using the CLUMP (Version 1.6 for DOS) and the MedCalc software (Version 4.20.006 for Windows 95/NT).


    Results
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 Patients and methods
 Results
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Characteristics of patients and healthy controls
Two hundred and ten (210) SLE patients and 158 healthy controls entered into the present study. The median age of patients at onset of the disease was 27 yr (range 4–66), 191 were females. Their clinical manifestations and immunopathological findings are shown in Table 1. The median age of the healthy controls was 33 yr (range 17–70), 143 were females.


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TABLE 1. Clinical and immunopathological characteristics of SLE patients under study (n = 210)

 
Allele distribution
We found four different alleles for the IL10.R and 11 different alleles for the IL10.G microsatellite, respectively. The most frequent alleles were IL10.R2 (68% in SLE patients vs 73% in healthy controls), IL10.R3 (30 vs 24%), IL10.G9 (41 vs 40%) and IL10.G13 (24 vs 29%). There were no significant differences comparing the allele distribution between SLE patients and healthy controls (Figs 1 and 2). Slightly increased frequencies of the alleles R3 (OR 1.32; 95% CI 0.94–1.83) and G9 (OR 1.07; 95% CI 0.79–1.44) in patients as well as of the alleles R2 (OR 0.79; 95% CI 0.57–1.08) and G13 (OR 0.78; 95% CI 0.56–1.09) in controls did not result in significant odds ratios.



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FIG. 1. IL10.R allele distribution in SLE patients and healthy controls (HC). Bars represent the allele frequencies in the respective population. Allele distribution was not significantly different between SLE patients and healthy controls [{chi}2 (T4 statistics) = 2.76; P = 0.2359].

 


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FIG. 2. IL10.G allele distribution in SLE patients and healthy controls (HC). Bars represent the allele frequencies in the respective population. Allele distribution was not significantly different between SLE patients and healthy controls [{chi}2 (T4 statistics) = 4.35; P = 0.6438].

 
Haplotype distribution
Using the PHASE software, we reconstructed a total of 22 different haplotypes containing the IL10.R and G microsatellite polymorphisms with an average phase probability of 99%. Ninety six per cent of the reconstructed phases had a probability greater than 95%. Linkage disequilibria between these two loci are presented in Table 2. The highest positive linkage was observed for R2-G13 and R3-G9, while R2-G9, R3-G13 and R3-G14 were negatively linked. The prevailing haplotypes were R2-G9 (15% in SLE patients vs 18% in healthy controls), R2-G13 (23 vs 29%) and R3-G9 (24 vs 19%). Again, the haplotype distribution was not significantly different between patients and healthy controls (Fig. 3). However, the R3-G9 haplotype tended to be more prevalent among patients (OR 1.34; 95% CI 0.94–1.92), whereas the haplotypes R2-G9 (OR 0.80; 95% CI 0.54–1.18) and R2-G13 (OR 0.74; 95% CI 0.53–1.04) were found more often in the control population.


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TABLE 2. Linkage disequilibria between IL10.R and G alleles

 


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FIG. 3. IL10.R-G haplotype distribution in SLE patients and healthy controls (HC). Bars represent the haplotype frequencies in the respective population. Haplotype distribution was not significantly different between SLE patients and healthy controls [{chi}2 (T4 statistics) = 10.63; P = 0.3059].

 
Gene dosage effect
Neither the hetero- nor the homozygous presence of the frequent alleles and haplotypes was associated with a significant risk for SLE (Table 3). However, homozygosity for the R3-G9 haplotype increased the odds ratio for SLE, whereas a lowered odds ratio in the case of homozygosity for R2-G9 and R2-G13 is suggestive of a protective effect. In contrast, the allele-based gene dosage analysis revealed no consistent correlation of the zygosity status with the strength of SLE association.


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TABLE 3. Gene dosage effect of IL10.R and G alleles and haplotypes in SLE patients and healthy controls (HC)

 
SLE manifestations and antibody profile
The comparison between subgroups of patients with major clinical and immunopathological findings revealed no association of IL-10.G and R microsatellite alleles/haplotypes with malar rash, photosensitivity, serositis, neuropsychiatric lupus, cardiopulmonary manifestations, nephritis, or anti-dsDNA antibodies. In contrast, after stratification for anti-Sm antibody positivity significant differences in the IL10.G allele [{chi}2 (T4 statistics) = 17.11; P = 0.0056] and haplotype distribution became evident [{chi}2 (T4 statistics) = 17.11; P = 0.0310]. In detail, positive associations were found for the G14 and G15 alleles and the R2-G14 and R2-G15 haplotypes with the presence of anti-Sm antibodies (Table 4).


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TABLE 4. Association of IL10.G alleles and IL10.R-G haplotypes with anti-Sm antibody status

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The present analysis of the IL-10 promoter microsatellite polymorphisms IL10.R and G in German Caucasian patients with SLE revealed no significant association of individual alleles or haplotypes with the disease susceptibility. As far as IL10.R is concerned, these results confirm two earlier published reports [24, 27]. In contrast, individual IL10.G alleles have previously been shown to be associated with SLE susceptibility [24, 25, 27, 34]. However, those data are inconsistent as the association referred to different alleles. Eskdale et al. [24] described an association of SLE in Scottish Caucasians with G13, whereas Mehrian et al. [25] found G10 to be elevated in Mexican patients, and in two Italian cohorts the G11 allele was found to be associated with SLE. In contrast, Alarcón-Riquelme et al. [26] reported no association of IL10.G alleles with the disease susceptibility in a large series of 330 Mexican SLE patients. Our results are corroborative of the latter study and extend these findings to a German Caucasian population. Further evidence against a predominant role of IL-10 microsatellite polymorphisms in SLE susceptibility derives from linkage analysis in multiplex SLE families [26].

These discrepant findings may be explained by the hypothesis that the IL10.G microsatellite is rather a marker of disease susceptibility due to linkage disequilibrium than the primary causative genetic polymorphism. A further reason may be the considerable genetic heterogeneity between different ethnic groups. The prevailing IL10.G allele in all populations cited is G9 with a frequency between 40 and 50%. However, in Italians its frequency was described to be as low as 30%. A second common allele, G13, was found in the European populations with a frequency ranging between 20 and 30%, while in Mexicans it was clearly less frequent at about 10 to 15%. This is paralleled by an increase in G10 to 15–20% in Mexicans, which is an allele with a frequency of less than 10% in Europeans. Thus, interethnic variations account for substantial differences in the allele distribution, a problem for association studies that can only partly be overcome by ethnically matched controls. Due to the characteristic allele distribution of the IL10.G locus discrepancies are unlikely to derive from the different typing technology and allele nomenclature used in the published studies.

Several reasons may account for a lack of association of IL-10 microsatellite polymorphisms with SLE. First of all, on the assumption that the microsatellite polymorphisms actually influence IL-10 production, it may be possible that altered IL-10 levels are not relevant to the pathophysiology of SLE. However, the well-documented immunological properties of IL-10, the elevated IL-10 serum levels in SLE that correlate with the disease activity and the effects of anti-IL-10 treatment render this explanation rather unlikely [6, 11, 15]. Another reason may be that IL-10 abnormalities in SLE patients are not primarily genetically determined, but driven by other (e.g. environmental) factors. This hypothesis is supported by the finding of elevated numbers of IL-10 secreting cells not only in relatives but also in spouses of patients with SLE [17, 35, 36]. However, the known inheritance patterns of differences in IL-10 production in SLE patients and their relatives make this explanation improbable. Thus, it is more likely that the polymorphisms under study, at least in German Caucasians, do not influence IL-10 production to such an extent that it affects susceptibility to SLE. Indeed, further polymorphic elements like single-nucleotide polymorphisms (SNP) in the IL-10 promoter have been identified. The analysis of three SNPs in the proximal IL-10 promoter revealed no association with SLE susceptibility, but with certain disease manifestations [37–39]. More recently, further SNPs have been demonstrated in the distal IL-10 promoter close to the IL10.R microsatellite [40]. The distal IL10 SNP haplotypes segregate with differences in IL-10 production and have been found to be associated with SLE in a smaller series of 60 African-Americans. Thus, it may well be that these distal IL-10 promoter polymorphisms are more relevant for susceptibility to SLE, although this awaits further confirmation in larger patient populations and other ethnic groups.

The combination of polymorphic elements from the IL-10 promoter in the form of haplotypes has repeatedly been demonstrated [23, 41]. The analysis of haplotypes may be more informative when trying to find a functionally relevant sequence variation than studies of single microsatellite allele associations. We reconstructed haplotypes using a Bayesian, coalescent theory-based method [31, 32]. By means of this well-established method the most frequent haplotypes were R2-G9, R2-G13 and R3-G9, matching previously published data of Eskdale et al. [23] who deduced haplotypes from analysis of family genotyping data. In our SLE patient population we observed a tendency towards an elevated frequency of the R3-G9 haplotype, though not resulting in a significant odds ratio. This trend was confirmed in the following genotype analysis, as the odds ratio for SLE increased with homozygosity, although again not reaching significance. Thus we do not believe that this haplotype constitutes a major risk factor for SLE.

Subgroup analysis stratified for major clinical and immunopathological findings revealed associations of the alleles G14 and G15 and the haplotypes R2-G14 and R2-G15 with the presence of anti-Sm antibodies. Significance of the allelic imbalance held true after Bonferroni correction for multiple comparisons (Pc = 0.0448). Due to a negative linkage disequilibrium between R3 and G14/G15 there were no differences in the strength of association between the individual G alleles or the R-G haplotypes, respectively, and anti-Sm status. The polyclonal anti-Sm autoimmune response is directed against protein components of the small nuclear ribonucleoprotein (snRNP) particles [42]. Anti-Sm antibodies are present in approximately 30% of the patients with SLE and are highly specific for the disease. Thus, anti-Sm antibodies have been incorporated as a diagnostic tool into the ACR criteria for SLE [28]. The anti-Sm response has features typical of an antigen-driven immune response. Current work is attempting to identify the circumstances that predispose the common core proteins of snRNP particles to become autoantigens in vulnerable individuals. Our data provide evidence that IL-10 promoter haplotypes that have previously been linked to IL-10 secretion capacity partly determine the genetic background predisposing individuals to the elaboration of anti-Sm autoantibodies. Interestingly, the haplotype R2-G14 has been reported to be associated with the highest IL-10 secretion rate in vitro [23]. Thus, a constitutionally high IL-10 production may favour the anti-Sm immune response. To our knowledge, little is known about the relationship of IL-10 levels and production of anti-Sm antibodies. However, treatment of SCID mice transplanted with mononuclear cells from SLE patients with an immunomodulator known to reduce IL-10 production resulted in a significant decrease in anti-Sm antibody serum levels [43]. Admittedly, whether this reflects a more general reduction in B-cell activity or a specific mechanism for anti-Sm antibodies remains elusive [9].

In summary, our data provide no evidence for an association of the IL10.R and G microsatellite polymorphisms with overall susceptibility to SLE in German Caucasians. However, the identification of genetic risk factors such as the IL-10 high-response haplotype R2-G14 predisposing for the SLE-specific immune response against small nuclear ribonucleoproteins may help to clarify the conditions that lead to the development of the disease. Further polymorphic elements of the IL-10 promoter and their assigned haplotypes have to be included in future studies of SLE patients to delineate the effect of genetic variation within the IL-10 locus on disease susceptibility and presentation.

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 5 May 2004; revised version accepted 6 July 2004.



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