The role of interleukin-10 promoter polymorphisms in the clinical expression of primary Sjögren's syndrome
J. Font1,,
M. García-Carrasco1,3,
M. Ramos-Casals1,
A. I. Aldea2,
R. Cervera1,
M. Ingelmo1,
J. Vives2 and
J. Yagüe2
1 Systemic Autoimmune Diseases Unit and
2 Department of Immunology, Clinical Institute of Infection and Immunology, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, School of Medicine, University of Barcelona, Barcelona, Spain and
3 Department of Rheumatology, School of Medicine, Benemérita Universidad Autónoma de Puebla, Puebla, México
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Abstract
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Objective. To analyse the role of polymorphisms of the interleukin-10 promoter region in the epidemiologic, clinical and immunologic characteristics of patients with primary Sjögren's syndrome (SS).
Methods. Sixty-three consecutive patients (59 women and four men; mean age 57 yr; range 2083 yr) were studied in our Unit. All patients fulfilled four or more of the modified diagnostic criteria for SS proposed by the European Community Study Group in 1996. As controls, 150 healthy volunteers were recruited from the medical and laboratory staff working in our hospital. All the samples from patients and controls were analysed by PCR amplification and direct sequencing.
Results. The frequency of the interleukin-10 (IL-10) GCC haplotype was higher (0.48 vs 0.34, P=0.006) and the frequency of the IL-10 ACC haplotype lower (0.25 vs 0.39, P=0.005) in patients with primary SS compared with healthy controls. In the genotype analysis, the frequency of the GCC/ATA genotype was higher (29 vs 11%, P=0.001) and that of the ACC/ACC genotype lower (3 vs 12%, P=0.044) in patients with primary SS compared with healthy controls. GCC-carriers showed an earlier onset of the disease (48.06±14.98 yr vs 57.53±14.20 yr, P=0.034). The existence of systemic involvement (defined by cutaneous vasculitis, peripheral neuropathy, renal and/or pulmonary involvement) was more frequent in carriers of the GCC haplotype, although the difference did not reach statistical significance (40 vs 27%, P=0.278). No significant differences in the haematologic (hypergammaglobulinaemia, elevated ESR) and immunologic (ANA, RF, anti-Ro/SS-A and anti-La/SS-B antibodies) parameters were observed in carriers of the GCC haplotype.
Conclusion. We describe an abnormal distribution of IL-10 promoter haplotypes in patients with primary SS compared with healthy controls. This consists of a predominance of the GCC haplotype, mainly related to a higher frequency of the heterozygote haplotype GCC/ATA. The presence of the GCC haplotype does not originate a different immunologic pattern but leads to an earlier onset of primary SS.
KEY WORDS: Interleukin-10, IL-10 polymorphisms, Primary Sjögren's syndrome.
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Introduction
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Sjögren's syndrome (SS) is an autoimmune disease that mainly affects exocrine glands and usually presents as persistent dryness of the mouth and the eyes due to functional impairment of the salivary and lachrymal glands [1]. In the absence of an associated systemic autoimmune disease, patients with this condition are classified as having primary SS. The histological hallmark is a focal lymphocytic infiltration of the exocrine glands, and the spectrum of the disease extends from an organ-specific autoimmune disease (autoimmune exocrinopathy) [2] to a systemic process with diverse extraglandular manifestations [39]. Due to this heterogeneity, attempts have been made to identify subsets of patients who would permit more reliable prediction of the course of primary SS in affected individuals [1012].
Although the cause of primary SS is unknown, many arguments favour environmental factors, especially viruses, that might act in patients with genetic susceptibility. Genetic predisposition coded by the major histocompatibility complex (MHC) appears to play a role in determining an individual susceptibility to the development of SS, which has been shown to be associated with HLA-B8, DR2, DR3, DQ1 and DQ2 haplotypes [1315]. Recently, some studies have focused on the association between interleukin-10 (IL-10) polymorphisms and genetic susceptibility for systemic lupus erythematosus (SLE) [1620], a systemic autoimmune disease with many similarities to primary SS. Thus, it is possible that IL-10 polymorphisms may be responsible for a proportion of the non-HLA genetic predisposition to primary SS or in determining different patterns of disease expression. The objective of the present study was to analyse the role of polymorphisms of the interleukin-10 promoter region in the epidemiologic, clinical and immunologic characteristics of patients with primary SS.
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Patients and methods
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Patients
We investigated 63 consecutive patients (59 women and four men; mean age 57 yr; range 2083 yr) in our unit. All patients were white and fulfilled four or more of the modified diagnostic criteria for SS proposed by the European Community Study Group in 1996 [21]. We considered age of SS onset as the point when symptoms and/or signs clearly attributable to SS were first objectively determined. None of the patients presented clinical or immunological evidence of other systemic autoimmune disease.
All patients underwent a complete history and physical examination, and diagnostic tests for SS were applied according to the recommendations of the European Community Study Group [22]. Twenty-nine (46%) patients had disease limited to the exocrine glands, and the remaining 34 (54%) had one or more of the following extraglandular manifestations: articular involvement in 21 (33%) patients, cutaneous vasculitis (demonstrated by cutaneous purpura and skin biopsy) in 12 (19%), pulmonary involvement (demonstrated by the clinical picture, altered chest radiography and/or spirometry) in 11 (18%), autoimmune thyroiditis (positive autoantibodies and altered thyroid function) in 11 (18%), Raynaud's phenomenon in seven (11%), peripheral neuropathy (demonstrated by the clinical picture and nerve conduction tests) in seven (11%) and renal involvement (serum creatinine
2 mg/100 ml, renal tubular acidosis or interstitial nephritis/glomerulonephritis demonstrated by biopsy specimen) in three (5%) patients.
Controls
As controls, 150 ethnically matched healthy volunteers were recruited from the medical and laboratory staff working in our hospital.
DNA extraction
DNA was extracted either from lymphocytes or from whole blood according to standard methods, using a salting out technique [23] or a commercial QIAamp® Blood Mini Kit (Qiagen, Germany). The samples were stored at -70°C until used.
DNA amplification
PCR amplification was performed on a Perkin Elmer 2400 thermal cycler in 50 µl reaction mixtures containing 50 ng of test DNA, Taq polymerase buffer [20 mM TrisHCl, pH 8.0, 150 mM MgCl2, 100 mM KCl, 50% glycerol (Boehringer Mannheim, Germany), 200 µM each dGTP, dATP, dTTP and dCTP (Gibco BRL), and 1 U Taq DNA polymerase ExpandTM High Fidelity PCR System (Boehringer Mannheim)]. Primers were used which amplified the segment of the IL-10 gene promoter from -1120 to -533. The primers used were upstream IL10.1, 5'-ATCCAAGACAACACTACTAA-3' and downstream IL10.2, 5'-TAAATATCCTCAAAGTTCC-3'. The following cycling conditions were used: 95°C for 5 min; 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, 72°C for 1 min [23].
Sequencing of the IL-10 gene promoter
Initially, 2 µl of PCR product generated from genomic DNA using the primer pair described above were sequenced with ABI prismTM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, following the kit protocol and with 2.5 pmol of the upstream IL10.1 primer. Cycle sequencing was: 96°C for 1 min; 25 cycles of 96°C for 30 s, 50°C for 15 s and 60°C for 4 min. The sequencing reactions were analysed by the ABI Prism 377 system.
Polymorphism analysis
Three polymorphisms of the IL-10 gene promoter have been previously described [24]. The first, located at -1082, constitutes a G to A substitution, the second, at -819, is a C to T substitution and the third, at -592, is a C to A substitution. The allele polymorphism was assigned to each sample from the nucleotide sequence obtained from the promoter region located between -1120 and -533 for each sample. The putative haplotypes were GCC, ACC and ATA, corresponding to the allele usage at the -1082, -819, -592 polymorphisms, as previously described [25, 26]. All the samples from patients and controls were analysed by PCR amplification and direct sequencing, and were assigned according to these three haplotypes and none of them presented a cross-haplotype pattern.
Laboratory studies
Immunological tests included antinuclear antibodies (ANA) (indirect immunofluorescence using mouse liver/kidney/stomach as substrates), antibodies to dsDNA (determined by Farr's technique), precipitating antibodies to the extractable nuclear antigens Ro/SS-A and La/SS-B (ELISA) and rheumatoid factor (RF) (ELISA). Complement components (C3 and C4) were estimated by the radial immunodiffusion method and CH50 by Lachmann's haemolytic technique. Serum cryoglobulinaemia was measured after centrifugation; serum supernatant was removed, incubated at 4°C for 8 days and examined for cryoprecipitation.
Statistical analysis
Gene frequencies were calculated by gene counting and expressed as the ratio of the number of each allele observed within a locus to the total number observed in that locus. We used conventional
2 and Fisher's exact test to compare gene frequencies between SS patients and controls, Student's test for the comparison of means in large samples of similar variance, and the non-parametric MannWhitney U-test for small samples. Values of quantitative variables are expressed as mean±standard error of the mean (S.E.M.). A value of P<0.05 was taken to indicate statistical significance. This statistical analysis was performed by means of the SPSS program using the information stored in the database program.
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Results
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Polymorphisms of the IL-10 promoter in SS patients and controls
The genotype distribution of IL-10 polymorphisms, the haplotype carrier rate and the haplotype frequencies in primary SS patients and control subjects are summarized in Table 1
. The frequency of the IL-10 GCC haplotype was higher (0.48 vs 0.34, P=0.006) and that of the IL-10 ACC haplotype lower (0.25 vs 0.39, P=0.005) in patients with primary SS compared with healthy controls. Patients with primary SS and control subjects showed the same frequency of the IL-10 ATA haplotype (0.27). In the genotype analysis, the frequency of the GCC/ATA genotype was higher (29 vs 11%, P=0.001) and that of the ACC/ACC genotype lower (3 vs 12%, P=0.044) in patients with primary SS compared with healthy controls.
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TABLE 1. IL-10 gene polymorphisms in primary SS: individual IL-10 genotypes, haplotype carrier rate and haplotype carrier frequencies in healthy controls and in patients with primary SS
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Polymorphisms of the IL-10 promoter and SS features
Table 2
summarizes the main epidemiologic, clinical, haematologic and immunologic SS features according to the presence of the GCC haplotype (GCC carriers). GCC carriers showed an earlier onset of the disease (48.06±14.98 yr vs 57.53±14.20 yr, P=0.034). Systemic involvement (defined by cutaneous vasculitis, peripheral neuropathy, renal and/or pulmonary involvement) was more frequent in GCC carriers, although the difference did not reach statistical significance (40 vs 27%, P=0.278). Likewise, no significant differences in the haematologic (hypergammaglobulinaemia, elevated ESR) and immunologic (ANA, RF, anti-Ro/SS-A and anti-La/SS-B antibodies) parameters were observed in carriers of the GCC haplotype.
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TABLE 2. Frequency of the main epidemiologic, clinical, haematologic and immunologic SS features according to the presence of the GCC haplotype
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Discussion
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IL-10 is a major immunoregulatory cytokine with diverse immunomodulating effects on the immune system. IL-10 suppresses type 1 T-helper lymphocytes and also has potent in vitro stimulatory effects on B lymphocytes, which include increased MHC class II expression, production of immunoglobulins and DNA replication [27]. The gene encoding IL-10 has been mapped to chromosome 1 [28] and the capacity for IL-10 production appears to be genetically encoded [29]. Several polymorphic sites within the promoter region of the IL-10 gene have been described, and three biallelic polymorphisms, at positions -1082, -819 and -592 from the transcription initiation site, have been recently identified [24]. The three dimorphisms exhibit strong linkage disequilibrium and appear in three potential haplotypes: GCC (G at position -1082, C at position -819, C at -592), ACC and ATA. IL-10 production is associated with these haplotypes, and so these individuals can be genotyped for higher or lower levels of production of this cytokine [24]. The ability of individuals to produce high levels of IL-10 is evidently controlled by a G at position -1082, as this variant is found in the highest producers [24].
In primary SS, the increased frequency of the disease among first-degree relatives and the increased risk in siblings of SS patients emphasizes the importance of genetic influence on disease predisposition. Among the genetic factors, the role of HLA molecules has been widely investigated in SS patients [1315]. Recently, there was an increased interest focusing on the study of the non-HLA genetic susceptibility to systemic autoimmune diseases. Specifically, there have been many studies investigating possible associations between IL10 promoter polymorphisms in patients with SLE [1620] or rheumatoid arthritis [25, 30]. In primary SS, high levels of IL-10 have been observed in peripheral blood mononuclear cells and in salivary glands of patients with primary SS [31], and two recent studies have analysed the IL-10 promoter polymorphisms in patients with primary SS [32, 33].
Two major findings of this study are noteworthy. One is the differentiated distribution of genotype frequencies of patients with primary SS compared with the controls, with a clear predominance of the GCC haplotype (48.4%) and a lower frequency of the ACC haplotype (24.6%). This confirms the results recently published by Hulkkonen et al. [33] in 62 Finnish patients, who also described an increased frequency of the GCC haplotype (42.9%) and a decreased frequency of the ACC haplotype (35.1%) (Table 3
). In accordance with Hulkkonen et al. [33], we also found a higher frequency of the GCC/ATA genotype and a significant decrease in the prevalence of the ACC/ACC genotype in our patients with primary SS. Nevertheless, the presence of the haplotype GTA cannot be ruled out but its frequency is so low as to make it unlikely that its occurrence would alter the statistical evaluation.
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TABLE 3. Analysis of the IL-10 promoter polymorphisms in patients with primary SS: main results of the different studies
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The second major finding of our study was the earlier onset of primary SS in patients carrying the IL-10 GCC haplotype. This genetic susceptibility towards early development of the disease may be of prognostic significance in some patients, since an increased risk for lymphoma development in patients with early onset SS has been previously suggested [10]. Conversely, no differences in the clinical and immunologic SS features were observed in our patients with respect to the different IL-10 polymorphisms, confirming the results of previous studies [32, 33]. Hulkonnen et al. [33] found no correlation between extraglandular manifestations and IL-10 haplotypes, and Rischmueller et al. [32] also described no significant differences in the analysis of some clinical (parotidomegaly, Raynaud's phenomenon, purpura or arthralgias), analytical (leucopenia, lymphopenia) and immunologic (anti-Ro/SS-A, anti-La/SS-B, low C4 levels) features. Thus, the IL-10 promoter polymorphisms seem to have little influence on the clinical and immunologic expression of primary SS, although the altered distribution of IL-10 haplotypes (increase of GCC, decrease of ACC) may constitute a predisposing genetic factor for primary SS. However, it should also be pointed out that, although our data indicate that the human IL-10 locus may be an important component of the genetic background for primary SS in Caucasian patients, we have not yet uncovered a direct relationship between the allelic and genotypic imbalance and the deregulated IL-10 secretion seen in this disease. It is clear that the IL-10 genotype cannot account for all the specific disease features observed in SS, and that other genetic factors contribute to the clinical spectrum of this disease.
In conclusion, we describe an abnormal distribution of IL-10 promoter haplotypes in patients with primary SS compared with healthy controls. This consisted of a predominance of the GCC haplotype, mainly related to a higher frequency of the heterozygote genotype GCC/ATA. The presence of the GCC haplotype influences the clinical expression of primary SS, not by originating a different immunologic pattern, but by leading to an earlier onset of primary SS. Our results suggest that the IL-10 polymorphisms may be an important component of the genetic background in the susceptibility to and clinical expression of primary SS in Caucasian patients.
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Acknowledgments
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Supported by grants Marato TV3 no. 3110 and FIS 99/0280 from the Fondo de Investigaciones Sanitarias. M.R.-C. is a Research Fellow sponsored by a grant (2000FI, 00332) from the Generalitat de Catalunya (Comissionat per a Universitats i Recerca, CIRIT). A.I.A. is supported by grant Marato TV3 no. 3110.
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Notes
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Correspondence to: J. Font, Unitat de Malalties Autoimmunes Sistèmiques, Hospital Clínic, C/Villarroel, 170, 08036 Barcelona, Spain. 
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Submitted 14 May 2001;
Accepted 25 March 2002