Department of Rheumatology and
2 Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands and
1 Department of Rheumatology, GKT School of Medicine, Guy's Hospital Campus, King's College, London SE1 9RT, UK
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Abstract |
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Methods. The association between interleukin 10 (IL-10) production and joint destruction was studied by comparing IL-10 mRNA content in synovial biopsies from seven patients with destructive joint disease and six patients with non-destructive joint disease. The IL-10 mRNA content was 0.4 ± 0.6 arbitrary units in erosive joints compared with 2.3 ± 1.2 arbitrary units in non-erosive joints (P < 0.03, MannWhitney U-test). As this difference suggested that IL-10 production was associated with joint destruction, we tested whether the IL-10 locus determined the extent of joint damage.
Results. Innate differences in IL-10 production are locus-dependent. In line with these data, we showed that innate differences in IL-10 protein production were also present as differences in IL-10 mRNA levels. We tested if polymorphisms in the promoter of IL-10 were associated with the extent of joint damage.
Discussion. In a cohort study of female rheumatoid arthritis patients followed for 12 yr, the extent of joint destruction differed significantly between patients with different IL-10 genotypes. In patients with the -1082AA genotype who were studied prospectively, the mean increase in radiographic damage score (modified Sharp score of X-rays of hands and feet) during the first 6 yr was 9 ± 9 per yr vs 19 ± 16 per yr for patients with the genotype -1082GG (P < 0.02). In line with these data, cultures of endotoxin-stimulated whole blood from 158 donors showed that the presence of the allele associated with less joint destruction correlated with slightly higher IL-10 production.
Conclusions. Both the immunogenetic and the synovial biopsies suggest that a variation in IL-10 production is associated with joint destruction.
KEY WORDS: Interleukin 10, Joint damage, Genotypes.
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Introduction |
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In healthy individuals, the amount of IL-10 produced in a standard assay as a result of a standard amount of endotoxin varies between individuals [10]. A heritability analysis in monozygotic twins showed that genetic factors account for 75% of the interindividual variation in IL-10 secretion [10]. These heritable differences in IL-10 secretion could not be explained by differences in TNF secretion [11]. Moreover, the heritable differences in IL-10 production have been shown to be clinically relevant. In a study of first-degree relatives of patients with meningococcal disease, families characterized by high production of IL-10 had a 20-fold higher chance of a fatal outcome than families with low production of IL-10 [10]. In line with these data, the probability of a fatal outcome in patients with pyrexia presenting to the first-aid department was greater in patients with a high plasma level of IL-10 than in those with a low plasma level [12]. Studies of systemic lupus erythematosus (SLE) patients reported that these patients exhibited greater IL-10 production than controls [13]. First-degree family members of SLE patients also exhibited greater IL-10 production than controls, providing further evidence that a high innate level of IL-10 production is associated with SLE [14].
Genetic characterization of the IL-10 production phenotype was based on results from whole-blood cultures stimulated with lipopolysaccharide (LPS). The ability to secrete IL-10 was demonstrated to be associated with haplotypes of the IL-10 locus defined by two microsatellite loci in the 4 kilobases (kb) immediately upstream of the IL-10 transcription initiation site [15]. This suggests that innate differences in IL-10 production are locus-dependent. Recently, it was demonstrated that patients with juvenile RA with more than four joints affected were more likely to have a particular IL-10 genotype than patients with fewer than four joints affected [16]. We now demonstrate that differences in endotoxin-induced IL-10 protein production are also present at the level of mRNA. The association between interindividual differences in joint destruction and genotypes of the IL-10 promoter [17] was studied in a cohort of RA patients. Moreover, mRNA levels encoding IL-10 were measured in synovial biopsies of patients suffering from non-destructive chronic polyarthritis and in patients who were suffering from destructive polyarthritis.
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Patients, materials and methods |
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Cross-sectional group of RA patients
To study the association between IL-10 DNA polymorphisms and the occurrence of RA, blood was collected from 291 consecutive RA patients who attended the out-patient clinic of the Leiden University Hospital in 1994, after they had given informed consent and after approval had been obtained from the Ethics Committee of the Leiden University Hospital, as described previously [18]. All patients fulfilled the ACR criteria of 1987 for RA. DNA was isolated from these 291 patients to test whether the frequency of IL-10 polymorphisms in them differed from that in the general population.
Female RA patient cohort
To study the relationship between IL-10 genotypes and joint damage over time, a cohort study was performed. None of the patients in the female RA patient cohort was also present in the above-mentioned cross-sectional group of RA patients. The cohort started with a group of 138 incident female RA patients who were identified between 1982 and 1986 [19]. All patients fulfilled the 1958 criteria for definite RA. Of these 138 patients, 112 were assessed completely after an average follow-up period of 12 yr (range 1014 yr) for details see [1921]. For technical reasons (refusal of patients to have DNA isolated, failure of DNA isolation and failure of IL-10 genotyping), no genomic data could be generated for 21 of the 112 patients, yielding 91 patients. A detailed comparison of disease characteristics for the patients in which IL-10 haplotype testing was performed and those for which IL-10 haplotype testing failed revealed no differences. For example, the damage score (X-rays of hands and feet, modified Sharp score) at 3 yr was 49 ± 60 (mean ± S.D.) for all patients tested and 50 ± 66 for the patients for whom no IL-10 genotype was available. The damage score at 6 yr of tested patients remaining in the cohort was 81 ± 82 and that of the patients for whom no IL-10 genotype was available was 86 ± 92. The damage score at 12 yr was 141 ± 125 for the tested patients and 147 ± 130 for the patients for whom no IL-10 genotype was available. The 26 patients who were missing from the original cohort had baseline characteristics similar to those of the 112 patients available at 12 yr. The data used in the present analysis were obtained at study entry and after 3, 6 and 12 yr. At each visit, radiographs were taken of the hands and feet. The hand and feet radiographs were scored according to Van der Heijde's modification of Sharp's method [22]. This method reflects erosions and joint-space narrowing in 44 joints. The principal measure, the total score, is the sum of erosion and narrowing scores, and ranges from 0 to 448. The modification is, in principal, the inclusion of scoring of the feet. For the hands, one site of erosion (os triquetrum) and three sites of joint narrowing (radio-ulnar joint, lunar-triquetrum joint, first interphalangeal joint) are excluded. The erosion score is 1 if erosion is discrete, 2 or 3 if the erosion is larger, and 5 if it extends over the imaginary middle of the bone. The X-rays were scored per patient, randomly through time.
Whole-blood cultures
Blood was collected from healthy donors between 8.00 and 12.00 in the morning to limit the effect of circadian variation in cytokine production [10, 23]. Blood samples were obtained in endotoxin-free heparin tubes (Chromogenix, Molndal, Sweden) and were immediately diluted once with endotoxin-free RPMI 1640 (Flow Laboratories, Rockville, MD, USA). Aliquots of diluted blood (1 ml) were incubated for 24 h with 0, 10 and 1000 ng/ml endotoxin from Escherichia coli OIII:B4 (Sigma, St Louis, MO, USA) at 37°C and 5% CO2. Actinomycin D (Merck, Darmstadt, Germany) was added after 18 h of incubation with 1000 ng LPS/ml to determine the half-life of the mRNA. Each half hour a sample was harvested and centrifuged (10 min at 600 g). The resulting supernatant was collected and stored at -70°C until assay. The concentration of IL-10 was determined by enzyme linked immunosorbent assay (ELISA) (CLB, Amsterdam, The Netherlands). mRNA was isolated from the pellet as described above.
IL-10 RNA quantification in synovial biopsies
RNA harvested from synovial biopsies was reverse-transcribed into cDNA in a 20- µl volume at 45°C for 1 h using 1 µl M-MLV reverse transcriptase (200 U/ml), 5 x first-strand buffer, 0.1 M dithiothreitol, oligo dT1218 (20 ng/ µl) (GIBCO BRL) and dNTP mix (100 mM) (Pharmacia, Peapack, NJ, USA). The polymerase chain reaction (PCR) was performed using specific IL-10 and GAPDH primers, as described [24, 25], that give rise to products of 432 and 711 base pairs (bp) respectively. PCR was carried out using a cycling profile of 94°C for 1 min; 55°C (GAPDH) or 55°C (IL-10) for 2 min, and 72°C for 2 min with a final extension at 72°C for 8 min. To establish the primer-specific exponential region of the PCR, reactions were sampled every two cycles at the critical stage of the reaction. Five microlitres of the sample was taken and stored at 4°C until it was used. The PCR products were electrophoresed in 1% agarose, stained with ethidium bromide and blotted onto Hybond N-nylon membrane (Amersham Life Science, Amersham, UK). Gene-specific 24-mer internal probes (392 DNA/RNA Synthesizer, Applied Biosystems, Middletown, CT, USA) were prepared and labelled using polynucleotide kinase (200 U) (Pharmacia) and gamma-[32P]ATP (Amersham). Filter hybridizations were performed overnight at 57°C for GAPDH and 50°C for IL-10 and washed according to standard protocols. The resulting autoradiograms were quantified with a Bio-Imager densitometer and the results were expressed as intensity of optical density (IOD) values. The IOD values were plotted on a logarithmic scale against the number of PCR cycles. The final value for specific mRNA transcripts was expressed as the ratio IOD adhesion molecule/IOD control gene, using the mid-values in the exponential part of the curve.
IL-10 RNA quantification in whole-blood cultures
For 11 donors, mRNA was isolated from the pellet from whole-blood cultures stimulated for 18 h with LPS. Fifteen to twenty nanograms of RNA was separated by 1% agarose electrophoresis and transferred to a Hybond N (Amersham) membrane. A 32P-labelled dCTP probe encoding IL-10 (0.7 kb BglIIHindIII fragment of pH15C, ATCC no. 68192) was used to hybridize blots at 42°C for 18 h. The blots were washed, then exposed to Biomax film (Eastman Kodak Company, Rochester, NY, USA). Bands were quantified by means of a phosphor-imaging system (Molecular Dynamics, Sunnyvale, CA, USA). As an alternative method, semiquantitative PCR was performed on cDNA made from RNA from five of these 11 donors. The amount of cDNA was quantified relative to the amount of a multispecific internal standard, plasmid PQB3 (a generous gift of Dr D. Shire, Sanofi Research, Paris, France), essentially as described previously [26]. The PCR products were separated on an agarose gel and quantified by ethidium bromide staining using an Eagle Eye II (Stratagene, La Jolla, CA, USA).
DNA isolation
DNA was isolated from sodium dodecyl sulphate-lysed and proteinase-K-treated peripheral blood cells by phenolchloroform extraction. Fragments of the IL-10 promoter were amplified using the primer combination GTG CTG AGC TGT GCA TGC CT+51 and TTC CCC AGG TAG AGC AAC AC-1217. PCR fragments were subsequently dot-blotted on Hybond N+ membranes and hybridized according to described methods [27]. Sequences of the oligonucleotides and washing temperatures were as follows: -1082A, ACT TCC CC T TCC CAA AGA A, 52°C; -1082G, TTC TTT GGG A GG GGG AAG, 51°C; -819C, CAG GTG ATG TAA CAT CTC TGT GC, 62°C; -819T, GCA CAG AGA T AT TAC ATC ACC TGT, 63°C; -592C, CCG CCT GT C CTG TAG GAA, 50°C; -592A, TTC CTA CAG TTAC AGG CGG G, 52°C.
Statistical analysis
The IL-10 mRNA content in synovial biopsies, the number of joints affected by erosions in the hands after 3 yr and IL-10 production in whole-blood cultures were not normally distributed and the differences were therefore compared by the use of non-parametric tests (MannWhitney U-test and KruskalWallis H-test). The IQR represents the 25th and 75th percentiles of the distribution. Repeated measurements of the damage score over time were analysed by repeated-measures ANOVA after log transformation of the data. P < 0.05 was considered statistically significant.
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Results |
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IL-10 promoter polymorphism and joint damage in RA
The distribution of IL-10 genotypes in the cross-sectional group of RA patients was as follows: 1082AA, 20.3% (n = 59); -1082GA, 54.3% (n = 158); -1082GG, 25.4% (n = 74). This is not different from the genotype distribution in the population of The Netherlands [28] and that of the UK (P > 0.2) [17, 29, 30]. These data indicate that the -1082 polymorphism does not play a role in disease susceptibility.
The -1082G/A polymorphism is associated with the rate of radiographic damage in a longitudinally followed cohort of female RA patients
In this prospectively followed cohort of female RA patients, the increase in radiographic damage score for patients homozygous for the genotype -1082AA was 11 ± 16 per yr for the first 3 yr compared with 21 ± 20 per yr for patients with the genotype -1082GG (P < 0.02) (Fig. 2). [Repeated-measures ANOVA on the comparison between -1082AA and -1082GG (two groups, 1 degree of freedom) yielded F = 6.2, P = 0.017 (on log-transformed data F = 4.9, P = 0.03). Repeated-measures ANOVA on the comparison of -1082AA, -1082GA and -1082GG (three groups, 2 degrees of freedom) yielded F = 5.6, P = 0.005 (on log-transformed data F = 4.1, P = 0.002)]. In the first 6 yr, the increase in damage score was less in RA patients with the genotype -1082AA (9 ± 9 per yr) than in patients with genotype -1082GG (19 ± 16 per yr) (P < 0.02). Analysis of the rate of joint destruction between 3 and 6 yr of follow-up yielded similar results (Table 1
). However, there were no significant differences observed between 6 and 12 yr in the rate of change of radiographic damage between -1082AA and -1082GG patients (difference in rate of joint destruction between -1082AA and -1082GG patients, P > 0.1; MannWhitney U-test). A similar trend was observed for all individual joints (results not shown). The lower number of erosions in RA patients with the -1082AA genotype could not be explained by other determinants of radiographic damage score, such as an increased level of rheumatoid factor, the presence of DR4 or the radiographic damage score at onset (Table 2). Subgroup analysis to define the haplotypes further by including the 819 and 592 mutations yielded insufficient patient groups to allow sensible comparisons.
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IL-10 variation in protein production is reflected at the mRNA level
The data discussed above indicate that a polymorphism in the IL-10 promoter is associated with differences in joint damage. In a previous study, we have demonstrated that haplotypes of the IL-10 locus are associated with differences in IL-10 protein production [15]. Here we studied whether innate differences in IL-10 production are also present at the level of mRNA. In the LPS-induced whole-blood stimulation assay, the maximum levels of IL-10 mRNA were found after 18 h of incubation. Using this time point, the association between the amount of IL-10 mRNA and the amount of IL-10 protein was determined in 11 donors. The correlation between the relative amount of mRNA, as measured by Northern blot analysis, and the amount of protein was highly significant (n = 11, correlation 0.85, P < 0.001) (Fig. 3). Semiquantitative PCR yielded similar results in five of the donors tested.
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The half-life of IL-10 mRNA is similar in individuals with high and low production of IL-10
Differences in the amount of mRNA can be due to variability in gene transcription, differences in its half-life, or both. The half-life of mRNA encoding IL-10 was assessed in 12 donors by the addition of actinomycin D and measurement of IL-10 mRNA by Northern blot analysis. Figure 4 shows that there was no correlation between the time that mRNA encoding IL-10 was degraded in the presence of actinomycin D and the level of IL-10 protein production in different individuals.
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Different IL-10 promoter genotypes express different levels of protein
Three polymorphisms (-1082A/G, -819C/T and -592A/C) were determined in the IL-10 promoter. The IL-10 production of individuals with different IL-10 genotypes was measured by performing whole- blood cultures from 158 healthy individuals from 61 families. Segregation analysis in the families revealed that the IL-10 promoter polymorphisms give three haplotypes: -1082A/-819C/-592C, -1082A/-819T/ -592A and -1082G/-819C/-592C. IL-10 production was 3033 ± 252 pg/ml for the -1082A/-819C/-592C haplotype, 2931 ± 312 pg/ml for -1082A/-819T/-592A and 2601 ± 138 pg/ml for -1082G/-819C/-592C. The IL-10 production associated with the -1082G allele was 2682 ± 137 pg/ml vs 3166 ± 183 pg/ml for the -1082A allele (P < 0.02, MannWhitney U-test).
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Discussion |
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In arthritis, IL-10 can exhibit several functions. IL-10 down-regulates the synthesis of a broad spectrum of proinflammatory cytokines by monocytes/macrophages and neutrophils [31]. Moreover, IL-10 inhibits the inflammatory processes mediated by Th1-cells. The in vivo functions of IL-10 in immune homeostasis have been shown in studies with IL-10 knockout mice. In these mice the production of IL-12 and interferon is uncontrolled, which leads to autoimmune disease mediated by CD4+ cells [32]. In RA, both an imbalance between excessive production of the proinflammatory and anti-inflammatory cytokines and skewing of the T cells to a Th1-like response have been implicated. The assumption that differences in IL-10 production may result in differences in the regulation of inflammation in RA is thus attractive. Joint damage is induced by inflammation and may therefore be regarded as a result of the dysregulation of inflammation. Added to this, IL-10 decreases the production of matrix metalloproteinases and induces the production of tissue inhibitor of metalloproteinases 1 (TIMP-1) [33]. The metalloproteinases are responsible for cartilage degradation. Thus, a lack of IL-10 can lead to increased expression of metalloproteinases and reduced TIMP-1 expression, allowing joint destruction. In line with these data, IL-10 clearly inhibited joint destruction in animal models, whereas anti-IL-10 increased joint destruction in these models [46].
In the observational study, the steady-state level of IL-10 mRNA was greater in synovial biopsies of patients with non-destructive arthropathy than in synovial biopsies from patients with destructive RA. Two possible confounding factors may have influenced this result. First, the steady-state level of IL-10 may be influenced by immunosuppressive drugs. Two of the six patients with non-destructive arthropathy used second-line anti-rheumatic drugs that may affect IL-10 production (gold and methotrexate respectively) vs none of the patients with RA. However, the non-destructive patients still had higher levels of IL-10 mRNA. Secondly, a microsatellite at the IL-10 locus that is associated with high IL-10 production is over-represented in RA [15, 34]. Because the IL-10 mRNA content in the biopsies from the patients with non-destructive disease (three with RA and three with chronic polyarthritis with concomitant psoriasis) was higher than in those from the patients with erosive RA, selection of patients in our casecontrol study on high IL-10 levels by possible immunogenetic differences between psoriatic arthritis and RA would have led to results opposite to those observed. Therefore, we feel that the results of the biopsy study suggest that differences in IL-10 mRNA production are associated with joint destruction. These results are in line with those of Verhoef et al. [35], who reported that IL-10 production by peripheral blood mononuclear cells stimulated with anti-CD3/anti-CD28 of individual RA patients correlated strongly with their joint destruction.
The observation that RA patients with different IL-10 promoter genotypes had a different disease course was made in the prospective female RA patient cohort. Joint damage was evaluated in these patients at the same time and with the same techniques, which minimized methodological errors. In the prospective cohort, and in all groups of joints tested, patients with the -1082AA genotype developed less joint damage than patients with the -1082GG genotype. An alternative explanation for the difference in joint damage in patients genotyped for -1082 is skewing of other prognostic factors in these patients. However, no significant differences in prognostic factors indicative of severe RA were identified that could explain the difference in joint damage between -1082AA and -1082GG patients. The joint damage at onset was slightly lower in the -1082GA group than in the -1082AA and -1082GG groups, and there was a small difference between the -1082AA and -1082GG patients. This difference could not explain the different rates in an analysis in which joint destruction at onset was used as an independent variable to predict joint destruction.
Clinical data from patients with the genotype -1082GA were also available in the cohort study. The rate of joint damage of -1082GA RA patients was more similar to that of the -1082AA patients than to that of the -1082GG patients, although the median Sharp score in the -1082GA group was higher than that in the -1082AA group. The differences in joint damage were most striking in RA patients with a relatively short disease duration. This might be explained by the method of determination of joint damage. Determination of joint damage by the modified Sharp method is hindered by a ceiling effect. If a joint is eroded more than 40%, it is obviously possible that progression in joint destruction can occur. The progression will not be reflected in a change in Sharp score, because Sharp score 3 can only increase to collapse of the joint [36]. However, it might be that progression of joint destruction becomes similar in RA patients with long-standing disease irrespective of their level of IL-10 production. This observation can be explained by the hypothesis that prolonged inflammation leads to an alteration in the phenotype of the synovial fibroblasts that cause joint destruction by mechanisms that may be inflammation-independent [37, 38]. The proportions of joint damage induced by inflammation-dependent and inflammation-independent mechanisms may shift towards the latter during the course of the disease. If this last hypothesis was true, the mechanisms responsible for the rate of joint destruction would be influenced primarily by polymorphisms in the IL-10 promoter if the disease duration was less than 6 yr.
The use of promoter polymorphisms to investigate the relationship of a gene with a disease is based upon the assumption that these polymorphisms are associated with differences in production of the particular cytokine in a given disease. This assumption is difficult to prove for IL-10 and RA, as the relevant stimulus for the production of IL-10 is not known. However, when LPS-induced whole-blood culture is used the innate differences in IL-10 protein production are reflected in similar differences in the production of IL-10 mRNA. The present study shows that the large inter-individual differences in IL-10 production are probably reflected at the mRNA level. The results of the experiments using actinomycin D suggest that the half-life of the IL-10 mRNA did not differ between high and low IL-10 producers. However, actinomycin D may also block translation, and could therefore theoretically block the translation of a protein involved in the degradation of IL-10 mRNA. The differences in IL-10 production between -1082GG and -1082AA patients were small compared with the large inter-individual differences. The fact that a small, but significant, difference was observed suggests either that these polymorphisms are functional or that a functional locus is in linkage disequilibrium with these haplotypes. In a recent study of 45 Caucasians by Crawley et al. [16], it was found that ATA-homozygous donors produced less IL-10 than controls with other genotypes. We have not been able to determine the cause of the difference in results between our study and the study of Crawley et al. Transfection studies using the 5' flanking region and a luciferase reporter gene have been performed by our group (V. Keijsers et al., submitted for publication) and others [16]. The data from our group did not reveal a difference in promoter activity between the -1413 to +31 fragments of the IL-10 promoter haplotypes in transient transfection to U937 or monomac 6 cells using LPS as the stimulus. However, Crawley et al. [16] demonstrated a slight increase in transcriptional activity of the GCC haplotype compared with the ATA haplotype using dibutyryl cAMP as the stimulus. As we do not know which stimulus drives IL-10 secretion in the joint, we cannot determine whether possible functional sites in the IL-10 5' region are by themselves responsible for the observed association between joint destruction and IL-10 genotype or whether a functional locus linked to the IL-10 promoter causes the disease associations.
A number of studies from different groups have demonstrated allelic imbalance of the IL-10 promoter haplotypes in the region -1082 to -592 in a number of diseases, including rheumatic diseases such as SLE and juvenile RA [16, 28, 3942. Recently, we have shown that alleles of the IL-10G microsatellite, located 1.2 kb upstream of the coding region, have similar distributions in RA patients and in healthy controls [34]. These data are in accordance with the present study, which showed a similar distribution of the -1082, -819 and -529 haplotypes in RA patients and controls. However, the IL-10R2 allele (located 4 kb upstream of the coding region) exhibits a skewed distribution in three ethnic groups of RA patients. Recently, our group demonstrated that the four core IL-10-haplotypes, R3-(-IL-10G)-GCC, R2-(IL-10G)-GCC, R2-(-IL-10G)-ACC and R2-(IL-10G)-ATA, contained more than 90% of the possible haplotypes in the Caucasian population [43]. The lack of association of the R2 microsatellite with any of 14 ATA/ACC/GCC haplotypes is apparently the explanation for the lack of association between the occurrence of RA and the presence of one particular ATA/ACC or GCC haplotype. Thus, haplotypes of the IL-10 promoter (from approximately -1500 to the transcription initiation site) seem to be associated with the severity of arthritis, and haplotypes of this region, of about -4 kb, are associated with susceptibility to RA. In a model of RA in rats (pristane-induced arthritis), it was demonstrated recently, by crossing experiments and subsequent linkage analysis, that susceptibility to and severity of disease were affected by different loci [44]. We hypothesize that the IL-10R2 allele is associated with the presence of RA and that the IL-10R2 genotypes are associated with joint destruction in RA.
In animals, intervention studies providing formal proof that IL-10 inhibits joint destruction [47, 9] have been published. In human RA these data do not exist. The present study provides observational data (IL-10 content in synovial biopsy specimens of erosive vs non-erosive arthropathies) and immunogenetic data. These data are only indirect ways to demonstrate the relationship between IL-10 and joint damage in RA. However, the indirect evidence presented here indicates that IL-10 inhibits joint destruction.
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Acknowledgments |
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Notes |
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References |
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