Departments of Immunology and Rheumatology, Hôpital Edouard Herriot, Lyon, France,
1 Division of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK and
2 Department of Medicine, Tufts University and New England Medical Center, Boston, Massachusetts, USA
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Abstract |
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Methods. Allelic frequencies for polymorphisms in the IL-4 [variable number of tandem repeat (VNTR) polymorphism in intron 3] and IL-4 receptor chain (transition at nucleotide 1902) genes were assessed in 335 RA patients and 104 controls. Clinical indices of disease activity, disability and joint destruction and plasma levels of IL-1ß, IL-1Ra and sCD23 were assessed to evaluate a possible functional effect.
Results. Carriage of the rare IL-4(2) allele was higher in patients with non-destructive RA (40%) than in those with destructive RA (22.3%; odds ratio = 1.9, 95% confidence interval 1.13.5, P = 0.0006) and in controls (26%, P = 0.002). Patients positive for this rare allele had significantly less joint destruction, assessed by the Larsen wrist index (P = 0.004) and a lower erythrocyte sedimentation rate (P = 0.04). A significantly higher carriage rate of IL-4(2) was seen in HLA-DR4/DR1- patients with non-destructive RA than in those with destructive RA. The IL-4 receptor polymorphism was not over-represented. Plasma levels of IL-1ß, IL-1Ra and sCD23, known to be modified by IL-4, were not different in individuals having different alleles.
Conclusion. This IL-4 VNTR gene polymorphism may be a protective factor for severe joint destruction in RA that could be used as a prognostic marker early in the course of the disease.
KEY WORDS: Interleukin 4, Rheumatoid arthritis, Gene polymorphism, Destruction.
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Introduction |
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Different types of T-helper (Th) cells have been distinguished on the basis of the cytokines they produce: Th1 cells produce interleukin 2 (IL-2) and interferon (IFN-
) and Th2 cells produce IL-4, IL-10 and IL-13 [1]. T-cell clones from RA synovium produce Th1-related cytokines (especially IFN-
), and thus they can be classified as Th1 cells [2]. On the other hand, attempts to detect IL-4 in RA synovium have shown its relative absence at this site, providing an explanation for uncontrolled proinflammatory cytokine production leading to joint destruction [3]. Conversely, treatment with IL-4 was able to reduce joint inflammation and destruction in vitro and in animal models [4, 5].
IL-4, the prototypic member of the Th2 cytokines, is a potent anti-inflammatory cytokine, which reduces the production of proinflammatory cytokines and destructive enzymes by monocytes as well as by RA synovium samples. This cytokine is also able to induce the synthesis and release of sCD23, the soluble form of the low-affinity IgE receptor, and of anti-inflammatory factors such as the IL-1 receptor antagonist (IL-1Ra) in vitro and in vivo [6, 7]. The IL-4 gene has been located on the long arm of chromosome 5, together with genes for other Th2 cytokines, such as IL-5 and IL-13. For many cytokines and their receptors, genetic variants have been described [for a review see 8]. In the IL-4 gene, a variable number of tandem repeat (VNTR) polymorphism has been described that is located in the third intron of the gene [9, 10]. The frequent allelic form and the first published sequence consist of three 70 base-pair (bp) repeats in intron 3; a rarer allele with two repeats has also been described, and there is a third, much rarer allele with four repeats.
The IL-4 receptor (IL-4R) is composed of multiple chains, including a specific chain and a c chain, which is common to several cytokine receptors. In the IL-4R
-chain gene, an A
G transition at nucleotide 1902, causing a change from glutamine to arginine at codon 576, has been described and the presence of this rare allele has been associated with familial hyper-IgE syndrome and atopy [11].
When considering a possible defect in IL-4 function and production in RA, we decided to investigate the IL-4/IL-4R system as candidate genes in RA, to better understand the contribution of the complex balance between Th1 and Th2 cytokines to joint disease.
In this study we tested the allelic distributions of these IL-4/IL-4R gene polymorphisms, and investigated their involvement in the erosive outcome of RA. In order to assess their function in vivo, we looked for correlations with plasma levels of IL-1ß, IL-1Ra and sCD23.
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Methods |
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The control population consisted of 104 anonymous blood donors of the Lyon blood bank, France, and had the same genetic background as the patients. Biological and genetic studies were performed blindly by scientists unaware of the clinical status of the sample donors.
Clinical and biological data were collected in a computer database. Clinical indices of disease activity and joint destruction were evaluated by a single observer. The clinical data included age, sex, disease duration, Ritchie articular index, Steinbrocker functional index and Larsen X-ray index for the right wrist. Biological data included erythrocyte sedimentation rate (ESR) and rheumatoid factor (RF) (tested by the RoseWaaler assay). HLA typing was performed by serological methods.
Clinical and biological parameters of chronic polyarthritis patients according to disease severity are presented in Table 1. As expected, patients with non-destructive arthritis (mean Larsen wrist X-ray index <1) had indices of disease activity and joint destruction that were significantly lower than those of patients with destructive arthritis (mean Larsen wrist X-ray index >2).
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DNA analysis
For each individual enrolled in the study, a 7-ml sample of venous blood was collected in EDTA. DNA was extracted from uncoagulated blood by a modification of the salt-out technique (Nucleon; Scotlab, Glasgow, UK) and stored at a final concentration of 200 µg/ml until used for genotyping. Aliquots of plasma were freshly separated, and 0.5 ml aliquots of plasma were frozen at 20°C. A consecutive code number was assigned to each sample.
IL-4 VNTR.
A VNTR has been described in intron 3 of the IL-4 gene [9, 10]. Oligonucleotide primers were designed on the basis of the flanking region to the VNTR, and the size of the resulting polymerase chain reaction (PCR) products was directly diagnostic of number of repeats in the intervening sequence. Primer sequences and genotyping conditions were as described in Appendix 1.
IL-4R.
In the IL-4R gene, an AG transition at nucleotide 1902 of the mRNA sequence (accession number NM000418), causing a change from glutamine to arginine at codon 576, has been described [11]. We tested allelic frequencies at this locus by 5'-nuclease assay [TaqManTM allelic discrimination test (Norwalk, CT, USA)], a method that we have validated against our PCRrestriction fragment length polymorphism methods [17]. This test is based on the 5'-nuclease activity of Taq polymerase and the detection of the cleavage of two probes designed to match and hybridize with either allele copy during PCR. Double fluorescent probes were purchased from ABI-PE (Warrington, UK) and oligonucleotide PCR primers were synthesized on an ABI 373 synthesizer. Probe and primer sequences and cycling conditions are detailed in Appendix 2. Probes were labelled with carboxyfluorescein (FAM) and carboxy-4,7,2',7'-tetrachlorofluorescein (TET) fluorescent dyes at the 5' end, and with the quencher carboxytetramethylrhodamine (TAMRA) at the 3' terminus. Plates were scanned in a 7200 fluorimeter (Perkin-Elmer), and the resulting genotypes were manually entered into a spreadsheet and analysed.
Statistical analysis
Results of the gene polymorphism studies were analysed by comparison of allele frequencies (ratio of test allele to total alleles) and carriage rates (number of individuals with at least one copy of the test allele). Increase in allele carriage was analysed by the 2 test. Results are expressed as mean (S.E.M.). Categorical variables were compared using the
2 test and continuous variables with the unpaired `Student's t-test. Odds ratios (OR) were calculated for disease susceptibility or severity in carriers of specific alleles. The 95% confidence intervals (CI) for the OR were also calculated.
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Results |
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Allelic frequencies and disease severity
The patient population was divided into two groups according to joint destruction. As shown in Table 1, patients with non-destructive RA had indices of disease activity and joint destruction significantly lower than those of patients with destructive RA. As expected, frequencies of HLA-DR4 and RF, two classical markers of disease severity, were significantly higher in patients with destructive RA in comparison with patients with non-destructive RA [18]. Conversely, a higher frequency of HLA-DR3 was observed in the latter group, indicating a possible protective effect.
There was no difference in the frequencies of the rare IL-4R(2) allele and the more frequent IL-4R(1) allele amongst the groups of patients with non-destructive RA (39.2 and 97.9% respectively), the patients with destructive RA (34.7 and 95.8% respectively) and the controls (38.5 and 93.4% respectively). More interestingly, the carriage rate of the IL-4(2) allele was increased in non-destructive patients (40.0%; allele distribution: 1/1, n = 69; 1/2, n = 42; 2/2, n = 4), and decreased in destructive patients (22.3%; allele distribution: 1/1, n = 171; 1/2, n = 47; 2/2, n = 2). Thus allele 2 was over-represented in patients with non-destructive RA, who had disease with a better prognosis (non-destructive vs destructive RA: 2 = 11.7, df = 1, OR = 1.9, 95% CI = 1.13.5, P = 0 0.0006) (Table 2
). Conversely, the frequency of allele 1 was increased in destructive vs non-destructive RA (
2 = 12.1, df = 1, P = 0 0.0005).
To further assess this relationship, IL-4 gene polymorphism was analysed in relation to indices of disease activity and joint destruction. In particular, ESR values were lower in patients positive for IL-4(2) [23.5 (2.1) vs 32.3 (2.0) mm/h, P = 0.04]. Furthermore, carriage of the rare allele IL-4(2) was associated with a higher index of joint destruction [Larsen wrist index 1.29 (0.17) vs 1.93 (0.12), P = 0.004] (Fig. 1). The carriage rate of IL-4(2) was 40% when the Larsen index was <1 and 22.3% when it was >2 (P = 0.0006).
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Linkage with HLA-DR markers
We then compared IL-4 genotypes according to the HLA-DR4/DR1 genotype (Table 3), which has been associated with disease severity. In the HLA-DR4/DR1- subgroup, the carriage rate of the IL-4(2) allele was higher in patients with non-destructive than in those with destructive disease (45.1 vs 25.9%;
2 = 4.2, OR = 2.35, 95% CI = 1.05.3, P = 0 0.03). Among HLA-DR4/DR1+ patients, a higher carriage rate of the IL-4(2) allele was observed in those with non-destructive than in those with destructive RA (34.1 vs 19.1%;
2 = 3.9, OR = 2.2, 95% CI = 1.04.8, P = 0.05). HLA-DR3 was also analysed according to the carriage rate of the IL-4(2) allele. In the HLA-DR3+ subgroup, the carriage rate of IL-4(2) was similar in patients with non-destructive and destructive RA (22.6 vs 20%;
2 = 0.1, OR = 1.2; 95% CI = 0.34.7, not significant). Conversely, the carriage rate of the IL-4(2) allele was higher in HLA-DR3- patients with non-destructive disease than in HLA-DR3- patients with destructive disease (50 vs 23.5%;
2 = 12.5, OR = 3.3; 95% CI 1.76.2, P = 0 0.0004). When analysis of the two HLA DR4/DR1 and IL-4 genetic markers was combined, 76.7% of the DR4/DR1+ patients did not have the IL-4(2) allele (
2 = 4.4, P = 0.04). Thus, when the IL-4(2) allele was absent, an additive effect on destruction over that of HLA-DR4/DR1 was observed.
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Association with plasma levels of IL-4-regulated factors
In order to investigate the possible consequences of such polymorphism on IL-4 function, we looked at circulating levels of factors such as IL-1Ra and sCD23, which have been found to be increased in patients treated with IL-4 [7]. Plasma levels of sCD23, IL-1Ra and IL-1 were compared according to the various alleles. IL-1ß and IL-1Ra levels were not significantly different among RA patients with and without the IL-4(2) allele [2.03 (0.3) vs 2.33 (0.28) pg/ml, n = 139, and 0.43 (0.02) vs 0.56 (0.09) ng/ml, n = 234, respectively; not significant] and in patients with and without the IL-4(1) allele [2.23 (0.21) vs 2.41 (1.78) pg/ml, n = 139, and 0.53 (0.06) vs 0.31 ± 0.03 ng/ml, n = 234 respectively; not significant]. Plasma levels of sCD23 were not different in patients with and without the IL-4(2) allele [3.63 (0.18) vs 3.89 (0.32) ng/ml, n = 136, not significant) and those with and without the IL-4(1) allele [3.81 (0.23) vs 3.31 (0) ng/ml, P = 0, not significant]. No difference was observed according to IL-4R alleles.
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Discussion |
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This same IL-4 gene polymorphism has been related previously to multiple sclerosis [20]; it has been associated with the age at onset and to the overall severity of this disease, as for RA. More recently, a role in RA has been suggested [21]. In this study, the rare allele was found at a higher frequency in RA patients than in healthy controls. However, in contrast to our study, data for this polymorphism were not analysed according to disease severity, i.e. the degree of joint destruction. Our results show the protective effect on joint destruction, since only patients with non-destructive arthritis had an increased carriage rate of the rare IL-4 allele; it was not increased in those with destructive RA. This was confirmed by the greater joint destruction, as assessed by the Larsen index, in patients carrying this IL-4(2) allele. We focused on the wrist because it is one of the most common sites of joint involvement in RA [13, 14]. According to this simple classification, a group of RA patients with no or low-grade joint destruction is characterized by over-representation of the IL-4(2) allele. Our study therefore suggests that IL-4 gene variants affect the severity of RA rather than susceptibility to it. The same conclusion was reached for HLA-DR4 subtypes. It is of interest that more severe destruction was observed when the presence of DR4/DR1 was associated with the absence of IL-4(2), and vice versa. The two markers act independently of each other but have additive effects when combined.
The biological significance of these associations remains unclear. The association of IL-4(2) with less severe disease suggests that this gene marker is associated with a functional gene variant of IL-4 that determines a different level of biological activity. This could be either a protein dimorphism that changes the receptor affinity of the ligand, or a sequence variant in the regulatory regions, which would influence transcription or mRNA stability. Our study, of course, does not support speculation either way. Our experience has indicated that IL-4 measurements are difficult even when highly sensitive assays are used [22]. In order to assess the functional consequences of such polymorphism, we determined plasma concentrations of biological factors that reflect IL-4 activity, such as sCD23 and IL-1Ra, which are induced by IL-4, and IL-1, which is inhibited by IL-4. In addition, clinical studies of cancer patients with recombinant IL-4 have shown increased levels of IL-1Ra and sCD23 [7]. However, plasma levels of these factors did not differ between RA patients with and without the IL-4(2) allele. It is possible that plasma levels of these factors reflect the situation in blood and not that in the synovium, where the local concentration of IL-4 is known to be low [3, 22]. Additional polymorphisms in genes that, like IL-4 gene, are on the q arm of chromosome 5 (e.g. IL-3, IL-5, IL-9, IL-13, IL-15, GM-CSF) may be involved. The IL-4R chain gene polymorphism was strongly associated with IgE secretion and atopy in familial studies [11]. No such association was found in RA patients.
The present results indicate the possible contribution of IL-4 gene polymorphism to RA severity and in particular to joint destruction. It seems to be a weaker prognostic marker than HLA-DR4, but is additional to the latter. Indeed, IL-4 VNTR was more specifically predictive of reduced joint destruction in subjects negative for HLA-DR4 or HLA-DR1. Other confirmatory studies and functional experiments are needed to evaluate its use as a prognostic marker in erosive RA, and to aid patient selection for biomedicines such as human recombinant IL-4.
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Appendix 1 |
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Reverse primer: 5'-CAT CTT TTC CTC CCC TGT ATC TT-3'
PCR cycles: (95°C, 2 min) x1; (95°C, 1 min; 56°C, 1 min; 72°C, 30 s) x40; (72°C, 5 min) x1.
Allele 1 = 3 repeats = 342 bp PCR product; allele 2 = 2 repeats = 272 bp; allele 3 = 4 repeats = 412 bp.
PCR conditions: genomic DNA at 200 ng/25 µl reaction. MgCl2 at 2 mM and primers at 0.5 mM final concentration.
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Appendix 2 |
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Probe 2: 5''-C(TET) CAT GTA CAA ACT CCC GAT AGC CAC TGG (TAMRA)-3'
Forward: 5''-AGG CTT GAG AAG GCC TTG TAA-3''
Reverse: 5''-CCG AAA TGT CCT CCA GCA T-3''
Cycling: (50°C, 2 min) x1; (95°C, 1 min; 95°C, 15 s; 61°C, 1 min) x40.
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Acknowledgments |
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Notes |
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N. Buchs T. Silvestri, contributed equally to this work.
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References |
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