IL-1B and IL-1Ra gene polymorphisms and disease severity in rheumatoid arthritis: interaction with their plasma levels

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1 (2001) 2, Nature Publishing Group All rights reserved /01 $ IL-1B and IL-1Ra gene polymorphisms and disease severity in rheumatoid arthritis: interaction with their plasma levels N Buchs 1, FS di Giovine 2, T Silvestri 2, E Vannier 3, GW Duff 2 and P Miossec 1 1 Departments of Immunology and Rheumatology, Hôpital Edouard Herriot, Lyon, France; 2 Division of Genomic Medicine, University of Sheffield, UK; 3 Department of Medicine, Tufts University and New England Medical Center, Boston, MA, USA The balance between interleukin-1 (IL-1) and its competitive antagonist IL-1 receptor antagonist (IL-1Ra) may contribute to the pathogenesis of rheumatoid arthritis (RA). We analysed the frequency of different alleles in the IL-1B gene (at 511 and at +3954) as well as in the IL-1Ra gene (at +2018) in an association study involving 297 RA patients and 112 healthy controls from the same geographic area. We tested associations with RA susceptibility or severity, and with circulating levels of IL-1Ra and IL-1. Carriage of the rare IL-1B (+3954) allele 2 was increased in destructive arthritis (DRA) as compared to non-destructive arthritis (NDRA) (OR 1.7, 95% CI , 49.0% vs 35.9%) and controls (OR 1.7, 95% CI , 35.8%). Patients carrying this allele had a more destructive (Larsen wrist radiological index: mean ± s.e.m., 2.1 ± 0.2 vs 1.6 ± 0.1, P = 0.005; Steinbrocker functional index: 2.4 ± 0.1 vs 1.9 ± 0.1, P = 0.002) and active disease (Ritchie articular index: 8.1 ± 0.8 vs 5.3 ± 0.6, P = 0.002; erythrocyte sedimentation rate (ESR): 36.6 ± 2.9 mm/h vs 25.3 ± 1.8 mm/h, P = 0.002). This contribution was independent from that of HLA DR4/DR1 to severity. IL-1Ra plasma levels adjusted to ESR values were significantly lower in IL-1B2 (+3954) positive than negative RA patients (1.0 ± 0.1 vs 1.2 ± 0.1 ng/ml, P = 0.01). This IL-1B (+3954) gene polymorphism may be an important marker for the severity of joint destruction in RA and is associated with an imbalance in IL-1Ra production. As this genetic association was independent and additive to the risk of HLA DR4/DR1 status, it could be a useful addition to HLA-DR4/1 as a genetic prognostic marker early in the course of the disease. (2001) 2, Keywords: interleukin 1; interleukin 1 receptor antagonist; rheumatoid arthritis; gene polymorphism; destruction Introduction The aetiology of rheumatoid arthritis (RA) remains unknown, although susceptibility and severity to RA have been associated with several alleles of the HLA- DRB1, which can account for over 30% of the genetic component of the disease. 1 Despite this association, there are indications that other genes may be involved in susceptibility and severity of RA. 2 Three known genes have been described in the IL1 gene family, clustered in a 430-kb region on the long arm of human chromosome 2 (2q13). 3 IL1 and IL1 bind to the IL1 receptor type I, eliciting signal transduction. The product of the third gene, IL1 receptor antagonist (IL- 1Ra), also binds to the receptor but does not initiate intracellular signalling, acting as a competitive inhibitor. The pro-inflammatory cytokine IL-1 is produced by the Correspondence: Pr P Miossec, Clinical Immunology Unit, Departments of Immunology and Rheumatology, Hôpital Edouard Herriot, Lyon Cedex 03, France. miossec laennec.univ-lyon1.fr These studies have been supported in part by grants from the Hospices Civils de Lyon, the European Union (Biomed-2 program contract BMH4-CT ), the Association de Recherche sur la Polyarthrite (ARP) and the Arthritis Research Campaign for UK (ARC). Nicolas Buchs was supported by a fellowship from the Swiss National Science Foundation. Received 30 January 2001; revised and accepted 19 April 2001 inflammatory synovium and appears to play a predominant role in the destructive pattern associated with RA. 4 It is a known activator of collagenase and stromelysin and inhibits the synthesis of collagen and proteoglycan. Enhanced levels of IL-1 and IL-1 have been measured in RA synovial fluid. 5 Circulating levels of IL-1 have been shown to correlate with disease activity 6 and with radiographic progression. 7 Monocytes from both normal individuals and RA patients have been shown to vary considerably in the amount of IL-1 released in response to lipopolysaccharide stimulation. 8 This raises the possibility of a genetic component associated to a higher or lower IL-1 production in response to a given stimulus. Physiologic regulation of IL-1 occurs in part through the activity of IL-1Ra. RA outcome may be determined by the balance existing between IL-1 and IL-1Ra and various studies have shown deficient production of the IL-1Ra by RA synovium. 9,10 Polymorphisms for the IL-1B gene have been described within the promoter region (at 511) and in exon V (at +3954), 11,12 and related to IL-1 production 12,13 (Figure 1). For the IL-1Ra gene, polymorphisms include a VNTR in intron 2 14 and a single base variation (C/T) in exon 2 at position The importance of IL-1:IL-1Ra ratio has been underlined not only in RA but also in inflammatory bowel diseases and polymorphisms for the respective genes might regulate production and bioactivity of their

2 Figure 1 Allele description of the three IL-1 family gene polymorphisms. Refer to the method section for details. products. An association between alleles of IL-1 and IL- 1Ra polymorphisms has been described in inflammatory bowel diseases and associated with severity We analysed the frequency of these different alleles of IL-1 and IL-1Ra gene polymorphisms and their association looking for a possible impact on RA susceptibility and severity. We also looked at possible relationship between IL-1 and IL-1Ra plasma levels and these polymorphisms. Results IL-1B (+3954), IL-1B ( 511) and IL-1RN (+2018) allelic frequencies in controls and patients Characteristics of the patient population are shown in Table 1 according to the degree of joint destruction. The different alleles of IL-1 and IL-1Ra gene polymorphisms are described in Figure 1 and their frequencies in the patient population and controls are reported in Table 2, panel A. No association was found between the presence of a gene marker and that of RA. To look for a possible association with disease severity, the patient population was then divided into two groups, according to joint destruction as defined by the wrist Larsen index. 19 Indeed radiologic evaluation remains the gold standard to measure the rate of disease progression. 20 As expected and shown in Table 1, patients with non-destructive RA (NDRA) had indices of disease activity and joint destruction significantly lower than those with destructive RA (DRA). Similarly, frequencies of HLA-DR4 and RF positivity, recognised markers of disease severity, 21,22 were significantly higher in DRA patients in comparison with NDRA patients. Conversely, a higher frequency of HLA- DR3 was observed in the later, indicating a possible protective effect (Table 1). Allelic frequencies of the IL-1 gene cluster are indicated in Table 2, panels B (DRA vs NDRA) and C (DRA vs controls). The IL-1B2 (+3954) allele carriage rate was higher in DRA patients than in NDRA patients (P = 0.03, OR: 1.7, 95% CI , 49.0 vs 35.9%) and in controls (P = 0.03, OR: 1.7, 95% CI , 35.8%). There was no significant difference in allelic distribution in the other two markers tested (IL-1B 511 and IL1RN +2018). There was a significant association between IL-1B2 (+3954) positive and IL-1RN2 (+2018) negative status (P = 0.02, OR = 2.2, 95% CI = ), as it could be expected because of the linkage disequilibrium in this region. 15 To further assess the relationship between IL-1B (+3954) gene polymorphism and severity, we analysed its relationship with indices of disease activity and joint destruction. Erythrocyte sedimentation rate (ESR) values were higher in RA patients positive for IL-1B2 (ESR: mean ± s.e.m.: 36.6 ± 2.9 vs 25.3 ± 1.8 mm/h, P = 0.002). Furthermore, the carriage of the rare allele 2 was associated with a higher index of joint destruction (Larsen index: 2.1 ± 0.2 vs 1.6 ± 0.1, P = 0.005), clinical activity (Ritchie articular index: 8.1 ± 0.8 vs 5.3 ± 0.6, P = 0.002), and disability (Steinbrocker functional index: 2.4 ± 0.1 vs 1.9 ± 0.1, P = 0.002) (Figure 2). It should be noticed that there was no difference in disease duration between the group of patients positive and those negative for IL-1B2. Relationship between IL-1B(+3954) gene polymorphism and HLA-DR4/DR1 We looked at the occurrence of HLA-DR4/DR1/IL-1 composite genotypes in these populations (Table 3). As expected, there was an association between the DR4/DR1 markers and disease severity. Fifty-eight percent of HLA- DR4/DR1 positive patients vs 42% of those negative had DRA (OR 2.8, 95% CI , P = ). Of the DRA patients, 37% were positive for both HLA-DR4/DR1 and IL1-B2 (+3954), compared to 17% in the NDRA subgroup (OR 2.9, 95% CI , P = 0.001). The analysis of 223 Table 1 Clinical and biological parameters of RA patients according to disease severity Parameters Destructive RA Non-destructive RA P (n = 150) (n = 147) Sex (F:M) (n = 297) 113:37 118:29 NS Age (yr) (n = 296) 56.1 ± ± Disease duration (yr) (n = 294) 9.0 ± ± Ritchie articular index (n = 264) 8.7 ± ± Steinbrocker functional index (n = 230) 2.7 ± ± Right Larsen wrist index (n = 297) 3.2 ± ± ESR (mm/h) (n = 282) 39.8 ± ± Pts with RF (%) (n = 266) Pts HLA-DR4 + or DR1 + (%) (n = 249) Pts HLA-DR3 + (%) (n = 210) IL-1 (pg/ml) (n = 115) 2.9 ± ± IL-1Ra (ng/ml) (n = 276) 1.3 ± ± ESR adjusted IL-1Ra (ng/ml) (n = 271) 1.1 ± ± 0.1 NS ESR, erythrocyte sedimentation rate; Pts, patients; RF, rheumatoid factor; NS, not significant. Results are expressed as mean ± s.e.m. The number of patients in which data were available is indicated in brackets.

3 224 Table 2 Allelic distribution in controls and in patients with destructive (DRA) and non-destructive arthritis (NDRA) IL-1B ( 511) IL-1B (3954) IL-1RN (2018) 1,1 1,2 2,2 1,1 1,2 2,2 1,1 1,2 2,2 A RA Controls cont. table: 2 3 cont. table: 2 3 cont. table: 2 3 chi = 0.3 P = NS chi = 1.6 P = NS chi = 4.2 P = NS B DRA NDRA cont. table: 2 3 cont. table: 2 2(2+ vs 1,1) cont. table: 2 3 chi = 5.3 P = NS chi = 4.7 P = 0.03 chi = 0.3 P = NS OR = 1.17, 95% CI = C DRA Controls cont. table: 2 3 cont. table: 2 2(2+ vs 1,1) cont. table: 2 3 chi = 1.5 P = NS chi = 4.4 P = 0.03 chi = 2.5 P = NS OR = % CI = patients which were double positive for both HLA- DR4/DR1 and IL-1B2 (+3954) vs those that were negative showed a clear association with increased joint destruction (Larsen index: 2.4 ± 0.2 vs 0.8 ± 0.2, P = ), ESR levels (40.7 ± 4.0 vs 17.4 ± 2.3, P = ), Ritchie articular index (8.4 ± 1.0 vs 4.8 ± 1.3, P = ) and Steinbrocker functional index (2.4 ± 0.2 vs 1.8 ± 0.1, P = 0.003). Importantly, the IL-1B2 (+3954) positive status was associated with a more severe disease independently of the HLA-DR4/DR1 status. Indeed patients either HLA- DR4/DR1 negative or positive but IL-1B2 (+3954) positive had higher joint destruction (Larsen index: 1.6 ± 0.3 vs 0.8 ± 0.2 and 2.4 ± 0.2 vs 1.9 ± 0.2, P = 0.03 and P = 0.03, respectively), Ritchie articular index (8.2 ± 1.6 vs 4.8 ± 1.3 and 8.4 ± 1.0 vs 5.3 ± 0.7, P = 0.01 and P = 0.02, respectively) and Steinbrocker index (2.4 ± 0.2 vs 1.8 ± 0.1 and 2.4 ± 0.2 vs 2.0 ± 0.1, P = 0.01 and 0.03, respectively) than those IL-1B2 (+3954) negative. In fact, the associations of destructive disease with IL-1B (+3954) genotypes appeared very similar in both the HLA-DR4/DR1 positive and negative subgroups. This indicated an independent and additive effect over that of HLA-DR4/DR1. Link to plasma levels of IL-1 and IL-1Ra In order to investigate the possible consequences of such polymorphisms on IL-1 production and function, we looked at circulating plasma levels of IL-1Ra and IL-1. Both levels were positively correlated to ESR (r = 0.19, P = 0.001, n = 262, for IL-1Ra; and r = 0.40, P = , n = 105, for IL-1, respectively). Furthermore, IL1Ra plasma levels were positively correlated to those of IL1 (r = 0.58, P = , n = 112). We tested if IL-1 and IL-1Ra levels were associated with any particular genotype. Neither IL-1 nor IL-1Ra levels differed in patients grouped according to the IL- 1B ( 511), IL-1B (+3954) or IL-1RN genotypes (data not shown). IL-1Ra has been described as an acute phase protein, and like ESR its levels raise in the course of inflammation (hence the correlation with ESR. 23 Accordingly, we have corrected the individual levels of IL-1Ra data by normalising for the ESR levels measured at the same time. This was performed to take into account the contribution to IL-1Ra production given by the degree of inflammation. Corrected IL-1Ra plasma levels were lower in RA patients positive for IL-1B2 (+3954) than in those negative for this allele (1.0 ± 0.1 vs 1.2 ± 0.1 ng/ml respectively, P = 0.01, Figure 3). Discussion Taken together, these results support the concept that IL- 1 gene variants should be considered disease modifiers in RA. We report that the rare allele of the IL-1B (+3954) polymorphism is associated with the destructive pattern characteristic of chronic RA. The rare IL-1B (+3954) allele was over- represented in DRA patients, compared to NDRA individuals, rather than in the RA patients vs the control population. This suggests an implication of IL-1B gene variants in severity rather than susceptibility to RA. A recent study showed a similar increase in the IL-1B (+3954) rare allele in erosive as compared to non-erosive RA that was however not significant, possibly due to a smaller number of subjects analysed. 24 A previous genomewide scan 2 did not report evidence for linkage in this region (2q13), but this is not surprising because the phenotype considered was susceptibility to RA, rather than risk of erosive disease. We also studied, within the limits of the patient population available, if the IL-1B (+3954) status could be considered a risk factor on its own merit, similarly to HLA- DR4/1 and other genetic factors implicated in RA. We could show that patients who were both HLA DR-4/DR1 and IL1-B2 (+3954) positive, were mostly in the destructive erosive subgroup. Furthermore, within patients who were HLA DR4/DR1 negative, those carrying IL-1B

4 225 Figure 3 IL-1Ra circulating levels in RA patients concentration (ng/ml) according to the IL-1B (+3954) allele 2 carriage. IL-1Ra plasma levels were measured by RIA and were corrected for the ESR value. Results are expressed as mean ± s.e.m. and compared using the Mann-Whitney U test. Figure 2 Indices of joint destruction (Larsen wrist X-rays index, Steinbrocker functional index) and joint inflammation (ESR, Ritchie articular index) according to IL-1B (+3954) allele 2 carriage in RA patients. Results are expressed as mean ± s.e.m. and compared using the Mann-Whitney U test. Table 3 Association between IL-1B (+3954) and HLADR4/1 genotypes in patients with destructive (DRA) and non-destructive arthritis (NDRA) IL1B /2 or 1/2 1/1 genotype DRA NDRA DRA NDRA n = 58 n = 35 n = 57 n = 77 HLA-DR4/1 positive negative P = for HLA-DR4/1 and IL1B2 (+3954) positive DRA vs NDRA patients. (+3954) had significantly higher Larsen, Steinbrocker and Ritchie indices. This confirms that RA patients are more prone to develop destruction when additional genetic factors are present, as first described for HLA-DR4, 21 then for CTLA 4 in some 25 but not all populations. 26 The risk of severe arthritis associated with IL-1 locus genotypes had previously been reported in family 27 and association studies. 28 We clearly indicate in this study that the risk of severe disease conferred by the IL-1 locus is additional and independent of the HLA-DR4/DR1 status. Interestingly, the rare allele of IL-1B (+3954) has previously been associated with an IL-1 high production phenotype. 12,13 This rare allele may therefore represent a risk factor for joint destruction by being a marker for chromosomal events related to IL-1 production, shifting the cytokine profile toward a proinflammatory pattern. An impact of the rare allele of this polymorphism has already been shown in various diseases such as ulcerative colitis, insulin-dependent diabetes mellitus, myasthenia, periodontitis. 12,29 31 Pro-inflammatory events at the IL-1 locus would include modulation of production, secretion, or agonist bioactivity of IL-1, IL-1 or IL-1Ra. This is in line with the imbalance between IL-1 and IL-1Ra, with a relative excess in IL-1 over IL-1Ra described in RA. 9,10 In our study, the IL-1B (+3954) rare allele was not associated with an increased plasma level of IL-1 in contrast to other studies. 12,13 Data on the production of single cytokines in relation to single chromosomal markers in the IL-1 locus are highly controversial, and mostly plagued by experimental errors or shortcomings in a very complex system. In our case, we can only speculate that circulating levels do not reflect the situation at the level of the inflamed synovium, as we have shown in early studies. 32 Mechanisms other than direct co-regulation need to be invoked to explain the association of IL-1B (+3954) with lower plasma levels of IL-1Ra in RA patients. It is possible that this is related to a linkage disequilibrium with another IL-1 RN gene polymorphism (IL-1RN VNTR), which is in 100% linkage disequilibrium with IL-1RN (+2018), the one analysed in this study. This IL-1RN marker has been linked to an increase in IL-1Ra production for its rare allele in stimulated circulating monocytes 33 and in diabetic patients 34 but to a decreased mucosal production in the bowel. 35 The linkage disequilibrium between the rare allele of IL-1B (+3954) and IL-

5 226 1RN (+2018), and the possibility of tissue-specific gene regulation could in part explain our findings. The biological significance of the IL-1 locus genetic association with erosive RA remains unclear. The association of IL-1B2 (+3954) with greater disease severity may suggest that this is a genetic marker for an IL-1 haplotype which, by multiple pathways, determines different activities on the IL-1 receptor. The occurrence of specific IL-1 locus haplotypes may lead to a variation of the IL-1 /IL- 1Ra ratio in specific tissues, or making more IL-1 bioavailable intercellularly and leading for example to destructive RA. The functional correlates of IL-1 locus genetic variants are likely to play an important role in inflammatory diseases, but simple experimental approaches have to date failed to explain mechanisms of altered gene regulation. Simple functional hypotheses have generated contrasting studies and new experimental approaches are needed to unravel the functional significance of these genetic associations of the IL-1 gene variants. It is confirmed however that IL-1 gene markers can be very useful in predicting disease severity in RA, as risk factors which are independent and additional to that of DR4/DR1. Patients and methods Patients and controls A total of 378 patients with chronic polyarthritis were included. All were followed by the same investigators (NB, PM) at the same hospital in Lyon. Approval for this study was obtained from the local ethical committee. All patients fulfilled the commonly used criteria for RA (American College of Rheumatology (ACR)) criteria. 36 All patients had a disease duration of at least 2 years. Arthritis patients who had recent wrist X-rays (n = 297) were classified into two groups: those with destructive arthritis (DRA), ie with a mean Larsen wrist X-rays index 2 (3.21 ± 0.07, mean ± s.e.m., n = 150) and those without (NDRA), ie with Larsen wrist X-rays index 2 (0.18 ± 0.03, n = 147). The control population was composed of 108 anonymous blood donors of the Lyon blood bank, France, from the same geographic area as the patients. Biological and genetic studies were performed blindly by scientists unaware of the clinical status. 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 right Larsen wrist X-rays index. Biological data included: ESR, rheumatoid factor (RF) tested with Rose-Waaler assay. HLA typing for HLA-DR4 and DR1 was performed by a serologic method as described. 37 Clinical and biological parameters of RA patients according to the disease severity are presented in Table 1. As expected, patients with non-destructive arthritis (mean Larsen wrist X-rays index 2) had indices of disease activity and joint destruction significantly lower than patients with destructive arthritis (mean Larsen wrist X-rays index 2). Plasma levels of IL-1Ra were assessed with a specific radioimmunoassay (RIA) exactly as previously described. 38 Levels of IL-1 were measured with the human IL-1 beta Quantikine High Sensitivity ELISA from R&D (Abington, UK) according to the manufacturer s instructions. DNA analysis For each individual enrolled in the study, a 7-ml sample of venous blood was collected in EDTA. Plasma was freshly separated and stored in 0.5 ml aliquots at 20 C before cytokine determination. DNA was extracted from uncoagulated blood by a modification of the salt-out technique (Nucleon, Scotlab, UK) and stored at a final concentration of 200 g/ml until used for genotyping. The cytokine gene polymorphisms are presented in Figure 1. IL-1B ( 511): A single base variation (C/T) has been described in the promoter region of IL-1B gene at position 511(11). Allele 1 is C and allele 2 is T. Primer sequences and PCR conditions were: Forward primer: 5 TGG CAT TGA TCT GGT TCA TC-3 ; Reverse primer: 5 GTT TAG GAA TCT TCC CAC TT-3 ; PCR cycles: [95 C, 1 min] 1; [95 C, 1 min; 53 C, 1 min; 72 C, 1 min] 35; [72 C, 5 min] 1. The resulting products contained an Ava I restriction site in the frequent allele ( 1 ) and a Bsu 361 site in the rarer 2 allele. Products were analysed by PAGE, and allele 1 (C), Ava I digestion of PCR products yielded 190 and 114 bp fragments whereas for allele 2 (T), a single 304 bp product was observed. In contrast, Bsu 361 digestion produced 190 and 114 bp for allele 2, while allele 1 was uncut (304 bp). IL-1B (+3954): This polymorphism was described as Taq I RFLP of IL-1B (12). We sequenced the most likely region implicated and a C/T single base variation was found at in Exon V, explaining the RFLP. Allele 1 is C and allele 2 is T. PCR primers were designed to engineer a control Taq I site in the PCR product. Primer sequences and genotyping conditions were: Forward primer: 5 CTC AGG TGT CCT CGA AGA AAT CAA A-3 ; Reverse primer: 5 GCT TTT TTG CTG TGA GTC CCG-3 ; PCR cycles: [95 C, 2 min] 1; [95 C, 1 min; 67.5 C, 1 min; 72 C, 1 min] 35; [72 C, 5 min] 1. PCR products were size fractionated by PAGE. Taq I yielded a constant band of 12 bp and either two further bands of 85 and 97 bp (allele 1), or a single band of 182 bp (allele 2). IL-1-RN (+2018): A single base variation (C/T) has been described in Exon 2 at position (14). Allele 1 is C and allele 2 is T. Primer sequences and genotyping conditions were: Forward primer: 5 CTA TCT GAG GAA CAA CCA ACT AGT AGC-3 ; Reverse primer: 5 TAG GAC ATT GCA CCT AGG GTT TGT-3 ; PCR cycles: [96 C, 1 min] 1; [94 C, 1 min; 57 C, 1 min; 70 C, 2 min] 35; [70 C, 5 min] 1. PCR primers were designed to engineer two enzyme cutting sites on the two alleles. Alu I digestion of PCR products yielded 126 and 28 bp fragments for allele 1, whereas for allele 2, a single 154 bp product was observed. Msp I digestion produced 125 and 29 bp for allele 2, while allele 1 was uncut (154 bp). Statistical analysis The genotype distributions of the three IL-1B and IL1RN polymorphisms were compared between RA patients and controls, and between patients with destructive or nondestructive arthritis (Table 1). Heterozygous and homozygous odds ratios (ORhet and ORhom) were calculated

6 for each locus. These indicate the increased risk to an individual heterozygote or homozygote carrier for a specific allele compared with homozygous non-carriers, and are estimates of the genotypic relative risks (GRR). 36 On inspection of these, and comparison of observed vs expected values, data were either analysed in a 2 3 genotype table or a 2 2 table for the carriage of a specific allele (as indicated in the table). These contingency tables were tested using chi-squared analysis, and standard odds ratios with 95% confidence intervals were calculated. Nominal variables (eg HLADR4/DR1 positivity) were compared in different groups using the chi-square test, and continuous variables (like extent of erosions) by the unpaired Student s t-test or the Mann-Whitney U test. In order to show a possible correlation between the tested polymorphisms and IL-1Ra plasma levels, we have taken into account the positive impact of inflammation on IL- 1Ra plasma levels. 23 Accordingly, IL-1Ra plasma concentrations were corrected for the level of ESR, measured at the same time. Indeed a linear correlation was established in the patient population between IL-1Ra and ESR levels where IL-1Ra level = aesr level + b. Single IL-1Ra levels were then corrected for the level of inflammation for each sample, as measured by ESR, using the following formula: corrected IL-1Ra level = measured IL-1Ra level + mean IL-1Ra level in the population (aesr level+b). References 1 Deighton CM, Walker DJ, Griffiths ID, Roberts DF. The contribution of HLA to rheumatoid arthritis. Clin Genet 1989; 36: Cornelis F, Faure S, Martinez M et al. New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study. Proc Natl Acad Sci USA 1998; 95: Nicklin MJ, Weith A, Duff GW. 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