IL1RN haplotype (rs419598/rs315952/ rs9005=CTA) 0 1 or 2 copies Extended report Synovial ﬂuid analyte CRP (ng/ml) IL6 (pg/ml) 0 1 or 2 copies IL10§ (pg/ml) 0 1 or 2 copies IL1β§ (pg/ml) 0 1 or 2 copies IL1Ra¶ (pg/ml) 0 1 or 2 copies MCP (pg/ml) 0 1 or 2 copies MIP (pg/ml) COMP (ng/ml) 0 1 or 2 copies 0 1 or 2 copies N* Mean† SD p Value‡ 28 21 28 19 28 19 28 19 16 7 29 21 29 21 29 21 5195.3 5160.4 687.1 430.2 109.4 13.4 3.46 0.63 483 468.5 763.7 789.7 380.9 360.6 35020 30441 4991.4 4045.8 633.5 437.4 222.7 42.7 8.38 2.04 334.6 184.2 389 303.3 233.4 169.1 23280 20677 0.74 0.38 0.034 0.40 0.72 0.49 0.91 0.52 *Number of patients. †The mean of the mediator values of the two knees was used to represent the mediator value for the patient. ‡Non-parametric Wilcoxon test. §Values of these parameters that were below the lower limit of detection of the assay were assigned the value of 0.5 times the lower limit to facilitate data analysis. Fifty- two per cent of the IL10 values and 82% of the IL1β values were below the detection threshold limit. ¶aIL1Ra analysis was performed at a later time than analysis of the other analytes and fewer samples remained available with sufﬁcient volume for analysis. COMP, cartilage oligomeric matrix protein; IL1, interleukin 1; IL1Ra, interleukin 1 receptor antagonist; MCP, monocyte chemotactic protein-1; MIP, macrophage inﬂammatory protein-1β. levels of IL10 (tertiles 1 and 2) IL1Ra is increased in CTA carriers, as shown by the enrichment of CTA haplotype carriers com- pared with non-carriers in the highest quartile of IL1Ra (54.5% vs 16.7%; p=0.07). DISCUSSION Inﬂammatory mediators are produced by articular joint tissues in OA and have been implicated in disease pathogenesis.2 12 Of 15 SNPs in six inﬂammatory genes, variations in the gene for IL1Ra were signiﬁcantly associated with severe radiographic knee OA in two independent populations. One IL1RN haplo- type was associated with decreased risk for radiographic sever- ity, greater JSW, lower levels of SF inﬂammatory mediators and reduction of the age inﬂuence on severity. Although both populations were modest in size, these ﬁndings are unlikely to be false positives because (a) statistically signiﬁcant associa- tions were observed for multiple SNPs in the same gene, IL1RN, whereas no associations were found for SNPs in other genes; (b) a speciﬁc IL1RN haplotype was protective in two independent populations and with two different severity parameters; (c) in the combined dataset, associations with severity were highly signiﬁcant (p<0.0001). It is of interest that the IL1RN protective effect was only evi- dent in the medial compartment. Although power to show a protective effect for lateral knee OA dominant disease was less, these ﬁndings are consistent with previous reports of a stron- ger genetic effect on medial disease. In a female twins study1 changes over time in the medial compartment were clearly her- itable but changes in the lateral compartment were not, and an MRI-based study13 also found a stronger genetic inﬂuence for medial cartilage loss. The differential compartment effects 860 Table 4 Synovial ﬂuid inﬂammatory mediators relative to carriage of interleukin 1 receptor antagonist (IL1RN) haplotypes observed for genetic inﬂuence can presumably be attributed to the combination of genetic susceptibility and weightbearing load (70% concentrated in the medial compartment) which together are necessary and sufﬁcient to cause OA preferentially in the medial compartment. IL1 is well established as a regulator of cartilage degradation14 and resorption of bone.12 We and others have previously shown that imbalances in IL1 and IL1 antagonists, such as the IL1Ra and the type II decoy receptor, contribute to cartilage loss and increased inﬂammatory mediators in OA.2 Although blocking IL1 activity by intra-articular injections or by gene transfer of IL1Ra reduced cartilage and bone changes in animal models of OA,15 16 intra-articular IL1Ra injections in humans have pro- duced mixed results for symptomatic knee OA.17 The IL1 gene cluster region has been repeatedly associated with susceptibility to OA in various joints, but the results have been inconsistent.7–9 18–20 An extended haplotype including SNPs in the IL1A, IL1B and IL1RN genes has been associated with increased susceptibility to OA in various joints,9 19 but that haplotype is not consistent with the IL1RN haplotypes that con- tributed to knee OA severity in this study. Of the three IL1RN SNPs associated with severity in this study, two (rs419598, exon 3 Ala/Ala; rs315952, exon 5, Ser/Ser) are in the coding regions of the IL1RN gene but do not change the amino acid sequence. The third, rs9005, is in the 3′-UTR of exon 4. Blood levels of IL1Ra have been associated with vari- ous polymorphic loci within the IL1RN gene, but the ﬁndings have been inconsistent. In two recent studies of more than 1000 subjects,5 21 signiﬁcantly higher blood levels of IL1Ra protein were associated with allele T at one polymorphism (rs4251961) that is part of the broader haplotype marked by the CTA alleles included in this report. In addition, allele C at rs419598 of the CTA haplotype has been associated with increased peripheral blood mononuclear cell expression of IL1Ra and decreased risk for osteolysis after total hip arthroplasty.22 IL1β is among the ﬁrst genes activated with any tissue chal- lenge, including biomechanical stress, and activates down- stream cytokines including IL6 and IL10. The lower SF levels of IL10, IL1β and IL6 in the presence of the IL1RN CTA haplotype are consistent with lower IL1 biological activity, as would be expected in individuals with increased IL1Ra expression and a lower level of activation of local cytokine-producing cells. Although no simple relationship was seen between the SF levels of IL1Ra and carriage of the IL1RN CTA haplotype in all patients, this relationship was evident in patients who were not in the highest tertile of IL10 levels. IL1Ra expression is directly activated by IL10 in both monocytes and neutrophils.23 24 The ﬁnding of a higher level of ILRa in SF samples from patients car- rying the CTA haplotype only in the lower two tertiles of IL10 protein is consistent with a genetic effect that may be evident only under submaximal activation conditions. For example, if one assumes near-maximal activation of IL1Ra when IL10 is strongly elevated, as would be expected in tertile 3 of IL10 lev- els, a genotype effect on IL1ra expression may be less evident than at submaximal levels of IL10. This study has potential limitations, including the moderate sample size, yet consistent and signiﬁcant severity associations with the IL1RN loci were found in two independently ascer- tained populations. In addition, although the genotype asso- ciation is with severity, longitudinal data are not available to assess actual disease progression rates. In this study, age was associated with radiographic severity, and although adjusted for in the statistical models, age may have had other confounding effects on the results. It should be emphasised that the IL1RN Ann Rheum Dis 2010;69:856–861. doi:10.1136/ard.2009.113043