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Functional Correlates of the Interleukin-1 Receptor Antagonist Gene Polymorphism in the Colonic Mucosa in Ulcerative Colitis

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Functional Correlates of the Interleukin-1 Receptor Antagonist Gene Polymorphism in the Colonic Mucosa in Ulcerative Colitis Genes and Immunity (2004) 5, 8–15 & 2004 Nature Publishing Group All rights reserved 1466-4879/04 $25.00 www.nature.com/gene Functional correlates of the interleukin-1 receptor antagonist gene polymorphism in the colonic mucosa in ulcerative colitis MJ Carter1, S Jones1, NJ Camp4, A Cox2, J Mee2, B Warren3, GW Duff2, AJ Lobo1 and FS di Giovine2 1The Gastroenterology and Liver Unit, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK; 2Division Of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK; 3Histopathology Department, John Radcliffe Hospital, Oxford, UK; 4Genetic Epidemiology, University of Utah Medical School, UT, USA Association studies have identified the interleukin-1 receptor antagonist gene allele 2(IL-1RNn2) as a marker of susceptibility in ulcerative colitis (UC). This study investigated the significance of the IL-1RN genotype with respect to protein and mRNA expression in the colonic mucosa. Homogenates of rectal biopsies from 99 UC and 54 controls were assayed for cytokines IL- 1ra, IL-1a and IL-1b using ELISA. IL1RN, IL1A and IL1B genotypes were determined using restriction-enzyme analysis. The ability of the two IL1RN alleles to generate steady-state mRNA accumulation was assessed in the colonic mucosa of seven heterozygous patients. Stepwise linear regression demonstrated that IL-1RN genotype (P¼0.001), diagnosis (Po0.0001) and treatment (Po0.03) were independent factors associated with the IL-1ra protein level whilst IL1RN genotype (P¼0.005) and macroscopic inflammatory grade (Po0.0001) were associated with the IL-1ra/ total IL-1 ratio. The IL1RNn2 correlated with reduced IL-1ra and IL-1ra/IL-1 ratio with a gene dosage effect. In heterozygous UC patients the ratio of allele 1 mRNA / allele 2 steady state mRNA was always greater than 1 (range: 1.2–3.1) (P¼0.018). The IL-1RNn2 is associated with reduced levels of IL-1ra protein and IL-1RN mRNA in the colonic mucosa, providing a biologically plausible explanation for the observed association of the allele with the disease. Genes and Immunity (2004) 5, 8–15. doi:10.1038/sj.gene.6364032 Keywords: interleukin-1 receptor antagonist; cytokines; inflammatory bowel disease; ulcerative colitis; genetics Introduction trol association studies have demonstrated significant associations with particular alleles in a number of The causes of the chronic idiopathic inflammatory bowel candidate genes although lack of reproducibility may diseases (IBD), ulcerative colitis (UC) and Crohn’s further suggest genetic heterogeneity. disease (CD), remain unknown. Evidence suggests Cytokines have been implicated in the pathogenesis of however that disease susceptibility and severity are chronic inflammatory diseases and have a regulatory determined at least in part, by genetic factors.1 Recent and effector role in the mucosal immune and inflamma- epidemiological data, including family studies,2 twin tory response in IBD.11 Interleukin-1 (IL-1) alpha and studies3 and investigations in different ethnic popula- beta are major proinflammatory cytokines involved early tions,4 suggest that UC and CD represent related, in the inflammatory cascade. The interleukin-1 receptor complex genetic diseases. Associations with genetic antagonist (IL-1ra) is the inhibitor of these IL-1 agonists syndromes and autoimmune disorders, which have a and acts by competitively binding to IL-1 receptors known genetic component, further support this concept.5 without eliciting signal transduction.12 All three proteins Genome-wide linkage scans have identified a number of are coded by genes in the interleukin-1 gene cluster on susceptibility loci suggesting that several genes are the long arm of chromosome 2.13 associated with IBD.6–9 Some of these loci appear to be Enhanced production of IL-1 has been shown in the common to both CD and UC while others are unique to gut tissue of animal models of intestinal inflamma- either disease. Such genetic complexity is postulated to tion14,15 and in both the colonic mucosa16 and by cell arise from epistasis (gene–gene interactions) and from preparations isolated from the intestine of patients with gene–environment interactions.9,10 Individual case–con- IBD.17 Tissue levels correlate with disease activity but the IL-1ra/IL-1 ratio shows the closest correlation with inflammation.18 Rabbit immune complex colitis is atte- Correspondence: Dr MJ Carter, Flat 9, 156 Haverstock Hill, Belsize Park, nuated by the administration of IL-1ra19 and reduction of London NW3 2AT, UK. E-mail: Martyn. [email protected] IL-1ra levels by either gene knockout in mice20 or by This study was financially supported by a grant from The National antibody neutralisation in rabbits21 increases suscept- Association of Colitis and Crohn’s Disease and The Special Trustees ibility to the induction of colitis. An imbalance in the of the Central Sheffield Hospitals. Dr Carter is a Digestive Disorders Foundation Research Training Fellow. IL-1ra/IL-1 ratio may therefore contribute to the chronic Received 07 March 2003; revised 24 September 2003; accepted 26 inflammatory response in ulcerative colitis. The mole- September 2003 cular mechanisms underlying this imbalance in IBD are IL-1 receptor antagonist in ulcerative colitis MJ Carter et al 9 unknown. However, interindividual differences in cyto- flamed controls and UC patients, studied. In this small kine production may be under genetic control.22 cohort of patients, no statistical differences in allele The IL-1 gene cluster contains several polymorphic carriage rates or allelic frequencies were demonstrated genetic markers. The IL-1ra gene (IL-1RN) contains a although a trend was evident for IL-1RN, with carriage variable number of tandem repeats (VNTR) polymorph- of the allele 2 increased in patients with UC compared to ism in intron 2,23 which is in complete linkage disequili- controls was observed (53 vs 39%) as expected. These brium with a single-nucleotide polymorphism (SNP) in patients were genotyped as part of a large study exon 2 ( þ 2018).24,25 SNPs have also been identified in assessing the role of the IL-1RN in genetic susceptibility both the IL-1 beta (IL-1B) and the IL-1a genes (IL-1A).26 to UC.25 The variable degree of linkage disequilibrium that exists across the gene cluster between these markers has Cytokine concentrations in colitis vs normals recently been studied in detail.27 Table 2 shows the tissue cytokine levels (normalised for Polymorphisms within the IL-1 gene cluster have been DNA content) of biopsies from UC and control patients. associated with both susceptibility to and severity of a The tissue levels of IL-1a and IL-1b were low in the number of chronic inflammatory and autoimmune control biopsy specimens. Total IL-1b tissue levels were conditions.26 Allele 2 of the IL-1RN VNTR polymorph- approximately 5–10 fold higher than the IL-1a levels. IL- ism (IL-1RNn 2) was first reported to be associated with 1ra tissue levels were approximately 1000 fold higher UC in 1994.28 Although not all subsequent studies in than total IL-1 levels in biopsies from controls. The IL-1a, Caucasian Northern European patients have confirmed IL-1b and IL-1ra tissue levels were all increased in rectal this association29–34 their lack of statistical significance biopsy samples from patients with UC compared to could reflect inadequate power. This was supported by controls. The IL-1ra/IL-1 ratio in the mucosa was the significant association found in a further well- decreased in UC patients compared to the controls. powered study and an accompanying meta-analysis of this and the seven previous studies in Caucasian North 25 European patients. Two subsequent studies have been Table 2 Cytokine protein steady-state levels and IL-1ra/total IL-1 performed in Northern European populations. One of ratio in colonic mucosa in controls and UC patients (rg cytokine/ these studies confirmed the association35 while the other mg DNA)(mean7s.e.) did not.36 A further study did confirm the genetic association in Jewish and Hispanic populations.37 The UC patients Controls association appears to be greatest in patients with extensive disease and in those requiring surgery for IL-1a 144.8754.9 9.371.7 UC.32,38 Allelic associations of SNPs within the IL-1A IL-1b 1954.37653.3 48.177.5 7 7 and IL-1B with IBD have not been demonstrated, IL-1ra 158250.9 15396.1 43291.5 7337.8 IL-1ra/total IL-1 479.97103.4 1062.97196.7 although this may represent the limited power of these studies.28,32,34,39 However, a recent study did suggest that the IL-1B þ 3954 polymorphism might have a role in Colonic mucosal levels of IL-1 alpha, IL-1 beta, IL-1ra and IL-1ra/ 40 total IL-1 ratio of biopsies from UC and noninflammatory control determining disease behavior. 7 To further investigate the previously described genetic patients. Data are shown as mean ( SEM). Cytokine concentrations association with UC, the functional effect of the IL-1RNn2 are normalised for DNA content of the homogenate (to normalise for cell number). Cytokine concentrations and DNA content of and other polymorphic variants within the IL-1 gene tissue homogenates were measured using ELISA and a novel cluster have been examined in terms of steady-state protein fluorescent nucleic acid stain, Picogreens respectively, as described and mRNA expression in vivo in the colonic mucosa. in Materials and methods. Using stepwise linear logistic regression as described in Statistical methods, diagnosis of UC was indepen- dently significantly associated with IL-1a (P¼0.008), IL-1b Results (Po0.0001) and IL-1ra (Po0.0001) colonic protein levels. The IL- 1ra/total IL-1ra level was not significantly reduced in the colonic Patient characteristics/IL-1 gene cluster SNP genotypes mucosa in patients with UC compared to noninflammatory controls Table 1 shows the IL-1RN ( þ 2018), IL-1B (À511 and (P40.05) after degree of inflammation and IL-1RN genotype were þ 3954) and IL-1A ( þ 4845) genotypes for the nonin- accounted for by the regression analysis.
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