Interleukin-4 and Interleukin-4 Receptor Gene Polymorphisms in Inﬂammatory Bowel Diseases

Genes and Immunity (2001) 2, 287–289  2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene BRIEF COMMUNICATION Interleukin-4 and interleukin-4 receptor gene polymorphisms in inﬂammatory bowel diseases

W Klein1, A Tromm2, T Griga2, H Fricke3, C Folwaczny3, M Hocke4, K Eitner4, M Marx1, N Duerig1 and JT Epplen1 1Molekulare Humangenetik, Ruhr-Universitaet, 44780 Bochum, Germany; 2Abteilung fuer Gastroenterologie, Universitaetsklinik Bergmannsheil, 44789 Bochum, Germany; 3Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians Universitaet Muenchen 80336 Muenchen, Germany; 4Klinik fuer Innere Medizin I, Friedrich-Schiller Universitaet, 07743 Jena, Germany

Imbalances in the regulation of Th1 and Th2 lymphocytes are crucial in inflammatory bowel diseases. Interleukin-4 is secreted by Th2 lymphocytes and downregulates cytokine production from Th1 lymphocytes. Functionally relevant polymorphisms have been described in the interleukin-4 and the interleukin-4 receptor ␣ genes. Association of inflammatory bowel diseases with these polymorphisms has been reported recently suggesting high transcription and enhanced signalling activity in Crohn’s disease. Our study, comprising 211 patients with Crohn’s disease, 147 patients with ulcerative colitis and 446 healthy controls revealed significant association of Crohn’s disease with the −590 T allele of the interleukin-4 gene (P = 0.03). This allele entails reduced expression of IL-4. The reason for these contrasting findings may be discussed in the context of a putatively predisposing allele in linkage disequilibrium with the alleles of the interleukin-4 gene. Genes and Immunity (2001) 2, 287–289.

Keywords: colitis ulcerosa; Crohn’s disease; interleukin-4; interleukin-4 receptor; association study

The precise etiology of inflammatory bowel diseases (IL4R). IL4R consists of two subunits, the ␣ and the com- (IBD), Crohn’s disease (CD) and ulcerative colitis (CU) is mon ␥ chain, a component of several cytokine receptors. poorly understood and remains a field of intense investi- The IL-4 gene is located in the chromosomal region gation. IBD are characterised by imbalanced activation of 5q31–33 and the IL-4RA gene maps to 16p12.1. Linkage the intestinal immune system leading to the destruction has been shown for the two regions to CD.6,7 Conse- of the mucosa. T lymphocytes are known to play a funda- quently, these genes are good candidates both by position mental role in regulating the immune response caused by and by functional reasoning for genetic predisposition. distinct antigens. Imbalanced activation of Th1 and Th2 One of the single nucleotide polymorphism (SNP) with lymphocytes has been shown to be crucial for the functional relevance for transcription in the IL-4 gene has initiation and maintenance of destructive inflammation been described in the promoter (C/T at position −590).9– of the intestine.1–3 The importance of activated Th1 lym- 11 Several SNPs are located within the IL-4R␣ gene. Two phocytes and the consecutive production of proinflam- SNPs leading to an amino acid exchange (Ile50Val and matory cytokines like IL-2 and IFN-␥ has been estab- Glu576Arg) have been found to be associated with lished in mouse models for IBD.4 Th2 lymphocytes atopy.12,13 In order to evaluate the role of the aforemen- downregulate the activation of Th1 lymphocytes by tioned polymorphisms in the genetic predisposition for secreting cytokines like IL-4, IL-10 or IL-13.5 IBD, we genotyped of 215 patients with CD, 156 UC and There is increasing evidence from epidemiological and 446 healthy controls of Caucasian origin. genome-wide linkage studies that genetic factors play an Blood samples were collected at the universities of important role in the predisposition to IBD. Several Bochum, Munich and Jena. Diagnoses were confirmed by chromosomal regions have been identified to be linked clinical, endoscopical and histological parameters. Con- to IBD, however the respective genes are still unknown.6–8 trol samples have been described before.14 DNA was iso- Many functionally relevant polymorphisms in cytokine lated from EDTA-anticoagulated blood after standard genes have been described recently. Polymorphisms in protocols.15 The polymorphism of the IL-4 gene was genes involved in the regulation of Th1 and Th2 lympho- genotyped by RFLP analyses as described by Kawashima cytes are excellent genetic markers for association studies et al.11 Genotyping of the IL-4R␣ SNPs was performed by to define predisposing alleles. IL-4 is secreted by Th2 SSCP analyses lymphocytes and it interacts with the IL-4 receptor Comparison of the allele, genotype and carrier frequencies of the SNPs in the IL-4R␣ gene revealed no significant differences between patients and controls (see Correspondence: Dr. med. Wolfram Klein, Abteilung fuer Molekulare Table 1). This result is in accordance with the report of 16 Humangenetik, Universitaetsstrasse 150, 44801 Bochum, Germany. E- Olavsen et al. For the IL-4 gene the percentage of −590T mail: wolfram.kleinȰruhr-uni-bochum.de allele carriers was reduced among CD patients compared IL-4 and IL-4 receptor gene polymorphisms in IBDs W Klein et al 288 Table 1 (a) Genotype frequencies for the Q576R IL-4RA polymor- association studies reveal significantly contrasting results phism in IBD patients and controls in the way that the predisposing alleles exert opposite functional effects. The IL-4 gene is located within a cyto- Genotype RR or QR QQ P value kine gene cluster in the chromosomal region 5q22-q32. vs control Therefore, it cannot be excluded that our finding and the 17 = contrasting association identified by Aithal et al are due Controls (n 444) 171 (39%) 273 (61%) to a functionally relevant allele of a different gene in link- CD (n = 220) 90 (41%) 130 (59%) 0.55 UC (n = 156) 61 (39%) 95 (61%) 0.89 age disequilibrium with the polymorphisms examined. Different haplotype frequencies of this putative polymor- (b) Genotype frequencies for the I50V IL-4RA polymorphism in IBD phism with the IL-4 SNPs may cause the divergent associ- patients and controls ations in the British and German populations. Further investigations are necessary to clarify the contribution, if Genotype VV VI II any, of altered IL-4 expression to the development of IBD.

Controls (n = 410) 72 (18%) 207 (50%) 131 (32%) CD (n = 215) 42 (20%) 110 (51%) 63 (29%) References UC (n = 153) 28 (18%) 76 (50%) 49 (32%) 1 Parronchi P, Romagnani P, Annunziato F et al. Type 1 T-helper CD, Crohn’s disease; UC, colitis ulcerosa; OR, odds ratio; *95% con- cell predominance and interleukin-12 expression in the gut of fidence interval. A 10␮l polymerase chain reaction (PCR) mixture patients with Crohn’s disease. Am J Pathol 1997; 150: 823–832. containing 50 ng DNA, 1 U Taq polymerase (Genecraft), 0.2 mmol 2 Monteleone G, Biancone L, Wedel S, Pallone F. IL-4 hypores- of each nucleotide, 1 mm MgCl2 in 500 mm KCl, 100 mm Tris-HCl ponsiveness of Crohn’s disease mucosal T lymphocytes: a Ј (pH 8.3), 20 pmol of each primer (Q576R: sense 5 - response of polarised Th1 cells? Dig Liver Dis 2000; 32: 495–497. Ј Ј CTCTCTGAGCCAACCACTGT-3 , antisense 5 -CTCCACCGCATG 3 Niessner M, Volk BA. Altered Th1/Th2 cytokine profiles in the Ј Ј Ј TACAAAC-3 ; I50V: sense 5 -TGTCTGCAGAGCCCACAC-3 , anti- intestinal mucosa of patients with inflammatory bowel disease Ј Ј ␮ 32 ␣ sense 5 -CGCTGGGCTTGAAGGAG-3 ) and 0.2 l[ P] dCTP as assessed by quantitative reversed transcribed polymerase (10 mCi/ml) was run in a Thermocycler (Trio-Block, Biometra). chain reaction (RT-PCR). Clin Exp Immunol 1995; 101: 428–435. After two initial cycles at 6°C and 3°C above the annealing tempera- 4 Strober W, Ludviksson BR, Fuss IJ. The pathogenesis of mucosal ture, 25 cycles of 95°C, annealing temperature (Q576R. 56°C; I50V: 53°C) and 72°C were run for 0.5, 1 and 1 min, respectively. For inflammation in murine models of inflammatory bowel disease SSCP analyses 3 ␮l of PCR product was mixed with 95% formam- and Crohn disease. Ann Intern Med 1998; 128: 848–856. ide, 10 mm NaOH, 20 mm EDTA, 0.02% xylene cyanol and 0.02% 5 Kambayashi T, Jacob CO, Strassmann G. IL-4 and IL-13 modu- bromophenol blue. The samples were heated for 4 min at 95°C and late IL-10 release in endotoxin-stimulated murine peritoneal cooled on ice immediately. Three ␮l of each mix was run on 50 cm mononuclear phagocytes. Cell Immunol 1996; 171: 153–158. vertical polyacrylamide gels containing 10% glycerine at constant 6 Hugot JP, Thomas G. Genome-wide scanning in inflammatory 50W in a 4°C cold room for 4 h.20 Gels were dried and subjected bowel diseases. Dig Dis 1998; 16: 364–369. to autoradiography overnight. 7 Rioux JD, Silverberg MS, Daly MJ et al. Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci. Am J Hum Genet 2000; 66: 1863–1870. to controls (P Ͻ 0.03; see Table 2). Thus, our data suggest 8 Russel MG, Pastoor CJ, Janssen KM et al. Familial aggregation of that CD is associated with decreased production of IL- inflammatory bowel disease: a population-based study in South 4. Hence, these results are in contrast to the published Limburg, The Netherlands. The South Limburg IBD Study association studies17 which described an increased carrier Group. Scand J Gastroenterol Suppl 1997; 223: 88–91. − 9 Rosenwasser LJ, Klemm DJ, Dresback JK et al. Promoter poly- frequency of the T allele of a SNP at position 34 of the morphisms in the chromosome 5 gene cluster in asthma and − 18 IL-4 gene. The latter allele is tightly linked to 590T as atopy. Clin Exp Allergy 1995; 25 (Suppl 2): 74–78. also confirmed in our cohort (Klein, unpublished data). 10 Rosenwasser LJ, Borish L. Genetics of atopy and asthma: the 17 Aithal et al had suggested increased IL-4 signalling in rationale behind promoter-based candidate gene studies (IL-4 CD. and IL-10). Am J Respir Crit Care Med 1997; 156: 152–155. Such conflicting data may be explained by false- 11 Kawashima T, Noguchi E, Arinami T et al. Linkage and associ- positive association in one study by chance or due to dif- ation of an interleukin 4 gene polymorphism with atopic derma- ferences in the population stratification in the patient and titis in Japanese families. J Med Genet 1998; 35: 502–504. control cohorts. Many significant results have been 12 Mitsuyasu H, Izuhara K, Mao XQ et al. Ile50Val variant of IL4R described in association studies which have not been alpha upregulates IgE synthesis and associates with atopic asthma. Nat Genet 1998; 19: 119–120. reproduced.11,19 Yet, comparing the findings of Aithal et 17 13 Hershey GK, Friedrich MF, Esswein LA, Thomas ML, Chatila al and the present report, this is the first instance that TA. The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor. N Engl J Med 1997; 337: 1720–1725. Table 2 Genotype frequencies for the IL-4 polymorphism at pos- 14 Epplen C, Jaeckel S, Santos E et al. Genetic predisposition to ition −590 in IBD patients and controls Multiple Sclerosis as revealed by immunoprinting. Ann Neurol 1997; 41: 341–352. Genotype T/T or T/C C/C P value 15 Miller SA, Dykes DD, Polesky HF. A simple salting out pro- vs control cedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215. Controls (n = 446) 132 (30%) 314 (70% ± 4.2%*) 16 Olavesen MG, Hampe J, Mirza MM et al. Analysis of single- CD (n = 211) 45 (21%) 166 (79% ± 5.5%*) 0.03 nucleotide polymorphisms in the interleukin-4 receptor gene for OR = 1.55 association with inflammatory bowel disease. Immunogenetics = UC (n 147) 38 (26%) 109 (74%) 0.38 2000; 51: 1–7. 17 Aithal GP, Day CP, Leathart J, Hudson M. Association of single CD, Crohn’s disease; UC, colitis ulcerosa. nucleotide polymorphisms in the interleukin-4 gene and

Genes and Immunity IL-4 and IL-4 receptor gene polymorphisms in IBDs W Klein et al 289 interleukin-4 receptor gene with Crohn’s disease in a British 20 Jaeckel S, Epplen JT, Kauth M, Miterski B, Tschentscher F, population. Genes Immun 2001; 2: 44–47. Epplen C. Polymerase chain reaction-single strand conformation 18 Takabayashi A, Ihara K, Sasaki Y, Kusuhara K, Nishima S, Hara polymorphism or how to detect reliably and efﬁciently each T. Novel polymorphism in the 5Ј-untranslated region of the sequence variation in many samples and many genes. Electro- interleukin-4 gene. J Hum Genet 1999; 44: 352–353. phoresis 1998; 19: 3055–3061. 19 Hijazi Z, Haider MZ. Interleukin-4 gene promoter polymorphism [C590T] and asthma in Kuwaiti Arabs. Int Arch Allergy Immunol 2000; 122: 190–194.

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