inflammatory bowel disease: no association between allele combinations of the interleukin (il) 1β...

European Journal of Clinical Investigation (1998) 28, 214–219 Paper 277

Inflammatory bowel disease: no association between allelecombinations of the interleukin (IL) 1b and IL-1 receptorantagonist gene polymorphisms

U. T. Hacker, C. Bidlingmaier, M. Gomolka, E. Keller, A. Eigler, G. Hartmann, Chr.Folwaczny, H. Fricke, E. Albert, K. Loeschke and S. EndresUniversity of Munich, Germany

Abstract Background Interleukin 1 (IL-1) and its physiological antagonist interleukin-1 receptorantagonist (IL-1ra) play a crucial role in the pathogenesis of inflammatory bowel disease.Polymorphisms in the genes coding for these cytokines, the restriction enzyme TaqIpolymorphism for IL-1b and the variable number of tandem repeats (VNTR) polymorphismfor IL-1ra, have been shown to influence cytokine synthesis in vitro. Recently, an associationhas been described for distinct allele combinations of these two polymorphisms in patientswith ulcerative colitis and with Crohn’s disease but not in healthy control subjects.

Methods We studied 56 patients with ulcerative colitis, 64 patients with Crohn’s diseaseand 196 healthy control subjects. All were unrelated Caucasians of European ancestry. Afterpolymerase chain reaction (PCR) the amplification products were analysed on agarose gels.For the IL-1b polymorphism the PCR product was additionally digested using the restric-tion enzyme TaqI.

Results The allele and genotype frequencies as well as the carriage rates of the IL-1b TaqIpolymorphism in healthy control subjects were in agreement with previous findings in otherpopulations. Allele and genotype frequencies of the IL-1b polymorphism were not differentin inflammatory bowel disease patients compared with healthy control subjects. Comparingallele combinations of both polymorphisms no association could be identified either withinhealthy control subjects or in the groups of patients with ulcerative colitis or Crohn’s disease.

Conclusion Thus, we could not confirm the results of a previous study reporting anassociation between the IL-1ra and IL-1b gene polymorphisms in patients with inflamma-tory bowel disease.

Keywords Cytokine, immunogenetics, inflammatory bowel disease, interleukin 1b, poly-morphism, ulcerative colitis.Eur J Clin Invest 1998; 28 (3): 214–219

Introduction

It is well established that genetic factors contribute to theethiopathogenesis of the inflammatory bowel diseases(IBDs) ulcerative colitis and Crohn’s disease [1]. In

addition to ethnic differences in prevalence [2], a familialaggregation of inflammatory bowel disease has beendescribed [3]. First-degree relatives of both patients withulcerative colitis and Crohn’s disease have an estimatedten-fold increase in risk of developing an inflammatorybowel disease compared with the general population [4].Furthermore, twin studies indicate that genetic factors aremore important in Crohn’s disease than in ulcerative colitis[5].

In 1994 Mansfield et al. [6] first described an associationbetween a distinct allele of a polymorphism in the genecoding for the cytokine interleukin 1 receptor antagonist(IL-1ra) and ulcerative colitis; however, others [7] and ourown group [8] could not confirm this association. Ascytokines exert an essential role in acute and chronicinflammation, polymorphisms in cytokine genes and theirassociation to IBD are now being studied more extensively.

Q 1998 Blackwell Science Ltd

Medizinische Klinik, Klinikum Innenstadt, University ofMunich, Germany (U. T. Hacker, C. Bidlingmaier, A. Eigler, G.Hartmann, Chr. Folwaczny, H. Fricke, K. Loeschke and S.Endres); Laboratory for Immunogenetics, Kinderpoliklinik,Klinikum Innenstadt, University of Munich, Germany (M.Gomolka, E. Keller, E. Albert).

Correspondence to: S. Endres, Division of ClinicalPharmacology, Medizinische Klinik, Klinikum Innenstadt derUniversitat, Ziemssenstraße 1, 80336 Munchen, Germany.

Received 13 August 1997; accepted 7 November 1997

IL-1b and IL-1 receptor antagonist gene and IBD 215

Q 1998 Blackwell Science Ltd, European Journal of Clinical Investigation, 28, 214–219

The genes coding for the cytokines IL-1a, IL-1b and IL-1ra are located in a cluster on the long arm of chromosome2 [9]. Genetic polymorphisms have been identified in allthree genes [6,10–12]. In a biallelic polymorphism in theIL-1b gene (exon five) one allele defined by a silent T→Ctransition is digested by the restriction enzyme TaqI (allele1). The IL-1ra polymorphism is characterized by two to sixrepeats (variable number of tandem repeats, VNTR) of a86-bp-long segment in the second intron of the gene. Forthese two polymorphisms, an association of distinct alleleswith in vitro production of the respective cytokine afterstimulation has been reported. Mononuclear cells isolatedfrom donors carrying at least one copy of the less frequentIL-1ra allele 2 showed increased production of IL-1raprotein when stimulated with granulocyte–macrophagecolony-stimulating factor (GM-CSF) in vitro [13]. Mono-nuclear cells from donors carrying one or two copies ofallele 2 of the IL-1b TaqI polymorphism producedincreased amounts of IL-1b protein when stimulated withlipopolysaccharide (LPS) [14].

These observations raise the question whether thedescribed polymorphisms for IL-1b and IL-1ra play arole in the pathogenesis of IBD by influencing the ratiobetween the proinflammatory cytokine IL-1 and the anti-inflammatory cytokine IL-1ra. An association has beendemonstrated for the combination of distinct alleles ofthe IL-1b and IL-1ra polymorphisms in patients withCrohn’s disease and ulcerative colitis but not in healthycontrol subjects [15]. Patients who were non-carriers of theIL-1b allele 2 were more frequently carriers of the IL-1raallele 2 than expected for a balanced distribution. Incontrast, non-carriers of the IL-1ra allele 2 more oftenwere carriers of the IL-1b allele 2. This association wasstronger in Crohn’s disease than in ulcerative colitis. Takinginto account the results derived from the in vitro cytokinestimulation studies, the first combination of alleles wouldbe associated with high IL-1ra production and low IL-1b

production, leading to an attenuated inflammatoryresponse. In contrast, the second combination of alleleswould be associated with high IL-1b production and lowIL-1ra production, resulting in an enhanced inflammatoryresponse. Thus, the association reported by Bioque et al.[15] would not uniformly shift the IL-1b/IL-1ra balancetowards an increased inflammatory response.

In the present study, we attempted to confirm thisassociation between distinct allele combinations of thepolymorphisms in the IL-1ra and IL-1b gene in a southernGerman patient population with ulcerative colitis andCrohn’s disease.

Methods

Subjects

The study included 56 patients with ulcerative colitis and64 patients with Crohn’s disease. These patients make upthe groups previously reported [8], except for one patient

with Crohn’s disease and one patient with ulcerative colitisfor which genotyping of the IL-1b polymorphism was notavailable. The patient characteristics are shown in Table 1.All participants were unrelated Caucasians and the diag-nosis was confirmed by conventional clinical, endoscopicaland radiological criteria. A total of 196 healthy, unrelatedCaucasians served as a control group. Peripheral blood (5mL) from patients and healthy control subjects was col-lected in sterile tubes containing EDTA as anticoagulant.Genomic DNA was extracted using a salting out method asdescribed elsewhere [16].

IL-1ra VNTR polymorphism

The allele frequencies of the IL-1ra gene VNTR poly-morphism in these patients and control subjects havepreviously been reported [8]. In brief, a segment in thesecond intron of the IL-1ra gene containing between twoand six repeats of a 86-bp-long DNA sequence was ampli-fied using PCR. The product was analysed on a 2% agarosegel and genotypes were determined by comparing the sizeof the bands with a molecular weight ladder ranging insteps of 123 bp from 123 to 4182 bp. The exact protocol forthis procedure is shown elsewhere [8].

IL-1b Taq I polymorphism

Genomic DNA was amplified using PCR to study thepolymorphism in exon 5 of the IL-1b gene. It is character-ized by a silent T→C transition at the amino acid residue105 of the mature IL-1b protein [17]. The allele with arecognition sequence for the restriction enzyme TaqI(CCGA) has been named allele 1, and the other, which isnot cut by TaqI (TCGA), allele 2 [15]. The reaction volumewas 20 mL containing 750 ng of genomic DNA. Finalconcentrations of reagents were 20 mmol/L for dNTP(Gibco-BRL, Berlin, Germany) and 2 mmol/L for MgCl2

Table 1 Patient characteristics.

Ulcerative Crohn’scolitis disease

Total number of patients 56 64

SexFemale 31 46Male 25 18

Age (years 6 SD)Mean 40·3 6 11·8 37·5 6 10·7Median/range 40/17–66 33/19–69

Age at onset of symptoms (years)Mean 31·3 6 11·6 26·4 6 9·9Median 30 22

Duration of symptoms (years)Mean 9·3 6 6·3 11·3 6 7·8Median 8 9

216 U. T. Hacker et al.

in PCR Buffer II (Perkin Elmer, Weiterstadt, Germany).Primers 50-GTTGTCATCAGACTTTGACC-30 and 50-TTCAGTTCATATGGACCAGA-30 (Pharmacia BiotechEurope, Freiburg, Germany) as described [15] were usedin a final concentration of 1 mmol/L each. One unit ofAmpliTaq DNA polymerase (Perkin Elmer, Weiterstadt,Germany) was added. The PCR was performed using aPCR System 2400 (Perkin Elmer): Initial denaturation at958C for 5 min was followed by 958C (1 min), 518C (1min), 748C (1 min) for five cycles and 958C (30 s), 518C(30 s), 748C (30 s) for 30 cycles. Final extension was

carried out at 748C for 10 min. A fraction of the PCRproducts was analysed on a 2% agarose gel stained withethidium bromide before digestion to control correctamplification. Then, 10 mL of the PCR products wasincubated at 658C for 90 min with 0·125 mL of TaqI (1unit) restriction enzyme (Pharmacia Biotech Europe,Freiburg, Germany) in buffer (One-Phore-All, PharmaciaBiotech Europe) at a final volume of 15 mL. A positivecontrol was included in each series of reactions. Thesesamples were analysed by electrophoresis on a 3·5%agarose gel stained with ethidium bromide. Genotypeswere determined by comparing the size of the PCRproduct fragments with a 100-bp ladder of DNA (Phar-macia Biotech Europe). Three genotypes can be deter-mined (Fig. 1). The allele frequencies and carriage rateswere calculated from the numbers of genotypes. Forstatistical analyses the x2-test was performed on allelefrequencies, and genotypes and carriage-rates. The Bon-ferroni procedure was performed for multiple compari-sons.

Results

In the control group of 196 healthy subjects the allelefrequency of the IL-1b TaqI polymorphism was 72% forallele 1 and 28% for allele 2. This confirms the findings ofthe study by Bioque et al. [15] (77% for allele 1, 23% forallele 2) and by Pociot et al. [14] (74% for allele 1, 26% forallele 2). The genotype and allele frequencies as well ascarriage rates of the IL-1b TaqI polymorphism were similarin patients with ulcerative colitis and Crohn’s diseasecompared whit healthy control subjects (Table 2). Dividingthe patients with ulcerative colitis and Crohn’s disease intosubgroups by site of disease no significant differences inallele frequencies were determined (Table 3).

The genotype and allele frequencies of the IL-1ra poly-morphism in these patients and control subjects have beenreported [8]. We investigated the frequencies of allelecombinations of the IL-1b and IL-1ra gene polymorphism.In healthy control subjects, the combination non-carrier ofallele 2 of the IL-1b polymorphism and carrier of allele 2 ofthe IL-1ra polymorphism and, vice versa, the combinationcarrier of allele 2 of the IL-1b polymorphism and non-carrier of allele 2 of the IL-1ra polymorphism were over-represented. However, after correction for multiple


Figure 1 IL-1b TaqI polymorphism. Lanes 1–3 show the bandsobtained with amplified DNA on a 3·5% agarose gel of threedifferent individuals representing the three possible genotypes. Inlane 1, the 250-bp-long PCR product is not cut at the restrictionsite by the enzyme Taq I (i.e. homozygosity for allele 2). In lane2, the PCR product is completely cut into fragments of 114 and136 bp length by TaqI (i.e. homozygosity for allele 1). Lane 3shows the PCR product that is cut by TaqI only at one allele.Thus, an uncut fragment of 250 bp length and the two cutfragments of 136 and 114 bp length appear on the gel. Lane 4shows the negative control (i.e. PCR mix without template). Inlane 5 a DNA ladder in steps of 100 bp serves as a standard todetermine the size of the fragments.

Table 2 Genotype frequencies, allele frequencies and carriage rates of the IL-1b TaqI polymorphism in healthy control subjects and inpatients with inflammatory bowel disease.

Genotype frequency Allele frequency Carriage rate(n) (%) (%)

Alleles A1/A1 A1/A2 A2/A2 A1 A2 A1 A2

Control subjects (n ¼ 196) 100 84 12 72 28 94 49Ulcerative colitis patients (n ¼ 56) 29 23 4 72 28 93 48Crohn’s disease patients (n ¼ 64) 35 26 3 75 25 95 45

n, number of individuals; A1, allele 1, A2, allele 2.



comparisons, the significance level was not reached. Noassociation between distinct alleles was detected within thegroups of patients with ulcerative colitis and Crohn’s dis-ease. Also, compared with healthy control subjects thedistribution of allele combinations in these patient groupsshowed no differences (Table 4).

In the subgroup of patients with pancolitis ulcerosathere was an increased frequency of the allele combinationcarrier of allele 2 of the IL-1b polymorphism and non-carrier of allele 2 of the IL-1ra polymorphism (40% vs.30% in healthy control subjects) and a decreased fre-quency of the allele combination non-carrier of allele 2of the IL-1b polymorphism and carrier of allele 2 of theIL-1ra polymorphism (23% vs. 28% in healthy controlsubjects). In the small number of patients with pancolitis(n ¼ 31) this trend did not reach statistical significance (P¼ 0·156). In the other patient groups, i.e. patients withulcerative colitis not affecting the entire colon and inpatients with Crohn’s disease, these two combinationswere detected in frequencies almost equal to healthycontrol subjects (Fig. 2). Other subgroups determinedby the age of onset of IBD or by extraintestinal manifesta-tions showed no associations with distinct combinations ofalleles.

Discussion

Genetic markers within the HLA region on chromosome 6have been identified over the past years that are associatedwith ulcerative colitis and Crohn’s disease [18–20]. Forulcerative colitis, an association with HLA-DR2 wasdemonstrated. In Crohn’s disease the haplotype HLA-DR1/DQ5 [19] and a distinct haplotype of microsatellitemarkers covering the region of the tumour necrosis factorgene on chromosome 6 within the HLA region wereidentified more frequently than in control subjects [20].However, the results of a recent segregation analysis infamilies with inflammatory bowel disease suggest only aweak correlation between HLA haplotypes and IBD [21].

The proinflammatory cytokine IL-1 and its physiologi-cal antogonist IL-1ra play an essential role in the patho-genesis of inflammatory bowel disease. A decrease in theratio IL-1ra/IL-1 has been demonstrated in the mucosa ofpatients with IBD [22]. Furthermore, in an animal modelof rabbit immune colitis, administration of monoclonalantibodies against IL-1ra lead to exacerbated and pro-longed inflammation [23], whereas administration orrecombinant IL-1ra induced a decline in the activity ofinflammation [24].

In the present study we investigated associationsbetween ulcerative colitis or Crohn’s disease and the com-binations of alleles of two polymorphisms in the IL-1ra andIL-1b gene, both located within the IL-1 gene cluster onchromosome 2q. For the IL-1b Taq I polymorphism noassociation with either ulcerative colitis or Crohn’s diseasewas detected. These results are in accordance with otherstudies [15]. For the IL-1ra polymorphism an associationof allele 2 with ulcerative colitis has been described [6].However, in previous studies, we [8] and others [7] couldnot confirm this finding. Recent data suggest that thispolymorphism is important in distinct subgroups ofpatients with ulcerative colitis [25]. To investigate thepresence of an association between alleles of both poly-morphisms the patient groups and healthy control subjectswere divided into carriers and non-carriers of the less

Table 3 IL-1b allele frequencies in patients with ulcerative colitisand Crohn’s disease divided by site of disease.

A1 A2

Ulcerative colitisProctitis (n ¼ 4) 2 (50%) 2 (50%)Left-sided colon (n ¼ 48) 38 (79%) 10 (21%)Total colon (n ¼ 60) 41 (68%) 19 (32%)

Crohn’s diseaseIleum only (n ¼ 28) 24 (86%) 4 (14%)Ileum and colon (n ¼ 68) 47 (69%) 21 (31%)Colon only (n ¼ 32) 25 (78%) 7 (22%)

n, number of individuals; A1, allele 1; A2, allele 2.

Table 4 Frequencies of the IL-1b allele 2 carriage in control subjects and patients with ulcerative colitisand Crohn’s disease in relation to carriage of IL-1ra allele 2.

Non-carrier Carrier Carrier/non-carrierIL-1b A2 IL-1b A2 IL-1b A2n (%) n (%) n (%)

Control subjects (n ¼ 196)Carriers IL-1ra allele 2 (n ¼ 93) 55 (59% of 93) 38 (41% of 93) 93 (47% of196)Non-carriers IL-1ra allele 2 (n ¼ 103) 45 (44% of 103) 58 (56% of 103) 103 (53% of 196)

Ulcerative colitis (n ¼ 56)Carriers IL-1ra allele 2 (n ¼ 22) 14 (64% of 22) 8 (36% of 22) 22 (39% of 56)Non-carriers IL-1ra allele 2 (n ¼ 34) 15 (44% of 34) 19 (56% of 34) 34 (61% of 56)

Crohn’s disease (n ¼ 64)Carriers IL-1ra allele 2 (n ¼ 29) 16 (55% of 29) 13 (45% of 29) 29 (45% of 64)Non-carriers IL-1ra allele 2 (n ¼ 35) 19 (55% of 35) 16 (45% of 35) 35 (55% of 64)

n, number of individuals; A2, allele 2.

218 U. T. Hacker et al.

frequent allele 2 of both the IL-1ra and the IL-1b TaqIpolymorphism. No statistically significant associationbetween distinct alleles of these two polymorphismscould be determined within the groups of healthy controlsubjects, of patients with ulcerative colitis and with Crohn’sdisease. Also, between these groups the distribution ofallele combinations was not different. Thus, we could notconfirm the findings by Bioque et al. [15], who reported anassociation of distinct alleles in patients with Crohn’sdisease and in patients with ulcerative colitis but not inhealthy control subjects.

In the subgroup of patients with pancolitis ulcerosa atrend towards an increased frequency of the allele combi-nation carrier of IL-1b allele 2 and non-carrier ofIL-1raallele 2 (40% vs. 30% in healthy control subjects) wasobserved. This allele combination, based on in vitro data,favours an inflammatory response as it is associated withhigh secretion of proinflammatory IL-1b [14] and lowsecretion of anti-inflammatory IL-1ra [13]. However, thenumber of patients with pancolitis was rather small (n ¼

31) and the increase in this combination did not reachstatistical significance. No increased frequency of thiscombination was observed in patients with Crohn’s diseaseand in patients with ulcerative colitis not affecting the entirecolon (Fig. 2).

It has to be taken into account that anatomical andclinical subclassification of ulcerative colitis is problematicas the clinical pattern of disease may change over the timecourse. It is unlikely that methodological reasons areresponsible for the differences observed in the presentstudy and that by Bioque et al. [15] as the same primerswere used for genotyping under similar conditions.Furthermore, the stratification criteria used were similar.

Population sampling differences and disease heterogenicitymay be most relevant.

In summary, it appears unlikely that these polymorph-isms are important determinants of inflammatory boweldisease in a south German patient group.

Acknowledgments

The authors thank Dr Jochen Moeller, Desiree Zarskyi,cand. med. Uta Emmerich, cand. med. Anne Krug andcand. med. Britta Siegmund, for most helpful discussions.The experimental data of this study are part of the dis-sertation of C. Bidlingmaier (Medizinische Fakultat derLudwig-Maximilians-Universitat Munchen, in prepara-tion). This work was supported by grant En 169-2/3 andSFB 217 from the Deutsche Forschungsgemeinschaft.

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Figure 2 Carriage rates of allele combi-nations of the IL-1b TaqI and IL-1raVNTR polymorphisms for healthy con-trol subjects, patients with ulcerativecolitis not affecting the entire colon,patients with pancolitis and patients withCrohn’s disease.



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inflammatory bowel disease: no association between allele combinations of the interleukin (il) 1β...

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