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Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease
Immunology Letters 77 (2001) 113– 117
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Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn’s disease Hans Herfarth a, Beatrix Pollok-Kopp b, Michael Go¨ke c, Andreas Press d, Martin Oppermann b,* a Department of Internal Medicine, Uni6ersity of Regensburg, Regensburg, Germany Department of Immunology, Uni6ersity of Go¨ttingen, Kreuzbergring 57, 37075 Go¨ttingen, Germany c Department of Gastroenterology and Hepatology, Hanno6er Medical School, Hanno6er, Germany d Department of Internal Medicine, Uni6ersity of Go¨ttingen, Kreuzbergring 57, 37075 Go¨ttingen, Germany b
Received 17 January 2001; received in revised form 2 March 2001; accepted 5 March 2001
Abstract Crohn’s disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5D32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5D32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n= 346) and in CD patients (n= 235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5D32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy– Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, D =1 9.6%, P = 0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, D = 7.3%, P= 0.136) in carriers of the mutant CCR5D32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility. © 2001 Elsevier Science B.V. All rights reserved. Keywords: CCR2; CCR5; Chemokines; Crohn’s disease; Gene polymorphism
1. Introduction The aetiology of Crohn’s disease (CD), a chronic inflammatory disease of the intestine, is largely unknown with an underlying genetic susceptibility likely acting in concert with undefined environmental factors [1,2]. Patient studies as well as data derived from experimental animal models of inflammatory bowel disease (IBD) support a key role for CD4+ T helper-I (Th1) lymphocytes and macrophages in the immunopatho* Corresponding author. Tel.: + 49-551-395822; fax: +49-551395843. E-mail address: [email protected] (M. Oppermann).
genesis of this disease [3]. As yet, little is known regarding the mechanisms that lead to the recruitment of leukocytes from the circulation into the mucosa. Chemokines are critical for the attraction and local activation of specific leukocyte subpopulations through processes determined by ligand specificity and the expression patterns of the corresponding chemokine receptors on leukocytes [4]. CC chemokines and their respective receptors effect the migration and activation of monocytes, eosinophils and basophils as well as of T-lymphocytes. We hypothesized that CC chemokine receptors CCR2 and CCR5 might play a central role in the regulation of the intestinal immune response in CD, based on the following observations.
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First, the CC-chemokine receptors CCR2 and CCR5 are expressed on intraepithelial lymphocytes and lamina propria lymphocytes, which are believed to represent activated/memory T-lymphocytes recruited to the intestinal mucosa after activation in gut-associated lymphoid tissues [5]. Both receptors are also expressed on macrophages, and CCR2- and CCR5-deficient mice show impaired macrophage migration and activation in response to different microbial pathogens [6,7]. Moreover, CC-chemokines that bind to CCR2 (MCP-1, MCP-3) or to CCR5 (MIP-1a, MIP-1b, RANTES) are expressed in the small intestine and colon under normal conditions, and their expression is further augmented in IBD [8 – 10]. Second, direct evidence that CCR2 and CCR5 are involved in the pathogenesis of IBD was obtained in experimental animal studies, which demonstrated partial protection of CCR2- and CCR5-deficient mice from dextran sodium sulfate (DSS)-mediated colitis [11]. Administration of Met-RANTES, a CCR1/CCR5 receptor antagonist, was shown in a separate study to result in the significant reduction of tissue damage in the chronic phase of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats [12]. Third, many chemokine receptors such as CCR2 and, in particular, CCR5 serve as HIV-1 cell entry cofactors together with CD4. It has been observed occasionally that in patients with long-standing CD, remission of the disease is associated with immunodeficiency caused by HIV infection [13]. This suggests a regulatory role for CCR51CD4-expressing T-lymphocytes in the pathogenesis of CD. Fourth, systematic genome screening studies have identified several IBD susceptibility loci, and a microsatellite marker D3S1573 linked to IBD is located on chromosome 3 (3p21) within 5cM of the genes for chemokine receptors CCR2 and CCR5 [14]. In our study, we took advantage of the fact that polymorphic forms of both CCR2 and CCR5 receptors are present in the Caucasian population at high frequencies that significantly affect receptor functions. Individuals homozygous for a 32 bp deletion in the CCR5 gene (CCR5D32), which generates a non-functional receptor and non-responsiveness to certain CC chemokines, are highly resistant to HIV infection [15]. Furthermore, a single-point mutation in the CCR2 gene (CCR2-641) has been shown to be associated with significantly delayed disease progression in HIV-infected individuals [16]. We hypothesized that functional CCR5 and CCR2 receptors might be essential for cellmediated immunity and consecutive development of CD. Therefore, we determined the frequencies of the CCR5D32 and CCR2-641 alleles in CD patients and investigated genotype– phenotype correlations in patient subgroups, which were defined by well-established outcome-related disease parameters.
2. Materials and methods
2.1. Study population Blood samples were obtained from 235 Caucasian patients resident in Germany who were diagnosed with CD according to standard diagnostic criteria [17]. Patients were retrospectively allocated into different subgroups according to a recently adopted classification of CD (Vienna classification), which is based on outcomerelated objective variables [18]. According to the Vienna classification, age at diagnosis was defined as Al (B40 years) or A2 (\ 40 years), and the location was defined as L1 (terminal ileum), L2 (colon), L3 (ileo-colonic) or L4 (upper gastrointestinal tract). The behaviour of CD was defined as B1 (non-penetrating, non-stricturing), B2 (stricturing) or B3 (penetrating). Only patients with adequate documentation who met classification criteria (n= 216, 93%) were entered into one of the Vienna classification phenotypes. All CD patients were genotyped for the CCR5 polymorphism, whereas due to a shortage of genomic DNA, only 145 patients could be genotyped for the CCR2 polymorphism. Ethnically matched healthy blood donors (n= 322) and staff members (n= 24) served as the control group.
2.2. CCR2 and CCR5 genotyping Genotypes were determined by polymerase chain reaction (PCR) or PCR restriction fragment length polymorphism (PCR-RFLP) using a modification of published protocols [19,20]. Briefly, genomic DNA was prepared from whole venous blood using a commercially available DNA isolation kit (Qiagen, Hilden, Germany). The genotype of CCR5 was determined by DNA amplification by a 40-cycle PCR with Taq polymerase (forward primer 5%-GCGTCTCTCCCAGGAATCATC-3%; reverse primer 5%-GGTGAAGATAAGCCTCACAGCC-3%). The PCR products were separated by 4% agarose gel electrophoresis and stained with ethidium bromide. The wild type and mutant A32 allele were detected as 242- and 210 base pair fragments, respectively. The CCR2-641 allele was detected with a PCR-RFLP assay using a BsaBl site introduced into the PCR forward primer next to the C–T transition that encodes the CCR2-641 polymorphism. Amplification (forward primer 5%-TTGTGGGCAACATGaTGG3% has a cytosine substituted with an adenine [in lower case]; reverse primer 5%-CTGTGAATAATTTGCACATTGC-3%) yielded a 183 bp fragment, which was digested by BsaBl (3 U at 60°C for 18 h) into 18- and 165 bp fragments when an isoleucine was present instead of a valine at position 64 in CCR2.
H. Herfarth et al. / Immunology Letters 77 (2001) 113–117
2.3. Statistical analysis Chi-square and Fisher’s exact tests were used to determine statistical significance of differences in genotype and allele distributions among the different subgroups. Differences were considered significant if P was B 0.05.
3. Results
3.1. Gene frequencies of CCR5 and CCR2 in patients and control subjects The CCR5 genotype was determined in 346 healthy individuals and in 235 CD patients. CCR2-641 allelic frequency was determined in 342 healthy controls and in 145 patients. Phenotype and gene frequencies for CCR2 and CCR5 polymorphisms are summarized in Table 1. Gene frequencies of CCR2-641 (8.2%) and CCR5D32 (9.2%) in the control population were in accordance with previous reports [16,21]. The gene frequencies of CCR5D32 (9.8%) and CCR2-641 (7.6%) in patients did not differ significantly from healthy controls, nor did the observed ratios deviate from the predicted Hardy–Weinberg distribution.
3.2. Influence of CCR5 and CCR2 genotypes on the phenotype of CD Our CD study population from three German referral centers enclosed a high percentage of patients with young age at diagnosis (Al) and a frequent ileo-colonic disease location, whereas the disease behaviour was nearly equally distributed (Table 2). No significant differences in the phenotypic disease manifestastions in relation to the CCR5 and CCR2 polymorphisms were found (Tables 2 and 3). We observed a trend towards sparing of the upper gastrointestinal tract (carrier frequency 0% in L4 versus 19.6% in L1– 3, D =19.6%, P=0.079; gene frequency 0 versus 10.3%, D =10.3%, P= 0.080) and a more stricturing type of disease (carTable 1 CCR2-641 and CCR5D32 genotypes of healthy controls and CD patients
CCR2-641 CCR2 wt/wt CCR2 wt/641 CCR2 641/641 CCR5D32 CCR5 wt/wt CCR5 wt/D32 CCR5 D32/D32
Controls
Patients
(n= 342) 289 (84.5) 50 (14.6) 3 (0.9) (n=346) 287 (82.9) 54 (15.6) 5 (1.5)
(n = 145) 123 (84.8) 22 (15.2) 0 (0) (n= 235) 191 (81.3) 42 (17.9) 2 (0.9)
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Table 2 CCR5 gene polymorphism (genotypic and phenotypic frequencies) in CD subgroups Subgroup
CCR5D32 gene frequency (%)
CCR5D32 carrier (%)
Al (age B40; n =194/90%) A2 (age \40; n =22/10%) L1 (terminal ileum; n =42/19%) L2 (colon; n =48/22%) L3 (ileo-colon; n =114/53%) L4 (upper gastrointestinal tract; n =12/6%) B1 (non-penetrating; n =66/31%) B2 (stricturing; n = 68/31%) B3 (penetrating; n = 82/38%) All CD patients (n = 216/100%) Controls (n =346)
9.5 11.4 10.7
18.0 22.7 19.0
7.3 11.4 0
14.6 21.9 0
8.3
15.2
12.5 8.5 9.7
23.5 17.0 18.5
9.2
17.1
rier frequency 23.5% in B2 versus 16.2% in B1/B3, D= 7.3%, P= 0.136; gene frequency 12.5 versus 8.4%, D=4.1%, P= 0.126) in carriers of the CCR5D32 allele. However, none of these differences reached a level of statistical significance.
4. Discussion Chemokines and their cognate membrane-bound receptors fulfill important roles in the directed migration/ activation of leukocytes and have been implicated in the pathogenesis of various inflammatory diseases. In Table 3 CCR2 gene polymorphism (genotypic and phenotypic frequencies) in CD subgroups Subgroup
CCR2-641 gene (%)
A1 (age B40; 5.8 n =113/89%) A2 (age \40; n = 14/11%) 10.7 L1 (terminal ileum; 10.4 n = 24/19%) L2 (colon; n =24/19%) 2.1 L3 (ileo-colon; n =75/59%) 6.7 L4 (upper gastrointestinal 0 tract; n =4/3%) B1 (non-penetrating; 7.3 n = 41/32%) B2 (stricturing; 5.0 n = 50/39%) B3 (penetrating; 6.9 n = 36/28%) All CD patients 6.3 (n = 127/100%) Controls 8.2
CCR2-641 carrier (%) 11.5 21.4 20.8 4.2 13.3 0 14.6 10.0 13.9 12.6 15.5
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this study, we investigated the potential significance of the CC chemokine receptors CCR2 and CCR5 in the pathogenesis of CD by analysing the association of genetic polymorphisms of these two receptors with clinical outcome in CD patients. A 32 bp deletion in the CCR5 gene is common among Caucasians and results in the expression of a truncated, non-functional receptor [15]. A point mutation in the CCR2 gene (CCR2641) is found in many ethnic groups and results in a conservative substitution of valine 64 to isoleucine [16]. Both CCR mutations independently confer protection towards HIV. This protection is reflected either, in the case of CCR5D32 homozygozity, by resistance to HIV infection or, in the case of CCR2-641, by slower disease progression [22]. Several recent studies have reported a protective effect of either CCR5 or CCR2 mutations towards certain chronic inflammatory diseases, such as multiple sclerosis [23,24], rheumatoid arthritis [25,26] or pulmonary sarcoidosis [20,27]. At the same time, CCR2641 or CCR5D32 mutant alleles appear to be overrepresented in diabetic children [28] or associated with a more pronounced clinical course of the disease in pulmonary sarcoidosis [27]. Taken together, these studies reveal differential associations of CCR2 and CCR5 polymorphisms with disease activity in a number of chronic inflammatory diseases that are characterized by enhanced cell-mediated immunity. In our own study, we show that the CCR5D32 gene frequency in all CD patients did not differ significantly from healthy controls, nor did the distribution deviate from the Hardy–Weinberg predictions. These results are corroborated by Hampe et al., who recently performed a high-resolution mapping of the IBD susceptibility locus on chromosome 3p in order to identify potential genetic associations with this disease [29]. These authors found allele frequencies of 8.3 and 12% for the CCR2-641 and CCR5D32 mutant alleles in 353 affected sibling pairs with the diagnosis of CD or ulcerative colitis, who were recruited in a large European cohort. Association statistics revealed no significant influence of these mutations on the phenotype of patients with inflammatory bowel disease. In the study of Hampe et al., CD patients were not classified according to their individual disease phenotype. Since there is accumulating evidence to suggest that CD patients comprise a heterogenous group of individuals with different phenotype-determining genetic backgrounds [2], we also examined chemokine receptor polymorphisms in various patient subgroups [18]. Our data revealed no significant effects of CCR2 or CCR5 genotypes on the age of onset, location or behaviour of the disease. Although not statistically significant, we observed that carriers of the CCR5D32 mutant had upper gastrointestinal tract disease less often compared with other locations of the disease.
Carriers of the mutant CCR5 allele were also more prone to develop a stricturing form of disease. If these findings are corroborated in independent datasets, they may suggest that CCR5 deficiency protects from a more aggressive, i.e. penetrating/fistulizing disease course due to a reduced capacity to generate an inflammatory response in the absence of CCR5-expressing cells. Furthermore, they point to the possibility of a differential role for CCR5 expressing cells in large versus small bowel. Interestingly, a positive association between fistulizing or large bowel disease and carriage of the TNF2 allele, which directs higher levels of TNFa transcription, has been described in a Belgian population [30]. The findings of this and the current study may be linked by the ability of CCR5-expressing mucosal Th1-lymphocytes and/or macrophages to induce TNFa secretion. The results of our study are in accord with the outcome of experimental animal models of IBD in CCR5 knock-out mice. It was reported that CCR5and, to a lesser degree, CCR2-deficient mice are partially protected from DSS-induced colitis, an experimental disease that shares many features of acute and chronic intestinal inflammation [11]. While CCR5 deficiency did not prevent DSS-induced intestinal inflammation, both clinical and histological disease scores indicated a diminished inflammatory response in receptor knock-out mice. Moreover, CCR5-deficient mice developed a Th2 pattern of cytokine expression after DSS treatment, which was characterized by significantly decreased levels of IFN-g, while expression of IL-4, IL-5 and IL-10 mRNA was enhanced. In summary, CCR2 or CCR5 gene polymorphisms are unlikely to be important determinants of overall disease susceptibility in CD. CCR5 gene polymorphism may participate in determining disease course and location and thereby contribute to heterogeneity of CD. However, the observed effects are modest and await replication in independent studies. Acknowledgements This work was supported by grant SFB402 B7 from the Deutsche Forschungsgemeinschaft. H.H. is supported by Kompetenznetzwerk ‘Chronisch-entzu¨ ndliche Darmerkrankungen’ of the German Federal Department for Research and Education (BMBF). We are grateful to Katrin Kraft for expert technical assistance. References [1] R. Duchmann, M. Zeitz, in: P.L. Pearay, J. Mestecky, M.E. Lamm, W. Strober, J. Bienenstock, J.R. McGhee (Eds.), Mucosal Immunology, Academic Press, San Diego, CA, 1999, pp. 1055 – 1080.
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Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn’s disease Hans Herfarth a, Beatrix Pollok-Kopp b, Michael Go¨ke c, Andreas Press d, Martin Oppermann b,* a Department of Internal Medicine, Uni6ersity of Regensburg, Regensburg, Germany Department of Immunology, Uni6ersity of Go¨ttingen, Kreuzbergring 57, 37075 Go¨ttingen, Germany c Department of Gastroenterology and Hepatology, Hanno6er Medical School, Hanno6er, Germany d Department of Internal Medicine, Uni6ersity of Go¨ttingen, Kreuzbergring 57, 37075 Go¨ttingen, Germany b
Received 17 January 2001; received in revised form 2 March 2001; accepted 5 March 2001
Abstract Crohn’s disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5D32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5D32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n= 346) and in CD patients (n= 235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5D32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy– Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, D =1 9.6%, P = 0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, D = 7.3%, P= 0.136) in carriers of the mutant CCR5D32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility. © 2001 Elsevier Science B.V. All rights reserved. Keywords: CCR2; CCR5; Chemokines; Crohn’s disease; Gene polymorphism
1. Introduction The aetiology of Crohn’s disease (CD), a chronic inflammatory disease of the intestine, is largely unknown with an underlying genetic susceptibility likely acting in concert with undefined environmental factors [1,2]. Patient studies as well as data derived from experimental animal models of inflammatory bowel disease (IBD) support a key role for CD4+ T helper-I (Th1) lymphocytes and macrophages in the immunopatho* Corresponding author. Tel.: + 49-551-395822; fax: +49-551395843. E-mail address: [email protected] (M. Oppermann).
genesis of this disease [3]. As yet, little is known regarding the mechanisms that lead to the recruitment of leukocytes from the circulation into the mucosa. Chemokines are critical for the attraction and local activation of specific leukocyte subpopulations through processes determined by ligand specificity and the expression patterns of the corresponding chemokine receptors on leukocytes [4]. CC chemokines and their respective receptors effect the migration and activation of monocytes, eosinophils and basophils as well as of T-lymphocytes. We hypothesized that CC chemokine receptors CCR2 and CCR5 might play a central role in the regulation of the intestinal immune response in CD, based on the following observations.
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First, the CC-chemokine receptors CCR2 and CCR5 are expressed on intraepithelial lymphocytes and lamina propria lymphocytes, which are believed to represent activated/memory T-lymphocytes recruited to the intestinal mucosa after activation in gut-associated lymphoid tissues [5]. Both receptors are also expressed on macrophages, and CCR2- and CCR5-deficient mice show impaired macrophage migration and activation in response to different microbial pathogens [6,7]. Moreover, CC-chemokines that bind to CCR2 (MCP-1, MCP-3) or to CCR5 (MIP-1a, MIP-1b, RANTES) are expressed in the small intestine and colon under normal conditions, and their expression is further augmented in IBD [8 – 10]. Second, direct evidence that CCR2 and CCR5 are involved in the pathogenesis of IBD was obtained in experimental animal studies, which demonstrated partial protection of CCR2- and CCR5-deficient mice from dextran sodium sulfate (DSS)-mediated colitis [11]. Administration of Met-RANTES, a CCR1/CCR5 receptor antagonist, was shown in a separate study to result in the significant reduction of tissue damage in the chronic phase of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats [12]. Third, many chemokine receptors such as CCR2 and, in particular, CCR5 serve as HIV-1 cell entry cofactors together with CD4. It has been observed occasionally that in patients with long-standing CD, remission of the disease is associated with immunodeficiency caused by HIV infection [13]. This suggests a regulatory role for CCR51CD4-expressing T-lymphocytes in the pathogenesis of CD. Fourth, systematic genome screening studies have identified several IBD susceptibility loci, and a microsatellite marker D3S1573 linked to IBD is located on chromosome 3 (3p21) within 5cM of the genes for chemokine receptors CCR2 and CCR5 [14]. In our study, we took advantage of the fact that polymorphic forms of both CCR2 and CCR5 receptors are present in the Caucasian population at high frequencies that significantly affect receptor functions. Individuals homozygous for a 32 bp deletion in the CCR5 gene (CCR5D32), which generates a non-functional receptor and non-responsiveness to certain CC chemokines, are highly resistant to HIV infection [15]. Furthermore, a single-point mutation in the CCR2 gene (CCR2-641) has been shown to be associated with significantly delayed disease progression in HIV-infected individuals [16]. We hypothesized that functional CCR5 and CCR2 receptors might be essential for cellmediated immunity and consecutive development of CD. Therefore, we determined the frequencies of the CCR5D32 and CCR2-641 alleles in CD patients and investigated genotype– phenotype correlations in patient subgroups, which were defined by well-established outcome-related disease parameters.
2. Materials and methods
2.1. Study population Blood samples were obtained from 235 Caucasian patients resident in Germany who were diagnosed with CD according to standard diagnostic criteria [17]. Patients were retrospectively allocated into different subgroups according to a recently adopted classification of CD (Vienna classification), which is based on outcomerelated objective variables [18]. According to the Vienna classification, age at diagnosis was defined as Al (B40 years) or A2 (\ 40 years), and the location was defined as L1 (terminal ileum), L2 (colon), L3 (ileo-colonic) or L4 (upper gastrointestinal tract). The behaviour of CD was defined as B1 (non-penetrating, non-stricturing), B2 (stricturing) or B3 (penetrating). Only patients with adequate documentation who met classification criteria (n= 216, 93%) were entered into one of the Vienna classification phenotypes. All CD patients were genotyped for the CCR5 polymorphism, whereas due to a shortage of genomic DNA, only 145 patients could be genotyped for the CCR2 polymorphism. Ethnically matched healthy blood donors (n= 322) and staff members (n= 24) served as the control group.
2.2. CCR2 and CCR5 genotyping Genotypes were determined by polymerase chain reaction (PCR) or PCR restriction fragment length polymorphism (PCR-RFLP) using a modification of published protocols [19,20]. Briefly, genomic DNA was prepared from whole venous blood using a commercially available DNA isolation kit (Qiagen, Hilden, Germany). The genotype of CCR5 was determined by DNA amplification by a 40-cycle PCR with Taq polymerase (forward primer 5%-GCGTCTCTCCCAGGAATCATC-3%; reverse primer 5%-GGTGAAGATAAGCCTCACAGCC-3%). The PCR products were separated by 4% agarose gel electrophoresis and stained with ethidium bromide. The wild type and mutant A32 allele were detected as 242- and 210 base pair fragments, respectively. The CCR2-641 allele was detected with a PCR-RFLP assay using a BsaBl site introduced into the PCR forward primer next to the C–T transition that encodes the CCR2-641 polymorphism. Amplification (forward primer 5%-TTGTGGGCAACATGaTGG3% has a cytosine substituted with an adenine [in lower case]; reverse primer 5%-CTGTGAATAATTTGCACATTGC-3%) yielded a 183 bp fragment, which was digested by BsaBl (3 U at 60°C for 18 h) into 18- and 165 bp fragments when an isoleucine was present instead of a valine at position 64 in CCR2.
H. Herfarth et al. / Immunology Letters 77 (2001) 113–117
2.3. Statistical analysis Chi-square and Fisher’s exact tests were used to determine statistical significance of differences in genotype and allele distributions among the different subgroups. Differences were considered significant if P was B 0.05.
3. Results
3.1. Gene frequencies of CCR5 and CCR2 in patients and control subjects The CCR5 genotype was determined in 346 healthy individuals and in 235 CD patients. CCR2-641 allelic frequency was determined in 342 healthy controls and in 145 patients. Phenotype and gene frequencies for CCR2 and CCR5 polymorphisms are summarized in Table 1. Gene frequencies of CCR2-641 (8.2%) and CCR5D32 (9.2%) in the control population were in accordance with previous reports [16,21]. The gene frequencies of CCR5D32 (9.8%) and CCR2-641 (7.6%) in patients did not differ significantly from healthy controls, nor did the observed ratios deviate from the predicted Hardy–Weinberg distribution.
3.2. Influence of CCR5 and CCR2 genotypes on the phenotype of CD Our CD study population from three German referral centers enclosed a high percentage of patients with young age at diagnosis (Al) and a frequent ileo-colonic disease location, whereas the disease behaviour was nearly equally distributed (Table 2). No significant differences in the phenotypic disease manifestastions in relation to the CCR5 and CCR2 polymorphisms were found (Tables 2 and 3). We observed a trend towards sparing of the upper gastrointestinal tract (carrier frequency 0% in L4 versus 19.6% in L1– 3, D =19.6%, P=0.079; gene frequency 0 versus 10.3%, D =10.3%, P= 0.080) and a more stricturing type of disease (carTable 1 CCR2-641 and CCR5D32 genotypes of healthy controls and CD patients
CCR2-641 CCR2 wt/wt CCR2 wt/641 CCR2 641/641 CCR5D32 CCR5 wt/wt CCR5 wt/D32 CCR5 D32/D32
Controls
Patients
(n= 342) 289 (84.5) 50 (14.6) 3 (0.9) (n=346) 287 (82.9) 54 (15.6) 5 (1.5)
(n = 145) 123 (84.8) 22 (15.2) 0 (0) (n= 235) 191 (81.3) 42 (17.9) 2 (0.9)
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Table 2 CCR5 gene polymorphism (genotypic and phenotypic frequencies) in CD subgroups Subgroup
CCR5D32 gene frequency (%)
CCR5D32 carrier (%)
Al (age B40; n =194/90%) A2 (age \40; n =22/10%) L1 (terminal ileum; n =42/19%) L2 (colon; n =48/22%) L3 (ileo-colon; n =114/53%) L4 (upper gastrointestinal tract; n =12/6%) B1 (non-penetrating; n =66/31%) B2 (stricturing; n = 68/31%) B3 (penetrating; n = 82/38%) All CD patients (n = 216/100%) Controls (n =346)
9.5 11.4 10.7
18.0 22.7 19.0
7.3 11.4 0
14.6 21.9 0
8.3
15.2
12.5 8.5 9.7
23.5 17.0 18.5
9.2
17.1
rier frequency 23.5% in B2 versus 16.2% in B1/B3, D= 7.3%, P= 0.136; gene frequency 12.5 versus 8.4%, D=4.1%, P= 0.126) in carriers of the CCR5D32 allele. However, none of these differences reached a level of statistical significance.
4. Discussion Chemokines and their cognate membrane-bound receptors fulfill important roles in the directed migration/ activation of leukocytes and have been implicated in the pathogenesis of various inflammatory diseases. In Table 3 CCR2 gene polymorphism (genotypic and phenotypic frequencies) in CD subgroups Subgroup
CCR2-641 gene (%)
A1 (age B40; 5.8 n =113/89%) A2 (age \40; n = 14/11%) 10.7 L1 (terminal ileum; 10.4 n = 24/19%) L2 (colon; n =24/19%) 2.1 L3 (ileo-colon; n =75/59%) 6.7 L4 (upper gastrointestinal 0 tract; n =4/3%) B1 (non-penetrating; 7.3 n = 41/32%) B2 (stricturing; 5.0 n = 50/39%) B3 (penetrating; 6.9 n = 36/28%) All CD patients 6.3 (n = 127/100%) Controls 8.2
CCR2-641 carrier (%) 11.5 21.4 20.8 4.2 13.3 0 14.6 10.0 13.9 12.6 15.5
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this study, we investigated the potential significance of the CC chemokine receptors CCR2 and CCR5 in the pathogenesis of CD by analysing the association of genetic polymorphisms of these two receptors with clinical outcome in CD patients. A 32 bp deletion in the CCR5 gene is common among Caucasians and results in the expression of a truncated, non-functional receptor [15]. A point mutation in the CCR2 gene (CCR2641) is found in many ethnic groups and results in a conservative substitution of valine 64 to isoleucine [16]. Both CCR mutations independently confer protection towards HIV. This protection is reflected either, in the case of CCR5D32 homozygozity, by resistance to HIV infection or, in the case of CCR2-641, by slower disease progression [22]. Several recent studies have reported a protective effect of either CCR5 or CCR2 mutations towards certain chronic inflammatory diseases, such as multiple sclerosis [23,24], rheumatoid arthritis [25,26] or pulmonary sarcoidosis [20,27]. At the same time, CCR2641 or CCR5D32 mutant alleles appear to be overrepresented in diabetic children [28] or associated with a more pronounced clinical course of the disease in pulmonary sarcoidosis [27]. Taken together, these studies reveal differential associations of CCR2 and CCR5 polymorphisms with disease activity in a number of chronic inflammatory diseases that are characterized by enhanced cell-mediated immunity. In our own study, we show that the CCR5D32 gene frequency in all CD patients did not differ significantly from healthy controls, nor did the distribution deviate from the Hardy–Weinberg predictions. These results are corroborated by Hampe et al., who recently performed a high-resolution mapping of the IBD susceptibility locus on chromosome 3p in order to identify potential genetic associations with this disease [29]. These authors found allele frequencies of 8.3 and 12% for the CCR2-641 and CCR5D32 mutant alleles in 353 affected sibling pairs with the diagnosis of CD or ulcerative colitis, who were recruited in a large European cohort. Association statistics revealed no significant influence of these mutations on the phenotype of patients with inflammatory bowel disease. In the study of Hampe et al., CD patients were not classified according to their individual disease phenotype. Since there is accumulating evidence to suggest that CD patients comprise a heterogenous group of individuals with different phenotype-determining genetic backgrounds [2], we also examined chemokine receptor polymorphisms in various patient subgroups [18]. Our data revealed no significant effects of CCR2 or CCR5 genotypes on the age of onset, location or behaviour of the disease. Although not statistically significant, we observed that carriers of the CCR5D32 mutant had upper gastrointestinal tract disease less often compared with other locations of the disease.
Carriers of the mutant CCR5 allele were also more prone to develop a stricturing form of disease. If these findings are corroborated in independent datasets, they may suggest that CCR5 deficiency protects from a more aggressive, i.e. penetrating/fistulizing disease course due to a reduced capacity to generate an inflammatory response in the absence of CCR5-expressing cells. Furthermore, they point to the possibility of a differential role for CCR5 expressing cells in large versus small bowel. Interestingly, a positive association between fistulizing or large bowel disease and carriage of the TNF2 allele, which directs higher levels of TNFa transcription, has been described in a Belgian population [30]. The findings of this and the current study may be linked by the ability of CCR5-expressing mucosal Th1-lymphocytes and/or macrophages to induce TNFa secretion. The results of our study are in accord with the outcome of experimental animal models of IBD in CCR5 knock-out mice. It was reported that CCR5and, to a lesser degree, CCR2-deficient mice are partially protected from DSS-induced colitis, an experimental disease that shares many features of acute and chronic intestinal inflammation [11]. While CCR5 deficiency did not prevent DSS-induced intestinal inflammation, both clinical and histological disease scores indicated a diminished inflammatory response in receptor knock-out mice. Moreover, CCR5-deficient mice developed a Th2 pattern of cytokine expression after DSS treatment, which was characterized by significantly decreased levels of IFN-g, while expression of IL-4, IL-5 and IL-10 mRNA was enhanced. In summary, CCR2 or CCR5 gene polymorphisms are unlikely to be important determinants of overall disease susceptibility in CD. CCR5 gene polymorphism may participate in determining disease course and location and thereby contribute to heterogeneity of CD. However, the observed effects are modest and await replication in independent studies. Acknowledgements This work was supported by grant SFB402 B7 from the Deutsche Forschungsgemeinschaft. H.H. is supported by Kompetenznetzwerk ‘Chronisch-entzu¨ ndliche Darmerkrankungen’ of the German Federal Department for Research and Education (BMBF). We are grateful to Katrin Kraft for expert technical assistance. References [1] R. Duchmann, M. Zeitz, in: P.L. Pearay, J. Mestecky, M.E. Lamm, W. Strober, J. Bienenstock, J.R. McGhee (Eds.), Mucosal Immunology, Academic Press, San Diego, CA, 1999, pp. 1055 – 1080.
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