Genetic effect of CCR3 and IL5RA gene polymorphisms on eosinophilia in asthmatic patients

Article Outline

• Abstract
• Methods
  • Subjects
  • Sequencing analysis of the human CCR3 gene
  • Genotyping by means of single-base extension and electrophoresis
  • Measurement of CCR3 and IL-5Rα expression on peripheral blood eosinophils of asthmatic subjects
  • Statistical analysis
• Results
  • Association between CCR3 gene polymorphisms and development of asthma
  • Association between CCR3 and IL5RA gene polymorphisms and peripheral blood eosinophil count in asthmatic patients
  • Gene-gene interactions between CCR3 ht2 and IL5RA c.−5091 polymorphism on peripheral blood eosinophil number in asthmatic patients
  • Flow cytometric analysis for CCR3 and IL-5Rα protein expression on peripheral blood eosinophils from asthmatic patients
• Discussion
• Acknowledgment
• Appendix. Supplementary data
• References
• Copyright

Background

Eosinophilic infiltration and peripheral blood eosinophilia in asthma require the cooperation of eosinophil-specific cytokines and chemokines and their receptors.

Objective

We investigated the association of polymorphisms in CCR3 and IL5RA with asthma susceptibility or peripheral blood eosinophilia and the effects of the polymorphisms on receptor expression.

Methods

Polymorphisms in CCR3 and IL5RA were identified and genotyped in 576 asthmatic patients and 180 healthy control subjects. CCR3 and IL-5 receptor α (IL-5Rα) protein expression on eosinophils was measured by means of flow cytometry.

Results

Although polymorphisms in CCR3 were not associated with asthma susceptibility, the CCR3 haplotype ht2 showed a negative gene dose effect on the eosinophil count (P = .003–.009). IL5RA c.−5091G>A was weakly associated with eosinophil count. The effects of ht2 were greater when paired with IL5RA c.−5091A (P = .001–.002). CCR3 protein expression was higher on eosinophils of asthmatic patients without ht2 than in those with ht2. Asthmatic patients with the IL5RA c.−5091A allele showed higher IL-5Rα expression than those who were homozygous for the G allele.

Conclusion

The genetic association between CCR3 polymorphisms and the number of circulating eosinophils was revealed as a novel finding. These associations were more pronounced when the CCR3 polymorphisms were paired with polymorphisms in IL5RA. The protein expression levels of CCR3 and IL-5Rα on peripheral blood eosinophils are associated with the polymorphisms on their own genes.

Clinical implications

The identification of single nucleotide polymorphisms and haplotypes of CCR3 and IL5RA might be useful in developing markers for intermediate phenotypes of eosinophil number and in designing strategies to control diseases related to hypereosinophilia.

Key words: Asthma, eosinophilia, single nucleotide polymorphism, CC chemokine receptor 3, IL-5 receptor α

Abbreviations used: CCR3, CC chemokine receptor 3, IL-5Rα, IL-5 receptor α, SNP, Single nucleotide polymorphism, UTR, Untranslated region

Asthma is a common respiratory disease characterized by intermittent airway obstruction and respiratory symptoms caused by chronic airway inflammation and remodeling.¹, ² Excessive infiltration of eosinophils has been identified within the target tissues of allergic disorders.³, ⁴ The extent of eosinophilic inflammation is a determinant of the severity of asthma symptoms,⁵ and the number of peripheral blood eosinophils is correlated with restricted airflow.⁶, ⁷ The processes of eosinophilic infiltration and peripheral blood eosinophilia are dependent on the cooperative action of eosinophil-specific cytokines and chemokines, such as IL-5 and members of the eotaxin family.⁸, ⁹

CCR3 is a G protein–coupled receptor¹⁰ for chemokines, which provoke an eosinophilic response in the peripheral blood and airways.¹¹, ¹² CCR3 mRNA and protein levels are increased in the bronchial mucosa of asthmatic patients in association with airway hyperresponsiveness.¹³ The human CCR3 gene (MIM no. 601268) is located on chromosome 3p21.3,¹⁴, ¹⁵ which is linked to atopic dermatitis¹⁶, ¹⁷ and asthma.¹⁸

IL-5 is important for the terminal differentiation, activation, and survival of committed eosinophil precursors.¹⁹, ²⁰ It stimulates the release of eosinophils into the circulation and prolongs their survival through the IL-5 receptor (IL-5R).²¹, ²² The human IL-5R complex is a heterodimer consisting of a unique α subunit (IL-5Rα) and a β subunit.²³ The α subunit is required for ligand-specific binding.²⁴ The IL5RA gene (MIM no. 147850) is located on chromosome 3p26-p25, which is linked to asthma development (D3S3564).¹⁸ IL5RA mRNA-positive cells are increased in the airways of asthmatic patients.²⁵

Given the biologic effects of CCR3 and IL-5Rα and their linkage to the chromosomal loci of asthma susceptibility, CCR3 and IL5RA polymorphisms might be related to asthma development or peripheral blood eosinophilia in asthma. In this study we screened and genotyped single nucleotide polymorphisms (SNPs) in CCR3 and IL5RA and examined the genetic effects of the SNPs on the risk for asthma and eosinophilia in asthmatic patients. Furthermore, we tested the interactive effects between the polymorphisms in CCR3 and IL5RA on eosinophilia. We also validated the genetic effects of the SNPs on the expression of CCR3 and IL-5Rα protein on eosinophils.

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Methods 

Subjects 

We enrolled 576 Korean asthmatic patients and 180 healthy control subjects from 3 tertiary hospitals in Korea (Soonchunhyang University Seoul, Bucheon, and ChunAn hospitals). Among the 180 healthy subjects, 166 were spouses of asthmatic patients, and 14 were healthy volunteers who performed Heath Screening Check Up for Job Application. The institutional review boards of the hospitals approved the study, and all patients gave informed consent. All the patients with asthma met the American Thoracic Society definition of asthma.²⁶ Additional details are provided in the Methods section in this article's Online Repository at www.jacionline.org. The clinical characteristics of all the subjects studied are summarized in Table I.

Table I. Clinical characteristics of study subjects

FVC, Forced vital capacity.

Sequencing analysis of the human CCR3 gene 

We sequenced all 4 exons, 3 introns, and the promoter region (∼1.5 kb) of the CCR3 gene in the DNA samples from 24 Korean subjects using an ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, Calif). Four primer sets (CCR3) for the amplification and sequencing analysis were designed based on GenBank sequences (reference genome sequence, NT_022517). Information on the primers is available in Table E1 in this article's Online Repository at www.jacionline.org.

Genotyping by means of single-base extension and electrophoresis 

Amplifying and extension primers were designed for single-base extension to genotype the SNPs. Reactions were performed with a SNaPshot ddNTP primer extension kit (Applied Biosystems), according to the manufacturer's recommendations. Information on the primers is available in Table E2 in this article's Online Repository at www.jacionline.org. Data were analyzed with ABI Prism GeneScan 3.5.1 and Genotyper 3.6 software (Applied Biosystems).

Measurement of CCR3 and IL-5Rα expression on peripheral blood eosinophils of asthmatic subjects 

Eosinophil-rich polymorphonuclear leukocytes were purified from the peripheral blood of the asthmatic patients or subjects having peripheral blood eosinophil counts higher than 10% because of other diseases as described previously.²⁷ Purified cells were stained by using immunofluorescence with anti-human CCR3 (R&D Systems, Minneapolis, Minn) or IL-5Rα antibody (BD Biosciences, San Jose, Calif). Eosinophils were distinguished from neutrophils by using a gated autofluorescence/forward-scatter method.²⁷, ²⁸ Additional details are provided in the Methods section of the Online Repository at www.jacionline.org.

Statistical analysis 

To examine Hardy–Weinberg equilibria, the Fisher exact test was performed with a modified version of the Markov-chain random walk algorithm.²⁹ Linkage disequilibria between loci were quantified as |D′| and r² values ³⁰, ³¹ by using the program Arlequin (http://anthro.unige.ch/arlequin) and Microsoft Excel. The haplotypes and their frequencies were inferred by using the algorithm implanted in PHASE.³² The allele and haplotype frequencies between phenotypes were compared by using logistic regression analysis, controlling for age and sex as covariates. The number of peripheral blood eosinophils was log transformed. The differences in the log-transformed values among genotypes or haplotypes were examined by using a generalized linear model type III SS. Data were analyzed with SAS (version 8.1; SAS Institute, Cary, NC). The effective number of independent marker loci in CCR3 and IL5RA was calculated by using the software SNPSpD (http://www.genepi.qimr.edu.au/general/daleN/SNPSpD/) to correct for multiple testing.³³ Statistical significance was defined at the standard 5% level. The ratios of fluorescence intensity for CCR3 or IL-5Rα were analyzed with the Mann-Whitney U test.

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Results 

Association between CCR3 gene polymorphisms and development of asthma 

Direct sequencing of the CCR3 gene, including the −1500 bp of the 5′-flanking region, identified 4 SNPs. Of 10 SNPs (−22557G>A, −520T>G, −174C>T, +51T>C, +240C>T, +652T>A, +824G>A, +827G>C, +828C>G, and +1052T>C) reported in white and Japanese subjects,³⁴, ³⁵, ³⁶ the last 6 listed were not identified in the 24 Korean subjects. Therefore the other 4 SNPs were selected for larger-scale genotyping. The genotype distributions of the 4 SNPs were in Hardy–Weinberg equilibrium within all subjects (P > 0.05, see Table E3 in this article's Online Repository at www.jacionline.org). Strong linkage disequilibria (|D′| > 0.9) were noted between the other alleles, except between −22557G>A and −174C>T (Fig 1, B), and 7 haplotypes were inferred from unphased genotype data (Fig 1, C). Among them, one haplotype (ht4: AGCC) was equivalent to +51T>C. Three haplotypes (ht1: GTCT, ht2: AGTT, and ht3: GGTT) were analyzed for the association study, and the other 3 haplotypes were excluded because of low frequency (<1%). The haplotype distributions were in Hardy–Weinberg equilibrium within all subjects (P > 0.05, see Table E3 in this article's Online Repository at www.jacionline.org). None of the SNPs or haplotypes of CCR3 showed a significant association with asthma development on logistic regression analysis (see Table E4 in this article's Online Repository at www.jacionline.org).

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• Fig 1. 

  Gene map (A), linkage disequilibrium (LD) coefficients (B), and haplotypes (C) in CCR3. In the map (Fig 1, A), coding exons are marked by solid blocks and UTR by open blocks. The frequencies of SNPs were based on sequencing data (n = 24). The first base of the translation site was denoted as nucleotide +1.

Association between CCR3 and IL5RA gene polymorphisms and peripheral blood eosinophil count in asthmatic patients 

The asthmatic patients with rare alleles for SNPs of the CCR3 gene (−22557G>A and −174C>T) showed a trend toward having a smaller number of peripheral blood eosinophils than those with the common alleles among asthmatic patients (P = .014−.043, P_corr = .042–.129; Table II). The asthmatic patients with ht1 (GTCT) showed a trend toward having a greater number of eosinophils than those without ht1 (P = .049, P_corr = .125, recessive model). The asthmatic patients with ht2 (AGTT) showed a significantly smaller number of eosinophils than those without ht2 (P = .003, P_corr = .008, codominant model; P = .009, P_corr = .023, dominant model; P = .005, P_corr = .013, recessive model). ht2 (AGTT) showed a clear gene dose-dependent effect on the peripheral blood eosinophil count in all 3 models (Table II). The results for diplotype analysis were similar to the results obtained from haplotype analysis (see Table E5 in this article's Online Repository at www.jacionline.org). The genetic effect of ht2 on the peripheral blood eosinophil count showed the same trends in atopic asthmatic patients and in nonatopic asthmatic patients (see Table E6 in this article's Online Repository at www.jacionline.org). The asthmatic patients who were ht1 homozygous had the highest number of eosinophils, and ht2 homozygotes had the lowest number of eosinophils. The asthmatic patients with ht1/ht2 had an intermediate number of eosinophils (P = .004, codominant model; Fig 2).

Table II. Association of SNPs and haplotypes of the CCR3 gene with the number of blood eosinophils of asthmatic patients

Cod, Codominant model; Dom, dominant model; Rec, recessive model.

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• Fig 2. 

  The comparison of the number of eosinophils in peripheral blood between the subjects possessing ht1, ht2, or both of CCR3. The bars and error bars represent geometric means and 95% CIs, respectively. P values were obtained by means of linear regression analysis, controlling age and sex as covariates in the codominant model.

Among the polymorphisms identified in our previous publication,³⁷ 10 polymorphisms in IL5RA were selected for larger-scale genotyping based on location, linkage disequilibrium, frequency, and haplotype-tagging status. IL5RA can be parsed into 2 haplotype blocks. There are 6 and 5 common haplotypes in block 1 and block 2, respectively.³⁷ Block 1-ht1, Block 1-ht2, Block 1-ht5, Block 2-ht2, Block 2-ht3, and Block 2-ht5 were not analyzed because they were equivalent to c.−6173T>C, c.−5091G>A, c.−6783T>C, c.−3643G>A, c.−480_482insdelGTT, and c.82+2068T>C, respectively. Block 1-ht6 was not analyzed because of its low frequency. Among the 10 SNPs on the IL5RA gene, c.−5091G>A showed a gene dose-dependent trend in association with the number of peripheral blood eosinophils (Table III). The asthmatic patients with rare alleles at c.−5091G>A of IL5RA showed a greater number of peripheral blood eosinophils than those with the allele common among asthmatic patients (P = .031, P_corr = .287, codominant model; P = .010, P_corr = .091, dominant model; Table III). The genetic effect of IL5RA c.−5091G>A on the peripheral blood eosinophil count showed the same trends in atopic asthmatic patients and in nonatopic asthmatic patients (see Table E6 in this article's Online Repository at www.jacionline.org). IL5RA c.−480_482insdelGTT showed an association with the number of eosinophils (P = 0.005, P_corr = 0.045, codominant model) but did not show an apparent gene dose effect.

Table III. The association of SNPs and haplotypes of the IL5RA gene with the values of the number of blood eosinophils in asthmatic patients

Cod, Codominant model; Dom, dominant model; Rec, recessive model.

Gene-gene interactions between CCR3 ht2 and IL5RA c.−5091 polymorphism on peripheral blood eosinophil number in asthmatic patients 

The combined genetic effect of CCR3 and IL5RA on the number of peripheral blood eosinophils was analyzed. The genetic effect of CCR3 ht2 became more pronounced when the asthmatic subjects were stratified according to IL5RA c.−5091G>A (Table IV). The asthmatic patients possessing the rare allele at c.−5091G>A exhibited a stronger gene dose effect of CCR3 ht2 on the number of peripheral blood eosinophils; those having ht2/ht2 at CCR3 showed the lowest number of peripheral blood eosinophils, whereas those without ht2 had the highest number of peripheral blood eosinophils (P = .001–.002). The same trends of the genetic interactions were observed in atopic and nonatopic asthmatic populations (see Table E7 in this article's Online Repository at www.jacionline.org). The genetic effect of CCR3 ht2 on the peripheral blood eosinophil number did not change when the asthmatic subjects were stratified according to IL5RA c.−480_482insdelGTT (data not shown).

Table IV. The association of the combination between IL5RA c.−5091G>A genotypes and CCR3 ht2 with the number of blood eosinophils in asthmatic patients

Cod, Codominant model; Dom, dominant model; Rec, recessive model.

Flow cytometric analysis for CCR3 and IL-5Rα protein expression on peripheral blood eosinophils from asthmatic patients 

To investigate whether the sequence variants of CCR3 or IL5RA could affect the expression of CCR3 or IL-5Rα protein, we measured CCR3 and IL-5Rα expression on eosinophils from asthmatic patients (Fig 3, A). The level of CCR3 expression was significantly lower on eosinophils from asthmatic patients with ht2 (n = 10) than in those without ht2 (n = 22; P = .043; Fig 3, B and D). In addition, the eosinophils from asthmatic patients with the A allele at IL5RA c.−5091G>A (n = 27) showed greater IL-5Rα expression than those with the G allele (n = 6; P = .035; Fig 3, C and E). The similar trends were observed in subjects with peripheral blood eosinophilia of more than 10% caused by diseases other than asthma (see Fig E1 in the Online Repository at www.jacionline.org).

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• Fig 3. 

  The CCR3 and IL-5Rα expression on the peripheral blood eosinophils from asthmatic patients. A, Gating for eosinophils among peripheral blood granulocytes by using autofluorescence is shown. B and D, Levels of CCR3 expression were compared between eosinophils from asthmatic patients having ht2 and having other haplotypes. C and E, Levels of IL-5Rα expression were compared between eosinophils from asthmatic patients who were G or A allele homozygotes on IL5RA-5091G>A. Each datum was presented by the ratio of fluorescence intensities for each molecule to the values of isotype-matched control.

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Discussion 

Eotaxin plays an important role in the infiltration of eosinophils into target tissues in allergic disorders in a cooperative mode with IL-5.³⁸, ³⁹ Whole-genome scans have suggested that the chromosome region 3p21-24, which contains a gene cluster of CC chemokine receptors, including CCR3, is linked to asthma.¹⁸ D3S3567 on chromosome 3p was closely linked to the asthma phenotype in a founder population.¹⁸ CCR3 is located very close to D3S3567 (distance apart, 4.1 Mb), and IL5RA is close to D3S3564 (distance apart, 39 Mb). These results suggest that CCR3 and IL5RA gene polymorphisms might have an effect on genetic susceptibility to asthma and eosinophilia as a subphenotype.

In our study we examined the 10 CCR3 gene polymorphisms previously reported in white and Japanese subjects.³⁴, ³⁵, ³⁶ Only 4 of these (−22557G>A, −520T>G, −174C>T, and +51T>C) were found to be present in the Korean subjects. This might be attributable to racial differences, as well as to the extremely low frequency (0.01–0.005) of the variations (+240C>T, +824G>A, and +1052T>C) in white subjects.³⁶ The allele frequencies of −520T>G, −174C>T, and +51T>C in our study were comparable with those in the Japanese population.³⁵, ³⁶ The absence of an association between −520T>G, −174C>T, and +51T>C and asthma development in our study concurs with 2 studies in Japanese populations,³⁴, ³⁵ despite the report of a significant association in a British population.³⁴ The +51T>C C allele is more frequent in the British population (12.8%) than in Korean (5.5%) or Japanese (4.2%) populations. This indicates the presence of a racial difference in CCR3 gene polymorphisms between white and Asian subjects.

The CCR3 gene contains 4 exons with 4 mRNA species by means of alternative splicing.⁴⁰ Intriguingly, the 3 SNPs associated with eosinophil number in our study locate in introns. It remains to be determined whether the alternative splicing is dependent on the SNPs (−22557G>A and −174C>T) in the CCR3 introns. Additionally, Zimmermann et al⁴¹ recently reported areas of active chromatin remodeling in exon 1 of CCR3. Given that −22557G>A is located only 63 bp downstream of exon 1 and between HindIII restriction sites, which is the location of a fragment showing DNase I hypersensitivity,⁴¹ the effect of the −22557G>A polymorphism on chromatin remodeling in the CCR3 gene should be investigated.

The present association analysis revealed a trend in associations of 2 SNPs (−22557G>A and −174C>T) with eosinophil number in asthmatic patients (Table II). The ht2 haplotype displayed a negative gene dose effect on the eosinophils number, whereas ht1 or ht3 showed a positive effect (Table II). This result indicates the strong genetic association among these 3 completely linked SNPs (−22557G>A, −520T>G, and −174C>T) and peripheral eosinophilia, which we believe is a novel finding. In contrast, we could not find the association in healthy control subjects. A possible explanation for the lack of association might be that they have relatively small numbers of circulating eosinophils because of the low level of IL-5 in healthy subjects.

In our previous study on IL5RA, the G allele of c.−5993A>G showed a lower frequency in asthmatic patients than in healthy control subjects (0.48 vs 0.51, P = .04).³⁷ Among the 10 SNPs in IL5RA, the c.−5091G>A polymorphism and c.−480_482ins/del had a marginal association with the number of peripheral blood eosinophils (Table III).

To predict the effects of c.−5091G>A on the function of the 5′-untranslated region (UTR), we searched for functional elements in the 5′-UTR of IL5RA using UTRScan (http://www.ba.itb.cnr.it/BIG/UTRScan) but found no patterns of functional elements in the region. Furthermore, there were no putative binding sites for transcription factors (TFSEARCH: V1.3; putative score, >0.87). c.−5091G>A might affect the level of IL-5Rα expression on peripheral eosinophils through an interaction with an unknown pattern in the 5′-UTR followed by translational controls or through a linkage disequilibrium with other functional SNPs not identified in this study or located on other nearby genes.

On the basis of the association of eosinophil number with polymorphisms of key regulatory genes, as shown in this study, we evaluated the interactions between the CCR3 and IL5RA genes. As shown in Table IV, there was an intergenetic association between CCR3 ht2 and c.−5091G>A of IL5RA, such that the genetic effect of this combination lowered P values more than the effect of either condition alone. These results strongly suggest that different gene polymorphisms might act additively to influence the development of eosinophilia in asthmatic patients. To evaluate whether these interactions are real or rather the result of fluctuations caused by the small size of the population, we tested the statistical interaction by using multiple regression analysis with interaction terms and could find significant interaction (P = .001, data not shown). We cannot exclude the chance of type I error in our positive results of gene-gene interaction using stratification methods because of the small numbers of subjects. However, considering the biologic roles of CCR3 and IL-5Rα, it seems obvious that there are biologic interactions on eosinophil differentiation and infiltration between them.⁸, ⁹

The expression of the CCR3 and IL-5Rα proteins was measured by means of flow cytometric analysis and was compared among asthmatic patients according to genotype to validate the genetic effects of CCR3 ht2 and IL5RA c.−5091G>A (Fig 3). CCR3 expression was higher on eosinophils in asthmatic patients lacking ht2 than in those with ht2. Asthmatic patients with the IL5RA c.−5091A allele showed higher IL-5Rα expression than those with the G allele. Similar trends were observed in subjects with eosinophilia of more than 10% caused by other diseases than asthma (Fig E1). In contrast, c.−480_482insdelGTT had no effect on the IL-5Rα expression level on eosinophils (data not shown). These findings demonstrate that CCR3 and IL-5Rα protein expression is dependent on the ht2 haplotype of CCR3 and c.−5091A of IL5RA.

In summary, the genetic association between CCR3 gene polymorphisms and the number of peripheral blood eosinophils was revealed as a novel finding. In addition, these associations were more pronounced when the CCR3 polymorphisms were paired with polymorphisms in the IL5RA gene. The protein expression levels of CCR3 and IL-5Rα on peripheral blood eosinophils are associated with the polymorphisms on their own genes. This information on the genetic polymorphisms in the CCR3 and IL5RA genes might be useful in developing markers for the intermediate phenotypes of peripheral blood eosinophil number and in designing new strategies for the control of diseases related to hypereosinophilia.

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We thank Eun-Young Kim, Myung-Ran Lee, and Shin-Ock Lee for establishing and supplying EBV-transfected B-cell lines.

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Appendix. Supplementary data 

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