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The Journal of Immunology, 2005, 174: 1525-1531.
Copyright © 2005 by The American Association of Immunologists

A Single Nucleotide Polymorphism on the Promoter of eotaxin1 Associates with Its mRNA Expression and Asthma Phenotypes1

Hun Soo Chang*, Jung Sun Kim*, June Hyuk Lee{dagger}, Jung Il Cho*, Tai Youn Rhim{dagger}, Soo-Taek Uh{dagger}, Byung Lae Park{ddagger}, Il Yup Chung2,*, Choon-Sik Park2,{dagger} and Hyoung Doo Shin{ddagger}

* Division of Molecular and Life Sciences, Hanyang University, Ansan, Gyeonggi-do, Korea; {dagger} Asthma Genome Research Center, Soonchunhyang University Bucheon Hospital, Bucheon, Gyeonggi-do, Korea; and {ddagger} Department of Genetic Epidemiology, SNP Genetics, Inc., Chongro-Gu, Seoul, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eotaxin1 plays a pivotal role in eosinophil-associated inflammation. Previously, we demonstrated 14 single-nucleotide polymorphisms (SNPs) in the human eotaxin1 gene and the association between the EOT+67G>A allele and the level of IgE. In this study, we investigated the association between the SNPs and plasma eotaxin1 levels, peripheral blood eosinophil counts, and PC20 methacholine values in normal and asthmatic subjects, and the effects of SNPs on the process of eotaxin1 production. The EOT–576C>T and EOT–384A>G polymorphisms and haplotypes (ht1 and ht4) were significantly associated with plasma eotaxin1 levels in the asthmatics (p < 0.001–0.040). The log [plasma eotaxin1] values correlated with the log [serum total IgE] values in the asthmatics and the normal controls (p = 0.012 and p = 0.004, respectively), and with the log [PC20 methacholine] values in the asthmatics (p = 0.014). A DNA-protein complex was formed with EOT–384A>G, but not with the other SNPs of the promoter. The interaction was stronger with the minor allele than with the common allele, and was reduced upon TNF-{alpha} exposure. TNF-{alpha}-stimulated PBMCs from the asthmatics with the minor allele homozygote expressed significantly lower levels of eotaxin1 mRNA than those from individuals with the common allele. The EOT+67G>A polymorphism, which substitutes alanine with threonine, did not affect eotaxin1 production or activity. Our data suggest that the EOT–384A>G SNP participates in the regulation of eotaxin1 expression by providing a potential binding site for a repressor, and that the ANOVA of EOT-384A>G may predict asthma phenotypes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eotaxin1 CCL11, which is a member of the CC chemokine family, acts via CCR3 (1, 2) on Th2 lymphocytes, basophils, mast cells, and eosinophils (2, 3, 4, 5, 6, 7), which participate in allergic inflammation. The expression of eotaxin1 mRNA and protein is increased in the airways of chronic asthmatics (8, 9) and patients with acute exacerbations of asthma (10, 11). The elevated eotaxin1 levels are involved in airway eosinophil infiltration, bronchial hyperreactivity, and impairment of lung function in asthmatics (8, 9, 10, 11) in concert with IL-5 (12, 13, 14). The human eotaxin1 gene (MIM (Mendelian Inheritance in Men) no. 601156) is located on chromosome 17q21.1 and consists of three exons and two introns (15). Gene mapping studies have established a strong link between asthma diagnosis and markers near the eotaxin1 gene (16). Previously, we reported 14 single-nucleotide polymorphisms (SNPs)3 in the eotaxin1 gene (17), including three known polymorphisms (18, 19, 20). Of the nine loci analyzed, the missense mutation EOT+67G>A showed a significant association with the total IgE levels and an absolute linkage disequilibrium (D' = 1 and d2 = 1) with EOT–488C>A in asthmatics (17). This indicates that the missense mutation caused by this SNP is linked to the altered transcription of eotaxin1 mRNA and/or functional alteration of the eotaxin1 protein. However, in vitro and in vivo results to support this hypothesis are currently lacking. We hypothesize that SNP in the promoter or coding region of the eotaxin1 gene alter the level of mRNA expression or the amount or function of the protein, which in turn regulate the intermediate phenotypes of asthma. To test this hypothesis, we evaluated the association of SNPs with plasma eotaxin1 levels, peripheral blood eosinophil counts, and a 20% fall in forced expiratory volume in 1 s (FEV1) (PC20) methacholine values, and examined the effects of SNPs on eotaxin1 transcription and translation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

The subjects were recruited from the Asthma Genome Research Center (Soonchunhyang University Hospitals, Bucheon, Gyeonggi-do, Korea). Ethical approval was obtained from the institutional review boards of the individual hospitals. The subjects were selected from patients with mild intermittent to moderate persistent asthma (21). All of the patients met the definition of asthma from the American Thoracic Society (22). Each patient showed inhalant bronchodilator-induced improvement of >15% in FEV1 and/or airway hyperreactivity of <10 mg/ml methacholine (23). The normal subjects were recruited among the patients’ spouses and the general population, who answered negatively to a screening questionnaire for respiratory symptoms (24) and had predicted FEV1 values of >75%, PC20 levels >10 mg/ml, and normal chest radiograms. Total IgE was measured using the CAP system (Pharmacia Diagnostics). Complete blood counts and differential cell counts were measured using a Coulter counter (Beckman Coulter). A battery of 24 common inhalant allergens was used for the skin prick test (25). Atopy was defined as having a wheal reaction equal to or greater than that induced by histamine or at least 3 mm in diameter.

Genotyping by single base extension and electrophoresis

The primer extension reactions were performed with the SNaPshot ddNTP Primer Extension kit (Applied Biosystems). To clean up the primer extension reaction, 1 U of SAP was added. The DNA samples, which contained the extension products, and the GeneScan-120 Liz Size standard were added to Hi-Di formamide (Applied Biosystems). The mixture was denatured and then electrophoresed in an ABI Prism 3100 Genetic Analyzer. The results were analyzed using ABI Prism GeneScan and Genotyper (Applied Biosystems) programs.

Measurement of eotaxin1 by ELISA

The amounts of eotaxin1 were measured by ELISA kits (OptEIA, BD Pharmigen). Since the detection limit of this analysis is ~6.5 pg/ml, samples with levels below this threshold were given the value of 0 pg/ml for the statistical analysis. The interassay and intraassay coefficients of variance were <10%.

Nuclear extract preparation and EMSA

Nuclear extracts were prepared from A549 cells that were untreated or treated with 10 ng/ml TNF-{alpha} (R&D Systems), as described by Matsukura et al. (26). EMSA was performed using the Gel Shift Assay System (Promega). The sequences of the DNA probes were as follows: EOT–1384G/A, 5'-AgACTAACCCACCg[g/A]gAATggAgCAggA-3'; EOT–576C/T, 5'-TggTgTgTTgTCCT[C/T]CCTggTTCAgAgA-3'; EOT–426C/T, 5'-AAggTTCTTAgATC[C/T]ACTCATCCCCCAg-3'; and EOT–384A/G, 5'-TgCTCCTTTCCCCg[A/G]CTACAggTgTTTC-3'. To determine the specificities of the DNA-protein complexes, 1.75 µM unlabeled probe was incubated with the nuclear extract for 10 min before addition of the labeled probe. For the supershift assay, 1 µg of goat anti-human Ikaros Ab (Santa Cruz Biotechnology) or mouse anti-human E2A Ab (BD Pharmingen) was incubated with the nuclear extract for 30 min before addition of the labeled probe.

Northern blot analysis for eotaxin1 mRNA expression by PBMC

PBMCs were isolated using Histopaque-1077 (Sigma-Aldrich), and cultured at 5 x 106 cells/ml in 10% FBS/RPMI 1640 medium with or without 10 ng/ml human TNF-{alpha} (R&D Systems). Total RNA was extracted using the modified guanidine thiocyanate precipitation method (Tri-Reagent; Molecular Research Center). Twenty micrograms of total RNA were resolved by electrophoresis in a 1% agarose-formaldehyde gel and transferred to a nitrocellulose membrane (Hybond-XL; Amersham Biosciences). The blot was hybridized with the [{alpha}-32P]dCTP-labeled probe using Ready-To-Go DNA Labeling Beads (Amersham Biosciences), then it was stripped and rehybridized with GAPDH probe.

Expression of the Ala- and Thr-type eotaxins

The full-length eotaxin1 gene, which has the EOT+67G/A alleles, was obtained by RT-PCR of allele-matched PBMCs. The PCR product was inserted into the pcDNA3.1 vector (Invitrogen Life Technologies) to construct the eotaxin1-expressing plasmid, or ligated into the pEGFP vector (Clontech Laboratories) to produce GFP fusion to the C terminus of eotaxin1. Transient transfection was performed using LF2000 (Invitrogen Life Technologies) into COS-7 cells that were cultured 10% FBS/DMEM (Life Technologies). As an internal control, we cotransfected pSV-{beta}-galactosidase control vector (Promega) with each eotaxin1-GFP fusion construct as per the manufacturer’s instructions. The expression levels of the eotaxin1-GFP fusion proteins were assessed by measuring the fluorescence intensity in flow cytometry (BD Biosciences) and using confocal microscopy (Bio-Rad).

Chemotaxis assay

Chemotactic activities of the culture media of COS7 cells that were transfected with either the Ala- or Thr-type eotaxin1 were measured using a 48-well Boyden’s chamber (Neuroprobe) as previously described (27). The human CCR3-transfected 4DE4 cell line was kindly donated by Dr. J. E. Pease (Imperial College School of Medicine, London, U.K.; Ref.28). Commercially available eotaxin1 (R&D Systems) was used as the control.

Statistics

The plasma levels of eotaxin1, eosinophil numbers, PC20 methacholine, and serum total IgE levels were log-transformed to normalize the right-skewed distribution. Differences in the clinical parameters between the asthma and normal control groups were compared using Student’s t test or the {chi}2 test. Linear regression analysis was used to assess the correlations between the parameters. The Fisher’s exact test was used to compare the observed numbers of each genotype with those expected for a population in Hardy-Weinberg equilibrium. The haplotypes were inferred using the algorithm developed by Stephens et al. (29). Missing genotype data were omitted for exact haplotyping. Genotype distribution of the SNPs and haplotypes among the asthmatics and normal subjects were analyzed with logistic regression models controlling age (continuous value), sex (male = 0, female = 1) and smoking status (nonsmoker = 0, ex-smoker = 1, smoker = 2) as covariables. Regression analyses of three alternative models (codominant, dominant, and recessive models) comparing log [plasma eotaxin1] and genotypes were calculated by multiple regression analyses, while controlling for age and sex as covariables. Rare allele was coded as dominant in the dominant model and recessive in the recessive model. Haplotype was also coded in same manner. The data are expressed as the means ± SEM. Differences between the two groups in the in vitro study were compared using the Mann-Whitney U test. Differences were considered to be statistically significant when the p value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Correlation of plasma eotaxin1 levels with peripheral blood eosinophil counts, total IgE concentrations, and PC20 methacholine values

Total IgE levels, eosinophil number, and positive skin test rates were significantly higher for the asthmatics than for the normal controls (p = 0.0001–0.003; Table I). The log [plasma eotaxin1] values were significantly lower for asthmatics who received inhaled corticosteroids than for those who received no medication (p = 0.012). However, the eotaxin1 levels were similar for the nonmedicated asthmatics and normal controls (p > 0.05). Thus, only the nonmedicated asthmatics were used to assess the association between eotaxin1 SNPs and plasma eotaxin1 levels and the correlation between eotaxin1 levels and intermediate phenotypes of asthma. The log [plasma eotaxin1] values were related to age in the asthmatics who received no medication and to sex in the normal controls (data available at <www.asthmagenome.re.kr/data/eotaxin001.htm>. The log [plasma eotaxin1] values correlated with the log [total IgE] values in the asthmatics and the normal controls (p = 0.012 and p = 0.004, respectively) and with the log [PC20 methacholine] values in the asthmatics (p = 0.014; Table II). There was no correlation between the log [plasma eotaxin1] and log [eosinophil number] of the two groups (Table II). These data present the evidence that eotaxin1 may have a role of IgE synthesis (30) and severity of asthma (8, 9, 10, 12).


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  		Table I. Clinical profile and laboratory findings of the study subjectsa

 

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  		Table II. Correlation between log [plasma eotaxin1 (pg/ml)] (eotaxin1), log [plasma total IgE] level (total IgE), log [blood eosinophil number (count/µl)] (eosinophil no.) and log [PC20 (methacholine)] (PC20) in asthmatics with no medication (n = 232) and normal controls (n = 211)a

 
Association of eotaxin1 SNPs and haplotypes with plasma eotaxin1 levels

SNPs in eotaxin1 were renamed by calculating from the translational start site to avoid confusion due to alternative nomenclature in previous studies (15, 17, 31, 32) and revised by human genome sequence (Ref. genome sequence: NT_010799, released on Jan. 2004; Table III). The distributions of all six SNPs in normal controls were in Hardy-Weinberg equilibrium (p > 0.05; data available at <www.asthmagenome.re.kr/data/eotaxin001.htm>. The allelic and haplotypic frequencies of the six SNPs and the two haplotypes were similar in the asthmatics and the normal controls (Table III). All of the SNPs and the two haplotypes were analyzed for a potential association with the log [plasma eotaxin1] values of the asthmatics (Table IV). Of the six loci, The polymorphic alleles of EOT-576C>T and EOT-384A>G showed significant associations with log [plasma eotaxin1] in the asthmatics (p = 0.001–0.04). The haplotypes ht1 and ht4 were also significantly associated with log [plasma eotaxin1] (p = 0.008–0.015). Asthmatics who carried the EOT-576C>T C* allele (CC and CT) had higher levels of eotaxin1 (mean log [eotaxin1] = 1.34 ± 0.42) than did individuals who had the homozygous EOT-576C>T TT allele (mean log [eotaxin1] = 1.11 ± 0.57; p = 0.001). EOT-384A>G showed the same pattern of genetic effect: asthmatics who carried the EOT-384A>G A* allele (AG and AA) had higher levels of eotaxin1 (mean log [eotaxin1] = 1.33 ± 0.42) than did individuals who had the homozygous EOT-384A>G GG allele (mean log [eotaxin1] = 1.08 ± 0.62; p = 0.002).


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  		Table III. Minor allele frequencies of SNPs in eotaxin1 in asthmatics (n = 348) and normal subjects (n = 211)a

 

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  		Table IV. Multiple regression analyses for age, and sex adjusted log [eotaxin1] with six SNPs and haplotypes in asthmatics without steroid therapy (n = 232)a

 
The effects of the two haplotypes (ht1 and ht4) on eotaxin1 production appeared to be significant (Table IV). The plasma eotaxin1 levels were dependent in a positive fashion on the number of ht1 haplotypes. Asthmatics with the ht1 haplotype (ht1+/+ and ht1+/–) had significantly higher levels of eotaxin1 (mean log [eotaxin1] = 1.34 ± 0.43) than individuals who lacked the ht1 haplotype (ht1–/–) (mean log [eotaxin1] = 1.17 ± 0.53; p = 0.028). The effect of the ht4 haplotype showed the inverse pattern to that of the ht1 haplotype. Individuals who carried the ht4 haplotype (ht4+/+ and ht4+/–) produced much lower levels of eotaxin1 (log [eotaxin1] = 1.10 ± 0.55) than did those who lacked this haplotype (ht4–/–) (log [eotaxin1] = 1.32 ± 0.45; p = 0.015).

The region of the promoter that contains EOT-384 is a binding site for a NF

To investigate whether the four loci on the promoter of eotaxin1 (-1384 G>A, –576C>T, –426C>T, and –384A>G) provide the binding sites for a transcription factor, gel shift assays were performed using the nuclear extract from A549. A DNA- protein complex was detected with the probe for EOT-384A>G, but not with those for –576C>T (Fig. 1) or the other SNPs (data not shown). The intensity of this complex was higher when hybridized with the probe for the minor allele (–384A>G G) than with the probe for the common allele (–384A>G A; Fig. 1) and decreased by treatment with TNF-{alpha}. This result indicates that the eotaxin1 promoter region around –384A>G may be the binding site for a transcription factor. Since certain candidate factors (Ikaros family and E2A) that potentially bind around –384 were observed in the TRANSFAC search, we performed a supershift assay for Ikaros and E2A. However, neither the Ikaros Ab nor the E2A Ab produced a supershift in EMSA (data not shown).



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  		FIGURE 1. Gel shift assay for SNP EOT–576 and EOT-384 using nuclear extract from A549. One million per milliliter of A549 cells were cultured for 24 h and then stimulated with or without 10 ng/ml TNF-{alpha} for 4 h. The nuclear extracts of A549 were incubated with the 32P-labeled probe, of which sequences include SNPs (see Materials and Methods). To identify specific binding complexes, nuclear extract from TNF-untreated A549 cells was preincubated with unlabeled probe before addition of labeled probe for competition (labeled lane as +Comp). The filled arrow indicates the specific DNA protein. This figure is representative of three positive results among six different experiments.

 
Effect of SNP (EOT–384A>G) on eotaxin1 mRNA expression in PBMC

To investigate how the allelic polymorphism of EOT–384A>G influence eotaxin1 mRNA expression, the levels of eotaxin1 mRNA were assessed by Northern blot analysis of the PBMCs from six asthmatics with each homozygote type (EOT-384A>G AA or EOT-384A>G GG). The level of mRNA expression was increased 4.3-fold by TNF-{alpha} treatment of PBMCs with the common allele homozygote, which was significantly higher than the 1.5-fold increase seen for the PBMCs with the minor allele homozygote (p < 0.05; Fig. 2).



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  		FIGURE 2. Association of SNP –384A>G with the expression of eotaxin1 mRNA. a, Northern blot analysis for eotaxin1 mRNA expression (upper panel) and GAPDH (lower panel) by PBMCs of asthmatics who carried the common allele homozygote (–384A>G AA) and minor allele homozygote (–384A>G GG). For inducing eotaxin1 expression, five million PBMCs treated with 10 ng/ml TNF-{alpha}. The result is representative of six different experiments in each group. b, Mean increased ratio of intensity of eotaxin1 mRNA after stimulation with TNF-{alpha}. The band intensity of eotaxin1 mRNA was measured by densitometer and normalized by GAPDH. (*, p < 0.05, n = 6).

 
Comparisons of the production rates and functional activities of the EOT+67G (alanine) and EOT+67A(threonine) eotaxins

Since the SNP on exon-1 (EOT+67G>A) causes a single amino acid change from alanine (EOT+67G>A G) to threonine(EOT+67G>A A) at the signal peptide cleavage site, the effects of EOT+67A>G on eotaxin1 production and secretion were investigated. The fluorescence intensities of the COS7 cells that were transfected with each type of eotaxin1-EGFP fusion construct were analyzed by flow cytometry and confocal microscopy. When COS7 cells were transfected with eotaxin1-GPF fusion constructs, the intracellular level of Thr-type eotaxin1 protein was apparently higher than that of Ala-type eotaxin1 protein as analyzed by confocal microscope and flow cytometry (Fig. 3a). Then we repeated this type of transfection experiment and measured both intracellular and secreted levels of each eotaxin variant by ELISA (Fig. 3b). Our results showed that the secreted levels of the two variants were comparable to each other in all time points tested, whereas the intracellular level of Thr-type eotaxin1 tended to be higher than that of Ala type, especially at later time points, yet without statistical significance (p > 0.05). However, the difference in the intracellular levels between the two variants was <0.7 pg ofeotaxin/µg of total protein at 48 h posttransfection, which seemed to be negligible as compared with the secreted levels (~12 pg of eotaxin/µg of total protein) of both variants. Hence the sum (14.61 ± 1.19) of intracellular and secreted amount of Thr type was similar to that (13.18 ± 1.20) of Ala type. Thus, although the intracellular level of Thr type of eotaxin 1 was higher than that of Ala type, the difference was not statistically significant and the influence of the difference on total amount of each variant produced was most likely marginal.



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  		FIGURE 3. The comparison of production and function of +67A and +67G eotaxin1. a, The flow cytometric analysis and confocal microscopic findings of COS7 cell line at 24 h after transfection with each type of eotaxin1. b, The time-course of the secreted eotaxin1 in cultured media by each type of eotaxin1 transfectant was measured by ELISA (n = 5, *, p < 0.05). c, The chemotactic activities of commercial eotaxin1 and Ala- and Thr-type eotaxin1 toward human CCR3-transfected 4DE4 cell line. Chemotaxis assays were performed using 48-well Boyden’s chamber (n = 5).

 
Since the N terminus of eotaxin1 is known to be critical for CC chemokine function (33, 34), we measured the functional activities of each type of secreted eotaxin1. Although culture medium that contained 100 ng/ml eotaxin1 was used for the assay, comparisons of the Ala-type and Thr-type eotaxins showed no differences in the levels of chemotactic activity to the human CCR3-transfected 4DE4 cell lines (Fig. 3c) and in shape changes of the peripheral blood eosinophils (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sequence variations in the promoter and coding region of the eotaxin1 gene are expected to alter the levels of mRNA or protein expression and/or the function of the protein, which are implicated in the expression of the intermediate phenotypes of asthma. In addition, substitution of alanine (EOT+67G>A G) with threonine (EOT+67G>A A) may lead to changes in the intracellular transport of eotaxin1 or the functional activity of eosinophils (19, 35, 36). The major aim of the study was to elucidate the association between SNPs and plasma eotaxin1 levels to confirm the functional effects of eotaxin1 gene polymorphisms on eotaxin1 synthesis. In this study, we demonstrated that SNPs in the promoter (EOT–576T>C and EOT–384A>G) correlated significantly with the plasma eotaxin1 levels in the asthmatics (Table IV). Haplotype-based analysis provides additional information on the associations between genotype and phenotype (37). In our study, the effect of each of the two haplotypes on eotaxin1 levels appeared to be significant (Table IV). The plasma eotaxin1 levels were dependent on the number of haplotypes of ht1. However, the effect of the ht4 haplotype showed the inverse pattern to that of the ht1 haplotype. These data suggested that analysis of the haplotypes might allow the definition of high and low plasma eotaxin1 groups. The implication of different levels of plasma eotaxin was investigated to correlate with the intermediate phenotypes of asthma. The eotaxin1 levels correlated with the total IgE levels in the asthmatics and the normal controls and with the PC20 methacholine values in the asthmatics (Table II). This data presents the evidence that eotaxin1 may have a role of IgE synthesis (30) and in the severity of asthma (8, 9, 10, 12). We also found that the EOT–384A>G locus on the promoter of eotaxin1, but not the other loci, provides the binding site for a NF. This result is in good agreement with the association analysis of SNPs with plasma eotaxin1 levels (Table IV). The association of EOT–576C>T with plasma eotaxin level might be result of strong linkage (complete linkage disequilibrium, D' = 1 and r2 = 0.77) with EOT–384A>G (17) when considering there was no transcriptional factor binding to EOT-576C>T.

To confirm that the EOT–384A>G allele is a determinant for eotaxin1 gene transcription, the mRNA expression levels in PBMC carrying polymorphic alleles at the EOT–384A>G loci were compared after stimulation with TNF-{alpha} which has been known to potent stimulator for eotaxin mRNA and protein production from eosinophils (38) as well as epithelial cells (26, 39). Asthmatics having the homozygotic wild alleles (–384A>G AA) showed significantly higher mRNA expression after stimulation with TNF-{alpha} than those having the minor allele homozygote (–384A>G GG) (Fig. 2). These results suggest that the factor bound to this locus may be a negative regulator of eotaxin1 gene expression. Since the candidate factors were Ikaros family and E2A, we performed a supershift assay for these factors. However, neither the Ikaros nor the E2A Abs showed supershifts in EMSA (data not shown). Therefore, the nature of the putative DNA-binding factor remains to be clarified.

The binding affinity of this DNA-binding factor was higher for –384G than for –384A with or without TNF-{alpha} stimulation (Fig. 1). In the steady state, this DNA-binding factor may bind constitutively to the promoter close to the –384A>G locus and repress eotaxin1 gene expression. Moreover, TNF-{alpha}-induced signaling may cause the release of this binding factor from the promoter, may reduce the amount of this factor, or may recruit different binding factor(s) from unstimulated state, which would allow eotaxin1 gene expression. In the case of guanine presence on –384, TNF-{alpha}-induced signaling may cause lower-level binding of this factor than –384A, which would inhibit eotaxin1 gene expression. Since –384A>G is located upstream of the NF-{kappa}B/STAT6-binding site, the factor that binds to the –384A>G locus may affect the transcription machinery for eotaxin1 gene expression, rather than directly regulating the expression of eotaxin1. Recently, it has been reported that pretreatment with IFN-{gamma} inhibits TNF-{alpha}-induced eotaxin1 expression (40); this inhibition is independent of IFN-{gamma}-responsive element activation and does not alter the binding of NF-{kappa}B. Furthermore, the inhibitory effect of IFN-{gamma} on TNF-{alpha}-induced eotaxin1 expression is dependent on the presence of the promoter region between –92 and –478. By all accounts, the promoter region adjacent to –384 provides the binding site for an unknown transcriptional repressor, the binding of which might be increased by IFN-{gamma} stimulation.

In this study, we did not observe any association between EOT-488C>T (allele of absolute linkage with EOT+67G>A) and serum eotaxin1 concentrations. This result is in contrast with that of Nakamura and coworkers (19), who reported that the minor allele homozygote of EOT+67G>A A* was associated with lower levels of plasma eotaxin1 than the wild allele homozygote. However, the allele frequency of EOT+67G>A was not different in asthmatics and normal controls. The absence of an association between the polymorphic alleles of +67G>A and asthma development corroborates the present result and our previous report (17).

Variation in the first exon of the eotaxin1 gene has been described in human dermal fibroblasts (32). The nonsynonymous SNP of EOT+67G>A substitutes the wild-type alanine (ALA) for a threonine residue (Thr) at the 23rd amino acid at the terminus of the leader peptide. The predicted site of the variant eotaxin1 is at the 24th amino acid (glycine), which possibly results in delayed trafficking and deterioration of function. Nakamura and coworkers (19) suggested that this variant Thr23 is associated with decreased secretion of eotaxin1 from PBMC, perhaps due to inefficient trafficking of the eotaxin1 protein (19). However, time-dependent changes in eotaxin1 protein secretion were not analyzed thoroughly in their study. In this study, we demonstrated that the substitution of the wild-type alanine with the variant-type threonine did not significantly alter the production and release of eotaxin1 until 48 h posttransfection. This suggests that the total amount of eotaxin1 released from the cells is similar for the wild-type and variant-type eotaxins. In terms of eotaxin1 function, both types of eotaxin1 showed equal abilities to induce chemotaxis of CCR3-transfected cell lines and to cause shape changes of the peripheral blood eosinophils (data not shown). These in vitro results support the in vivo finding in the present study; no difference in plasma eotaxin1 levels between subjects who carry the Ala- or Thr-type eotaxin1.

In our study, neither the eotaxin1 gene SNPs nor the plasma eotaxin1 levels showed significant associations with the peripheral blood eosinophil counts. It has been reported that this variant (+67A) is associated with both decreased eosinophil counts and higher lung function levels in subjects with asthma (19) and with total IgE in cases of atopic dermatitis (20). Despite the potent biological effect of eotaxin1 on eosinophils in vivo and in vitro, our data suggest that other factors, such as sequence variations in the multiple genes for eosinophil-activating cytokines and chemokines, which include eotaxin-2 and -3, may exert effects on the phenotypes of asthma, such as increased levels of blood eosinophils and IgE synthesis.

In conclusion, SNPs in the promoter (EOT–576T>C and EOT–384A>G) and different haplotypes (ht1 and ht4) are significantly associated with plasma eotaxin1 levels in a gene-dosage-dependent manner in asthmatics. This finding was confirmed by the presence of a DNA-binding factor differentially binding on the promoter region around EOT–384A>G and by differential mRNA expression patterns in PBMC that carried polymorphic alleles of EOT–384A>G. The plasma eotaxin1 levels were correlated with the parameters of the intermediate phenotype of asthma, such as serum total IgE and PC20 methacholine. These data suggest that analysis of nucleotide variance of EOT–384A>G may be used to predict plasma eotaxin1 levels and intermediate asthma phenotypes.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ3-PG6-01GN04-0003). Back

2 Address correspondence and reprint requests to Dr. Choon-Sik Park, Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, 1174 Jung-Dong, Wonmi-Ku, Bucheon, Gyeonggi-Do 420-020, Korea. E-mail address: mdcspark{at}unitel.co.kr; or Dr. Il Yup Chung, Division of Molecular and Life Sciences, Hanyang University, 1271 Sa-1 dong, Ansan, Gyeonggi-do 426-791, Korea. E-mail address: iychu{at}hanyang.ac.kr Back

3 Abbreviations used in this paper: SNP, single-nucleotide polymorphism; FEV1, forced expiratory volume in 1 s; PC20, a 20% fall in FEV1. Back

Received for publication June 15, 2004. Accepted for publication November 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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