Introduction

Asthma is a common and heterogeneous respiratory disease characterized by reversible airways obstruction caused by chronic inflammation of the airways. Bronchial hyperresponsiveness is a characteristic feature of asthma, and serum IgE levels are closely associated with asthma development. The development of asthma is determined by the interaction between host genetic susceptibility and a variety of environmental exposures (Ahmadi and Goldstein 2002; Burrows et al. 1989; Kim et al. 1999; Koh et al. 2000). Asthma is recognized as a T-helper type 2 (Th2) disease with a particular profile of cytokine release, including IL4 and IL5. However, increasing evidence indicates that other cytokines, which were classically considered to belong to Th1-type profiles, are also associated with the inflammatory response that characterizes human asthma (Chung and Barnes 1999). Cytokines play an important role in the coordination and persistence of the inflammatory process in chronic inflammation of the airways in asthma and many other diseases. Chronic and acute inflammatory changes observed in the asthmatic airway could result from excessive release of many types of cytokines (Broide et al. 1992; Chung and Barnes 1999; Robinson et al. 1992).

Several interleukins have been reported to be involved in asthma through the pathological and immunological pathways as lymphokines, proinflammatory cytokines, and anti-inflammatory cytokines (Bagley et al. 1997; Chouchane et al. 1999; Chung and Barnes 1999; Karjalainen et al. 2003a; Renauld 2001). The IL1 gene complex is involved in the regulation of IgE-mediated atopic reactions and eosinophil accumulation in vivo (Chung and Barnes 1999). IL2 enhances the production of GM-CSF in peripheral blood mononuclear cells from asthmatics and the production of IL5 from T cells in hypereosinophilic syndrome patients as a potent chemoattractant for eosinophil (Enokihara et al. 1989; Nakamura et al. 1993; Rand et al. 1991). IL3, together with IL5 and GM-CSF, are important modulators of eosinophilia and eosinophil function. The differentiation, migration, and pathological effects of eosinophils may occur through the effects of GM-CSF, IL3, and IL5. IL8 possesses chemotactic activity for primed eosinophil and induces accumulation of eosinophils (Shute et al. 1997; Warringa et al. 1991). IL4 and IL10 play an important role in modulating total serum IgE. IL4 has been shown to play a crucial role in the pathogenesis of allergic disease including bronchial asthma, and IL4 increases airway responsiveness by recruiting eosinophils into the airway in patients with allergic bronchial asthma. In addition, IL10 inhibits eosinophil survival and IL4 induces IgE synthesis (Jeannin et al. 1998; Shi et al. 1998). These functions of the two genes are deeply associated with their polymorphisms.

In the current research, we examined several genetic polymorphisms in important cytokine and receptor genes (IL1A, IL1B, IL2, IL3, IL4, IL8, IL10, and IL5RA) using data from a Korean asthma study that was established recently.

Materials and methods

Subjects

Subjects were recruited from the asthma genome research center that consists of four tertiary hospitals in Korea (Soonchunhyang University Hospital, Ajuo University Hospital, Choong-Ang University Hospital, and Ulsan University Hospital). Ethical approvals were obtained from the institutional review board of each hospital. All patients had met the definition of asthma by the American Thoracic Society (1987). Normal subjects were recruited from spouses of the patients and from the general population who answered negatively to a screening questionnaire for respiratory symptoms and had FEV1 greater than 75% predicted, PC20 methacholine greater than 10 mg/ml, and normal findings on a simple chest radiogram. Total IgE and specific IgE to Dermatophagoides farinae (Df) and D. pteronyssinus (Dp) were measured by the CAP system (Pharmacia Diagnostics, Sweden). Twenty-four common inhalant allergens, including dust mites (D. farinae and D. pteronyssinus), car fur, dog fur, cockroaches, grass, tree pollens, and ragweed, were used for the skin-prick test. Atopy was defined as having wheal reaction by allergen equal to or greater than that by histamine (1 mg/ml) or 3 mm in diameter and/or positive response of specific IgE to Dp and Df. Clinical parameters are summarized in Table 1.

Table 1 Clinical profile of study subjects
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Genotyping by single base extension (SBE) and electrophoresis

Primer extension reactions were performed with SNaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA, USA) (Table 2). To clean up the primer extension reaction, one unit of SAP was added to the reaction mixture, and the mixture was incubated at 37°C for 1 h, followed by 15 min at 72°C for enzyme inactivation. The DNA samples, containing extension products and Genescan 120 Liz size standard solutions, were added to Hi-Di formamide (Applied Biosystems) according to the recommendation of the manufacturer. The mixture was incubated at 95°C for 5 min followed by 5 min on ice, and electrophoresis was performed by ABI Prism 3100 Genetic Analyzer. The results were analyzed using the program of ABI Prism GeneScan and Genotyper (Applied Biosystems).

Table 2 Sequences of amplifying and extension primers for genotyping of polymorphisms of the IL1A, IL1B, IL2, IL3, IL4, IL5RA, IL8, and IL10 genes by single-base extension method
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Statistical analysis

We examined Lewontin’s D′ (|D′|) and LD coefficient r2 between all pairs of biallelic loci (Hedrick and Kumar 2001; Hedrick 1987). Haplotypes and their frequencies were inferred using the algorithm developed by Stephens et al. (2001). Logistic regression models were used for calculating odds ratios (95% confidential interval) and corresponding P values for single nucleotide polymorphism (SNP) sites and haplotypes controlling for age and gender as covariates.

Results

Nineteen SNPs in cytokine and cytokine receptor genes, including IL1A, IL1B, IL2, IL3, IL4, IL8, IL10, and IL5RA, were examined in a Korean asthma study (n=723). The allele frequencies in asthmatic and normal control subjects are shown in Table 5. Linkage disequilibria among SNPs in each gene were measured by calculating Lewontin’s D′ and r2 values (Table 3). The SNPs in absolute LD (|D′|=1 and r2=1): IL1A−1203 and IL1A−889 [=IL1A+4845 (Arg135Ser)], IL3−68 [=IL3+79 (Ser27Pro)], and IL10−819 (=IL10−592) were not used for further analysis (Table 3). Haplotypes in each gene were constructed as follows: three in IL1A, three in IL1B, three in IL2, two in IL3, four in IL4, four in IL8, and four in IL10 (Table 4). Haplotypes that had frequencies less than 3% and/or were almost equivalent to single SNPs were excluded in statistical analysis to avoid redundant statistical tests.

Table 3 Linkage disequilibrium coefficients (|D′| and r2) between SNP loci in the IL1A, IL1B, IL2, IL3, IL4, IL8, and IL10 genes in the Korean population (n=723)
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Table 4 Frequencies of haplotypesa constructed in the IL1A, IL1B, IL2, IL3, IL4, IL8, and IL10 genes in the Korean population (n=723)
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Genotype distribution of each SNP and common haplotypes of eight genes were compared (1) between asthma patients and the normal controls, and (2) between atopic and nonatopic subjects using logistic regression models controlling for age, gender, and smoking as covariates (Table 5). Among 19 polymorphisms tested in eight genes, one exonic (exon 1 in IL3) polymorphism that changes the amino acid Serine to Proline (IL3+79T>C; Ser27Pro) revealed significant association with the risk of asthma, whereas all other polymorphisms showed no noticeable difference in allele frequency. The positive associations of IL10 (IL10−592A>C) and IL4 (IL4−589T>C) polymorphisms with the severity or risk of asthma reported in Taiwanese (Hang et al. 2003), Caucasians (Hobbs et al. 1998), and Japanese (Noguchi et al. 2001, 1998) populations were not detected in the Korean population in this study (Table 5). The minor allele (Pro) of IL3+79T>C (Ser27Pro) showed a lower frequency in the asthmatic patient than in the normal control group (0.46 versus 0.54, P=0.01), and this association was even clearer in the nonatopic population (0.41 versus 0.62, P=0.0003).

Table 5 Minor allele frequencies of polymorphisms in the IL1A, IL1B, IL2, IL3, IL4, IL5RA, IL8, and IL10 genes, and distribution between bronchial asthma (BA) and normal control (NC) subjects according to atopic status in the Korean population
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The genetic influences of IL3 Ser27Pro (T>C) were further analyzed in four alternative models (reference, codominant, dominant, and recessive models) and subgroup analyses according to atopic status and diagnosis of subjects (Table 6). The reference analysis revealed that the protective genetic effects of Pro might be similar between heterozygotes and homozygotes (OR=0.80 versus 0.70 in all subjects and OR=0.34 versus 0.37 in nonatopic subjects), although much stronger associations were apparent in homozygotes than heterozygotes (P=0.40 versus 0.01 in all subjects and P=0.02 versus 0.0001 in nonatopic subjects). When considering referent analysis results, the effects of the Pro allele in the nonatopic group might be dominant, although no significant associations were found in the atopic group. The lack of a positive signal in the atopic group, as well as the stronger (P=0.02–0.0001) and more protective (OR=0.25–0.41) effects in the nonatopic subgroup, suggest that the association signal found in all subjects likely comes from the nonatopic subjects. Association analyses of the Pro allele with atopy were also performed. Although no positive signals were detected in all subjects and asthmatic patients, significant associations with atopy were apparent in normal controls.

Table 6 Association analysis of IL3+79T>C (Ser27Pro) with a risk of bronchial asthma and atopy development
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In conclusion, it could be suggested that the Pro allele of the IL3 Ser>Pro locus had a dominant and protective genetic effect on the risk of asthma development in nonatopic subjects rather than atopic subjects. It was also protective against atopy in normal controls rather than in asthmatic patients.

Discussion

Asthma is a polygenic and common respiratory disease involving genetic and environmental factors. Identification of genetic polymorphisms, which are involved in the development of asthma, might be clinically useful for both identifying patients at risk and preventing the occurrence of the disease (Townley et al. 1986). Cytokines are extracellular-signaling proteins. They act on target cells to cause a wide array of cellular functions including activation, proliferation, immunomodulation, and release of other cytokines or mediators. Several researchers have reported the functions of cytokines in asthma as well as association with their polymorphisms with asthma (Chouchane et al. 1999; Chung and Barnes 1999; Hobbs et al. 1998; Walley and Cookson 1996; Zhang et al. 2002).

It has been suggested that one or more genes on chromosome 5q31-q33, including IL3, IL4, IL5, IL9, IL13, and GM-CSF gene cluster, might cause susceptibility to asthma, and their polymorphisms may be associated with the risk of asthma (Postma et al. 1995). The IL3 lies on chromosome 5q31.1, together with GM-CSF, IL4, IL5, IL9, and macrophage colony-stimulating factor. IL3 is mainly produced from activated T cells and mast cells (Arai et al. 1990; Fung et al. 1984). An increase in number of cells expressing IL3 mRNA has been reported in patients with asthma (Robinson et al. 1992), and IL3 caused an increase in the number of mast cells and eosinophils around the airways (Du et al. 1999). Also, eosinophil has been associated with asthma and allergic diseases since increased numbers of cells are present in blood and airway samples, and these numbers can be related to disease severity (Bousquet et al. 1990; Gleich 1990).

The rolls of IL3 in asthma have not been relatively studied as yet. However, the increase in eosinophils in mild and allergen-induced asthma patients occur independent of IL3 (Du et al. 1999; Woolley et al. 1996). Because of the ability to accumulate eosinophils, IL3 has been implicated in chronic inflammatory conditions mediated by eosinophils, of which allergic inflammation such as asthma is one of the most studied. In bronchial asthma, eosinophils have long been recognized to play a central role as judged by their accumulation in the bronchoalveolar lavage fluid and presence in the sputum of eosinophil cationic proteins believed to contribute to local tissue damage (Owen et al. 1987). Moreover, IL3 has been linked to several other pathologic features of asthma including subepithelial fibrosis, mucus hyper-secretion, and eotaxin production (Zhu et al. 1999). Despite the potential importance of IL3 in the pathogenesis of asthma and allergic pathogenesis, the genetic influence of IL3 gene polymorphisms on asthma has not yet been reported.

In this study, we demonstrated that IL3+79T>C (Ser27Pro), which is in absolute LD with one promoter SNP (IL3−68T>C), was significantly associated with nonatopic bronchial asthma and also with atopy in the nonasthmatic population. Although asthma is a multifactorial condition, the strongest risk factor in the etiology of asthma is atopy. The atopic individuals have significantly greater probability of developing asthma, and people with a family history of atopic disease(s) are at greatest risk of asthma. The association of the IL3 polymorphism with the risk of asthma in nonatopic subjects in this study might present clues of different genetic etiology and pathogenesis of intrinsic from extrinsic asthma. Similarly, genetic association with the risk of atopy in normal controls might also provide the different genetic background of atopy other than asthma. Recently, one positive correlation between IL3 polymorphisms and rheumatoid arthritis (RA) has been reported (Yamada et al. 2001). Because no functional studies were performed on promoter polymorphisms (IL3−68T>C) and/or missense polymorphism (IL3+79T>C [Ser27Pro]), which were in absolute LD, it is not clear which site caused the phenotypic difference. However, the decreased risk of asthma and atopy might possibly come from IL3+79T>C (Ser27Pro) because of several plausible reasons, e.g., (1) amino acid substitution to proline could cause the conformational change of N-terminal subdomain and consequent alteration of function, (2) there was no putative transcription factor-binding motifs on and adjacent to IL3−68T>C (TFSEARCH Searching Transcription Factor Binding Sites V1.3. [http://molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html], putative score >0.85). Further study would be needed to elucidate the functions of the variants more extensively.

Although the sources of discrepancies among current study and previous reports—including the positive associations of IL10 (IL10−592A>C) and IL4 (IL4−589T>C) polymorphisms with the severity or risk of asthma reported in Taiwanese (Hang et al. 2003), Caucasians (Hobbs et al. 1998), and Japanese (Noguchi et al. 2001, 1998) populations were not detected in the Korean population in this study (Table 5)—might be hard to clarify, several factors could provide plausible explanations, e.g., (1) possible genetic difference among different ethnic groups, (2) small size of this study [especially normal controls (n=171)], and (3) different study designs and possible biases in recruiting case and controls among studies.

In summary, we have examined the genetic association of several interleukins, including IL1A and IL1B (proinflammatory cytokines), IL2, IL3, IL4 (lymphokines), IL8 (chemokines), IL10 (anti-inflammatory cytokines), and IL5RA, with the risk of asthma and atopy. Genetic association study revealed that the ser allele of IL3+79T>C (Ser27Pro) locus had a dominant and protective genetic effect on the risk of asthma development in nonatopic subjects rather than atopic subjects. It also was protective to development of atopy in normal controls compared to asthmatic patients. The results of this study could be helpful in understanding the important function of IL3 in asthma and atopy development and also could be a useful target for drug development to treat asthma and/or atopy.