In total, 373 bakery workers who participated in a questionnaire survey regarding work-related respiratory symptoms were enrolled from a single industrial site. The questions regarding respiratory symptoms included whether subjects had experienced upper respiratory symptoms such as nasal itching, runny nose, sneezing, or congestion, as well as lower respiratory symptoms, such as cough, sputum, shortness of breath or wheezing. The subjects who indicated that their symptoms were aggravated during the work, but improved after the work or during holidays were defined as having work-related respiratory symptoms. All bakery workers were given a skin prick test (SPT) that included common inhalant allergens (Bencard, Bretford, UK) and wheat flour allergen extract (4). Atopy was defined for subjects with more than one positive response to common inhalant allergens on the SPT. The total IgE concentration was measured using a UniCAP system (Pharmacia Diagnostics, Uppsala, Sweden) according to the manufacturer's instructions. The presence of a specific IgE, IgG1 and IgG4 antibodies to wheat flour was determined by enzyme-linked immunosorbent assay (ELISA), as described previously (4). Clinical demographies of all study subjects are shown in Table 1. All subjects were classified according to their dust exposure density as previously described (4) and characterized by prevalence of sensitization rate to wheat flour and inflammatory cytokines in Table 2.

Serum IL-18 level was measured in bakery workers using a Human IL-18 ELISA Kit (MBL, Nagoya, Japan), in accordance with the manufacturer's guidelines.

Genomic DNA was prepared from peripheral blood samples of all participants using a Puregene DNA purification kit (Qiagen, Germantown, MD, USA) according to the manufacturer's protocol. Genotyping of -607A/C (rs1946518) and -137G/C (rs187238) was performed using the SNaPshot assay according to the manufacturer's instructions (ABI PRISM SNaPshot Multiplex Kit; Applied Biosystems, Foster City, CA, USA). Genotyping of 8674C/G (rs5744247) was screened using the TaqMan fluorogenic 5' nuclease assay (Applied Biosystems). Nucelotide sequence information on primers used for SNP genotyping are described in Table 3. Linkage disequilibrium between all pairs of biallelic loci was measured using Lewontin's D'(|D'|) and r² in a linkage disequilibrium (LD) block (12).

The 752-bp fragment of the promoter of the human IL-18 gene, including -607A/C and -137G/C, was prepared by PCR amplification using each haplotype (ht1 [CG], ht2 [AG], and ht3 [AC]) human genomic DNA as templates using a pair of primers (forward primer, 5'-CTGAATTTTGGTATCCCTCTCCC-3'; reverse primer, 5'-GGAGACAATTCCTTGCTGACTG-3'). Each of the PCR products was gel purified using an agarose-gel purification kit (iNtRON, Daejeon, Korea) and ligated into the TOPO vector (Invitrogen Corporation, Carlsbad, CA, USA). These DNA plasmids were then cut with KpnI and XhoI restriction enzymes (Takara, Shuzo, Japan) and ligated into the KpnI-XhoI site of the pGL3-Basic Luciferase Reporter Vector (Promega, Madison, WI, USA) using T4 DNA ligase (Elpis Biotech, Daejeon, Korea). All constructs were verified by direct sequencing. DNA plasmids were prepared from these constructs using the Endo Free Plasmid Maxi Kit (Qiagen, Valencia, CA, USA) and then analyzed for quantity and integrity using UV spectrometry and agarosegel electrophoresis.

A549 (human alveolar basal epithelial cells, ATCC CRL-185) and BEAS-2B (human bronchial epithelial cell, ATCC CRL-9609) cells were maintained in RPMI 1640 (Gibco, Invitrogen Corporation) supplemented with 1% penicillin-streptomycin, 0.1 mM MEM non-essential amino acid (Gibco, Invitrogen Corporation) and 10% fetal bovine serum (FBS, Gibco, Invitrogen Corporation) in a 5% CO₂ incubator at 37℃. Cells were plated, and, after being grown to 70%-80% confluence, cells were transiently transfected by lipofectamine (Invitrogen Corporation) according to the manufacturer's protocol. Briefly, 1 × 10⁵ cells were seeded in 12-well plates and transfected with 1 µg of reporter construct, 5 ng of Renilla plasmid DNA, and 5 µL LipofectAMINE (Invitrogen Corporation). At 48 hr after transfection, the cells were lysed and assayed for firefly luciferase activity according to the manufacturer's instructions (Promega). Transfection and luciferase assays were repeated three times according to the method described above.

Genotype distributions at each polymorphism were evaluated for departures from Hardy-Weinberg equilibrium using the chi-squared goodness-of-fit test. Statistical analyses were performed using SPSS version 12 software (SPSS Inc., Chicago, IL, USA). Differences in clinical characteristics between groups were examined using Student's t tests and analysis of variance (for continuous variables) or the chi-squared test (for categorical variables). Risk factors were determined by logistic regression analysis after adjustment for age and gender covariates. Haplotypes of the IL-18 gene were analyzed using Haploview v4.0 (13).

The protocols used in this study were reviewed and approved by the Ajou University Institute Review Board. Written informed consent was obtained from each participant.

Table 1 shows the clinical demographics of study subjects. The mean age of the 373 workers was 35 ± 7.7 yr, and the mean working period in the bakery was 4 ± 3.5 yr. Atopy was detected in 34.6% of the subjects. Of the 373 workers, 51 (13.7%) complained of work-related lower respiratory symptoms, including cough, sputum, shortness of breath, or wheezing. The prevalence of wheat flour sensitization was 6.3% using the SPT and 6.4% by the presence of IgE specific to wheat flour. When the workers were categorized into three groups based on the extent of exposure to flour dust (low: 0.01 mg/m³, intermediate: 1.16 mg/m³, high: 3.04 mg/m³), in accordance with a previous study (4), serum IL-18 levels showed a significant rise with increased intensity of exposure to wheat flour (P < 0.05, data not shown).

When the workers were categorized into three groups based on the extent of exposure to flour dust (low; 0.01 mg/m³, Intermediate; 1.16 mg/m³, High; 3.04 mg/m³), in accordance with the previous study (4), the prevalence of work-related respiratory symptoms did not differ among workers with low, intermediate, and high exposure to flour dust. The prevalence of IgG specific to wheat flour increased with increasing intensity of exposure to wheat flour dust. In accordance with this finding, serum IL-18 level showed a significant increase (Table 2).

Three functional SNPs (-607A/C; rs1946518, -137G/C; rs187238, 8674C/G; rs5744247) were targeted in this study. The magnitude of LD between IL-18 SNPs, -137G/C and 8674C/G, showed a high value, with a pairwise LD value of D' = 1 and r² = 0.569. Among three SNPs of the IL-18 gene, the promoter polymorphisms at -137G/C showed a significant association with the rate of sensitization to wheat flour by the SPT (Table 4). The variant genotype (GC or CC) frequency of the -137G/C polymorphism was significantly higher in the positive SPT to wheat flour compared to that of the wild-type genotype (GG) (P = 0.002). Among the three haplotypes, haplotype 3, ht3 [ACC], showed a higher positive SPT to wheat flour (P = 0.005). The significance remained after multiple corrections. When we compared serum IL-18 levels according to IL-18 polymorphisms in bakery workers, -607C/A and haplotype 1, ht1 [CGC], showed an association with increased serum IL-18 levels.

For analysis of the functional variability of the two promoter SNPs (-607A/C and -137G/C), three plasmid constructs of the IL-18 promoter containing two SNPs, ht1 [CG], ht2 [AG], and ht3 [AC] were cloned into pGL3-Basic with a firefly luciferase gene reporter. The luciferase activities of ht1 [CG], which contains the -607 C allele, were significantly elevated compared to those of ht2 [AG] and ht3 [AC], which exhibit the -607A allele in two different airway epithelial cell lines (A549 and BEAS2B; Fig. 1). The variant genotype (AC or CC) of -607A/C also showed a significant association with serum IL-18 level; however, other SNPs (-137G/C; 8674C/G) were not associated (data not shown). These findings indicate that the -607 C allele may be important in transcriptional regulation of the IL-18 gene.