RESULTS

A. Identification of three genetic polymorphism in CysLTR1 gene

We investigated a genetic variation in 5’ upstream region of CysLTR1 gene in 43 normal healthy controls (NCs) by direct sequencing and found 3 SNPs in CysLTR1 gene promoter. The transcription start site of the human CysLTR1 transcript was determined by 5’-RACE and three polymorphic sites: C-634T, A-475C, A-336G were identified from the transcriptional start site.

Next, we investigated genetic association study of three genetic polymorphisms of CysLTR1 in three groups of study subjects classified as AIA (n=105), ATA (n=111), and NCs (n=125) by direct sequencing, in which the 2466bp promoter region of CysLTR1 gene was included. Nucleotide position on (+1) is transcription start site determined by 5’-RACE and the sequence is shown in Fig. 2. A graphic overview of 3 SNPs identified in relation to the exon / intron structure of human CysLTR1 gene is shown in Fig. 3A. The 3 SNPs were in strong linkage disequilibrium (LD) with each other and the linkage disequilibrium coefficients (1D’1) and r² among the SNPs were calculatedfor each SNP (Fig. 3B).

B. Genetic association study of CysLTR1 genetic polymorphism

1. Clinical characteristics of the study subjects

One hundred and five ASA-intolerant asthmatics, 111 ASA-tolerant asthmatics and 125 normal healthy control subjects were genotyped at the three CysLTR1 SNPs,

1 CATTTGGGAA TGGGTGAATC ACTGCCTTCC CCATTGCTCA AGAACAGAAA ATTAGCTAAT 61 GAAAGAAACA GGTAGGGGAG CACAAAGAAG AACCACAGGA AACACCCTCT CTGCCACAGC 121 TATTTCGGTG CCAACTTGAA GCTCAAGGAT TGAAGGGGGC CTGCCCCTCC ACGCCTGTGG 181 GTATTTCTCA TCAGGTGGTA CGAGAGACTG AGAAAAGAAA TGACACAGAG ACAAAGTATA 241 GAGAAAGAAC AGTGGGCCCA GGGGACCGGC AGCCTCAGCA TGTGAGGACC TGCACCAGCG 301 CTGGTCTCTG AGTTCCCTCA GTATTTATTG ATCATTATTT TTACTATCTT GGCAAGGGGA 361 GTGTAGCAGA GCAACAGGTG GGGAGAAGGT CAGCAGGAAA ACGTGAGCAA AGGATCTGTA 421 TCATGCATAA ATTCAAGGAA AGGTACTGTG CCTGGATGTG CATGTAGGCC AGATTTATGT 481 TTCACTTTAT ACAAACACCT CAGTGTAGCA AAGAGTAACA GAGCAGTATT TCTGCCAGCA 541 TATCTCGCCT CTAGCCACAG GGCGGTTTTA TCCTATCTCA GAATAGAAAG AATGGGAATG 601 GTTGGCTTTA CAGGAGACAT TCCATTCCCA GGGAGGAGCA GGAGACAGAA GCCTTCCTCT 661 TATCTCAACT GCAAAGAGAC CTCCCTCTTT CACTACTCCT CCTCAGCAGA GACCCTTTAC 721 GGGTGTTGTG CTGGGGTATG GTATCAAAGC AGAAACAATT TTTCCTGGTA CAGATCAAAA 781 TGGAATTTCT TGTGTCTTCC TTTTCTACAT AGACACAGTA ACAATCTGAT CTCTCTTATC 841 CCCACAAAGG ATCTAGCTCC TTTCTCTTTT CTTCATTTCC TGAGTATGAA TTGCCCTTCC 901 ACTGTAGCTC AAAATGGCTG TAACATTTCA GACTGGAGGA AAAGTGATAA TTCACTCTGT 961 GTCTCACTTA TAAATTTTCC TGTAGATATT TGGGGATCCA AACTCAGTTG AAGAAATACT 1021 AGTTTGAATG TCAGTTTAGT TACTAAAACT CCCTAGGAGA GGATTAGAAA GTCTAGTCTT 1081 GAATCCAAAT GTTAACATCC CTTCAAATGT GGTTGGATAG CCTTTTCCTC CAACTATCAC 1141 CATCACCACC ACAATCCAGA ACTCTGTGAA AAGAGATAAA GATTCAAGGG CCTTAAAGCG 1201 ATTGTCCCAG GTTGGTTGTG AGGTAATTCT TGTACCTAAA GATACAAAAT TTGAGTAAAT 1261 GACTTTGTTC CTACAGCAGG TAGCAAGCCA GAAGGCTACT GCTGGGCTCA TATTTTACAA 1321 GTACCTAAAA CTAGTTGTTA GTTATGGGGC ATGTTTACAA AGTGTTAACA TCAACATATT 1381 TATGCCCATC TCCATATAGA ATCTTCTTAA TATAACTCAA GATAATTGAT GTAACAAATT 1441 CACATCATGA TCTTACCAAT TTGGTTTTTA ACATCAAAGT GCTGCCCCAG GCTTCAATCA 1501 GCACATACCA GACTGCTATG TCCCTTTTCT CCAGAAGCCA TATTGAGCAC CCCACAGCTC (* C-634 T)

1561 TAACACCTCA GAGCCTAGCC AAAGGCCAAG AACACTTGCC TGATCCCCCA AATTTCCAAC 1621 ACAGGACCAT TGACAGCAAG CCCAGTCATT CCAGATCATC TTCAGTGGGA CAAAAAAAGA 1681 AAACAACAGT TTTAATCCAA TGGAGGCAAT TTATTGTTAT GAAGATTTCA GGAAACAAAG (* A-475C)

1741 CTCAAAAAGG AACCAGAAAG GAAAAGGCTT TTTAGAATGT GGATAGAGCC ACAAGTGTCA 1801 TTAAGGAGAG AGAGAGAGAA ACGGAGAGAT GAAATTTATG TTACTTAAGA TCAAGTTTCA (* A-336 G)

1861 CATAATGCCA GTTATATTAG CATATACTGG CAGGATTATC TTTCCCCACC TACAAATAGT 1921 CTAATGACCT CACAGTCACA GAAATCACAG AGAACTAAGC TGAAGAGAGA ACACTCGTCC 1981 CTGCTTCCCA TCTTAGAGCA GCTGAATAAT TTCCTGAGAA TTCTATTCCT GAAGCTAGGA 2041 AGAAAAGTTT ATTTATACAT ACACGCAACC TGCAAGTCTC CAGTTTCTAT TCTTCCTTCC 2101 TCTTTGACCC TTCCCCTCCC CCACTTTGCA CCAGAGAAGT CAGACTCCGG GAGTGCTTTA (5’ RACE/*+1) 2161 ACAGTTTGAA GGCTAATCTG AAAGAGGAAG AAGAATCTGT ATATCTGTAT ATATTGGCTA ( EXON I)

2221 GCAAATGTGC CCTGCTCTCT CCCCTCTTAA AAATAGCAGC AACCCATCTT TGCAAAGAAG 2281 CTTGCCTATA GAGCAGGCAC TCTGTGAATG GACTGTGCTT TTACGACCCT ACAGGGTATC 2341 AAGATACTGT GCAGCTCGCC AACAAGGATT AATTGCAAGG ACTGGTAGAT CGAATTTACT 2401 GAAGACTTGG AGCTTGCTTC TGAGAACAAA CGCAAAAGGA CAGTAAACTG TGGACCTTGA 2461 AGTTAGCAGCG

Fig. 2. 5’-RACE sequence and 3 SNPs location on the 2466bp fragment of CysLTR1gene. Transcription start site as denoted as +1 found from the 5’-RACE and presented in bold letter and under line (_). Sequences of three genetic variants (C-634T, A-475C, and A-336G) in promoter region were represented as aterisk (*).

A

B

r2 lD’l ^{C-634T A-475C A-336G}

C-634T 1 1

A-475C 0.9 1

A-336G 1 0.9

Fig. 3. Gene map and linkage disequilibrium (LD) coefficients in cysteinyl leukotrienes receptor 1 (CysLTR1). A. Gene map and 3 SNPs in CysLTR1 on chromosome Xq13-q21. Coding exons are marked by black blocks and 5’-and 3’- untranslated regions by white blocks. Transcription start site was denoted as nucleotide +1. (B), Linkage disequilibrium coefficient (lD’l) of CysLTR1 SNPs.

Exon 1 2 3 4 5

+1+1

+1+1 (5(5(5(5’ RACE) RACE) RACE) RACE)

***

C-634T, A-475C, and A-336G. The clinical characteristics of the study subjects are summarized in Table 1. There were no significant differences in the FEV1 % predicted, total IgE level, asthma duration and airway hyperresponsiveness to PC₂₀ methacholine between the AIA and ATA groups (p=0.37, 0.20, 0.09, 0.22, respectively). There were no significant difference in mean age, prevalence of sex, atopy status among three groups (p>0.05). Between the AIA and ATA groups, there were significant differences in percent in fall of FEV₁ during lysine-aspirin bronchoprovocation test (p<0.001). There were significant difference in the prevalence of nasal polyps between AIA and ATA, this results suggest that AIA patient (48%) were found to have significantly higher frequencies of nasal polyps than ATA patients (6.7%, p=0.003).

As the CysLTR1 gene is on the X chromosome, the distribution of the CysLTR1 promoter genotypes within male subjects deviated from Hardy-Weinberg equilibrium (p<0.01). All analyses were stratified by sex.

2. Genotype distribution of CysLTR1 3 SNPs

Next, we investigated a genetic association study of three genetic polymorphisms CysLTR1 in three groups of study subjects classified as AIA (n=105), ATA (n=111), and NCs (n=125). Allele and genotype frequencies of the 3 SNPs within the phenotypic groups are described in Table 2. Allele frequencies (q) were determined for each polymorphism. Male AIA patients were found to

have significantly higher frequencies of the minor alleles (T, C,G) of the CysLTR1 Table 1. Clinical characteristics of the study subjects

P value

AIA: ASA-intolerant asthma; NC: normal controls; ATA: ASA-tolerant asthma. N: number of patients;

NA: not applicable; NS: not significant; M: male. Data are expressed as the mean ±SD

The methacholine provocative concentration producing a 20% fall in forced expiratory volume in 1 second (PC20-FEV1) was calculated as an index of bronchial hyperresponsiveness (BHR).

promoter SNPs than male control subjects (p=0.031 for AIA vs. NC; p=0.022 for AIA vs. ATA). In contrast, there were no significant differences in allele and genotype frequencies among the three groups within female subjects (p>0.05).

Haplotypes of CysLTR1 gene were constructed by Bayesian algorithm with genotyped SNPs (Table 2) and 2 common haplotypes (frequency > 5%) were analyzed. Among males, AIA patients had a lower frequency of major haplotype, ht1 [C-A-A] (p=0.031, OR=0.36, 95%CI: 0.14~0.90 for AIA vs. NC; p=0.022, OR=0.34, 95%CI: 0.14~0.85 for AIA vs. ATA), and higher frequency of minor haplotype, ht2 [T-C-G] (p=0.031, OR=2.42, 95%CI: 0.98~5.98 in AIA vs. NC; p=0.031, (OR=2.57, 95%CI: 1.04~6.31 in AIA vs. ATA), than those in male control groups. Significant differences in the frequency of ht1 and ht2 were not found in female subjects. In addition, there were no significant differences in the haplotype distributions in female subjects by three alternative analysis models (co-dominant, dominant and recessive models).

3. Association analysis of AIA-associated quantitative phenotypes

Initially, AIA related phenotypes such as atopy, serum total IgE level, initial baseline FEV1 % predicted value and PC20 methacholine were evaluated for any association with the CysLTR1 C-634T SNP which is a tagging SNP of the 3 SNPs (Table 3).

In female AIA patients, with mutant genotypes of CysLTR1 promoter polymorphisms showed higher total IgE levels (p=0.003), while no significant associations were found in male subjects. No significant associations were found between CysLTR1 polymorphisms and other phenotypes such as airway

Table 2. Genotype frequencies of the three SNPs in CysLTR1 promoter

*Each P value was calculated with co-dominant, dominant and recessive models. q: minor allele frequency; AIA: ASA-intolerant asthma; NC: normal controls; ATA: ASA-tolerant asthma; N:

number of patients; NS: not significant. Values in bold indicate significant p value.

* C-634T is a tagging SNP of the three SNPs in CysLTR1 promoter. Significant associations (P<0.01) are printed in bold. P values are genotype-specific means ± SD.

Table 3. Association of CysLTR1 C-634T with atopy, total IgE, asthma duration, PC₂₀ methacholine, FEV₁ and fall of FEV₁.

Gender Genotype Age Atopy

Log IgE (IU/ml)

Asthma duration (year)

PC20

methacholine (mg/ml)

FEV1 (%)

Fall of FEV₁ (%)

Male C 43.16 ± 12.9 11(57.9%) 2.26 ± 0.63 8.4 ± 6.1 4.56 ± 8.78 84. 85 ± 25.1 20.3 ± 10.4 T 36.1 ± 12.2 11(55.0%) 2.34 ± 0.57 5.5 ± 4.2 8.81 ± 15.3 76.79 ± 23.8 21.5 ± 13.0

p 0.087 0.86 0.670 0.095 0.268 0.309 0.769

Female CC + CT 47.7± 12.9 22(40.7%) 2.08 ± 0.47 6.5 ± 6.1 4.01 ± 7.05 86.7 ± 20.2 23.2 ± 11.7 TT 42.6 ± 13.7 9(75%) 2.56 ± 0.55 7.2 ± 5.6 3.76 ± 4.83 77.3 ± 28.5 19.6 ± 7.1

p 0.226 0.031 0.003 0.716 0.929 0.182 0.438

hyperresponsiveness to metahcholine and pulmonary function in both sexes.

4. Effect of 3 SNPs on transcriptional activity

To explore the functional significance of the CysLTR1 promoter polymorphisms at the molecular level, promoter fragments containing all 2466bp CysLTR1 promoter haplotypes ht1 [C-A-A] and ht2 [T-C-G] were cloned into 5’

of the luciferase reporter gene within pGL3 basic vector. To perform this experiment, we screened several cell lines such as A549 cells, Hela cells, HL-60 cells, HUVEC, THP-1 cells, HEK-293T cells, U937 cells, Jurkat cells, and COS-7 cells by RT-PCR. Among them, endogenous expression of CysLTR1 was detected in HL-60 cells, Jurkat cells, U937cells, and A549 cells (Fig. 4). We performed transfection using lipofectaime into HL-60 and THP-1 cells and transfection efficiency of both cells was too low and therefore, we conducted transfection experiments in Jurkat cells, U937 cells, and A549 cells. Fig. 5A, 5B, and 5C summarized the data for the transient transfection and luciferase analysis in Jurkat cells, U939 cells, and A549 cells. The reporter activities were compared between two constructs containing either ht1 [C-A-A] or ht2 [T-C-G] at -634 bp, -475 bp, and -336 bp in the CysLTR1 promoter region (Fig. 5A, B and C). Luciferase activity was enhanced with the construct with minor haplotype, ht2 [T-C-G] (150%

increases) compared to the construct with major haplotype ht1 [C-A-A].

5. Identification of unknown nuclear protein binding by EMSA

To investigate whether the putative transcription factor binding sites on 3

Fig. 4. Expression of CysLTR1 mRNA in various cell lines. Four micrograms of total RNA was subjected to RT-PCR with the primers for CysLTR1. PCR products were separated on 1% agarose gel and stained with ethidium bromide.

A: A549 cells; B: Hela cells; C: HL-60; D: HUVEC cells; E: THP-1 cells; F:

Fig. 5. Promoter activity assay of human CysLTR1 promoter constructs. Data are mean values of independent mean ± SEM. (A) Transfection into Jurkat cell.

Luciferase activity assay was performed nine times (in total, n=27). (B) Transfection into U937 cell (n=9) and (C) transfection into A549 cells (Human lung carcinoma,

SNPs regions, we searched transcription factor binding site (TFSEARCH, Searching Transcription Factor Binding Sites ver. 1.3.) and signal scan database (www. bimas.cit.nig.gov/molbio/signal), but this region has not putative transcription factor binding sites.

We found that a shifted band was noted on- 475A, -336A, and -336G of CysLTR1 promoter in nuclear extracts of IL-4 primed THP-1 cells (10ng/ml for 30min) and PMA (100ng/ml for 4h) stimulated U937 cells (Fig. 6A. and 6B.). To investigate whether the genetic variants create a transcription factor binding site, each SNPs containing double strands oligonucleotide probe( C-634T, 475C, A-336G) was synthesized and labeled with [α-³²P]CTP as probe and incubated with each nuclear extracts of IL-4 primed THP-1 cells (10ng/ml for 30min) and PMA (100ng/ml for 4h) stimulated U937 cells. PMA stimulated U937 nuclear extract were represented in the formation of three specific protein-DNA complexes and contained nuclear proteins binding specifically to -475A and -336A and -336G region of CysLTR1 promoter. IL-4 stimulated THP-1 nuclear extracts revealed at least two major complexes. The formation of the band was inhibited completely by adding 50X excess of unlabeled - 475A oligonucleotide.

C. Characterization of CysLTR1 promoter in A549 cells

The finding of CysLTR1 expression in the A549 cells suggests that this cell line is a useful model system for studying the transcriptional regulation of human CysLTR1 gene. Various human CysLTR1 promoter-luciferase constructs were

1 2 3 -475A 50X -475C 50X NF-kB 50X

1 2

-634C 50X -634T 50X NF-kB 50X -336A 50X -336G 50X NF-kB 50X -475A 50X -475C 50X NF-kB 50X

A B

Fig. 6. Unknown factor bind to a -475A, -336A, and -336G region in the CysLTR1 promoter. (A), EMSA with each nuclear extracts prepared from unstimulated line (1, 6, 11) and PMA (100ng/ml for 4hr) stimulated line (2-5, 7-10, and 12-15) of U937 cell lines. EMSA revealed at least three specific protein-DNA complexes. An unlabeled oligo with -475A and -336A, and -336G competed for bands 2 but not band 1 and 3. (B), EMSA with nuclear extracts prepared from IL-4 stimulated (10ng/ml for 30min) THP-1 cell lines and the revealed two protein-DNA complexes. The formation of the bands 1 was specific inhibited completely by -475A unlabeled oligo. The Lane 1, 6, 11 were represented probe only.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

probe -634C -475A -336A - 634C -475A -336A

Competitor

-475A 50X -475C 50X NF-kB 50X -336A 50X -336G 50X NF-kB 50X

-634C 50X -634T 50X NF-kB 50X

transfected into the A549 cells to determine their promoter activities. The construct B567 (–256/+313)-Luc was found to have highest promoter activity in the transfected cells. The construct B981 (–670/+313)-Luc was found to have the second promoter activity and further 3′ deletion of this construct [to generate the B808 (–670/ +215)-Luc] could significantly abolish the promoter activity (Fig.

7A.), suggesting that the region within +215/+313 contains important transcriptional elememt(s) for the promoter function. IL-4 stimulation significantly increased luciferase activity in all tested construct (Fig.7B).

D. Transcriptional regulation of CysLTR1 gene by IL-4

1. Expression of CysLTR1

To investigate whether IL-4-increases at mRNA and protein levels of CysLTR1 gene, analysis of CysLTR1 mRNA expression was measured by real-time RT-PCR, and CysLTR1 cell surface expression was determined by flow cytometric analysis. IL-4 stimulation increased CysLTR1 mRNA in a time-dependent fashion, with the highest increase was observed after 24 h of incubation (Fig. 8A). IL-4 stimulation caused a increased in surface receptor expression, and maximum response was observed at 24 h of incubation (Fig. 8B).

2. Effect of genetic polymorphism on the transcription regulation by IL-4

CysLTs promoted generation of Th2 cytokines such as IL-4, IL-5, IL-13 and

Fig. 7. Deletion analysis of the human CysLTR1 promoter. (A), A549 cells were transfected by lipofectamin with five CysLTR1 promoter luciferase based reporter constructs as well as promoter- less vector (pGL3 basic), and allowed to recovery for 48hr. (B), a exposed to IL-4 or vehicle for 6h. Analysis of reporter gene expression using luminometry. Data are the mean ± SEM of six experiments; asterisk indicates (**) p<0.001and (*) p<0.01 versus promoterless vector (pGL3 basic vector). Numbers underneath refer to nucleotide up stream of the transcription start site.

0 mRNA, A549 cells were stimulated with IL-4 (10 ng/ml) for up to 24 h and CysLTR1 mRNA expression was measured by realtime RT-PCR analysis. Results are normalized to internal control (β-actin expression) and presented as fold increase from control values (treated cells vs. vehicle). The means SD of three different samples are shown.

(B), Flow cytometric analysis of CysLTR1 surface expression. A549 cells were stimulated with IL-4 (10 ng/ml) for 18 h and labeled with anti-CysLTR1 antibody or with isotype-matched control. Results of a single experiment, representative of at least three are presented. Dotted line represents isotype control antibody. Solid thick and thin lines represent labeling of medium- and IL-4-treated cells, respectively.

those cytokines also enhanced generation of CysLTs in macrophage, eosinophils, and monocytes by inducing the expression of CysLTR1 (Peters-Golden and Sampson, 2003; Thivierge and Pleszczynski, 2001; Thivierge and Rola-Pleszczynski, 2000; Chibana and Fukuda, 2003). We investigated whether IL-4 possess effect on transcriptional induction of CysLTR1 promoter, classified according to major haplotype, ht1 [C-A-A], and minor haplotype, ht2 [T-C-G].

There were no significant changes observed in the promoter activities in major haplotype, ht1 [C-A-A] by IL-4. In contrast, minor allele ht2 [T-C-G] had significantly higher promoter activity than untreated cells (p<0.001, Fig. 9.). We next determined to the effects of Th2 cytokine (IL-4), dexamethasone, and CysLTR1 specific antagonist (MK-571) on the CysLTR1 promoter activity in A549 cells.

E. Effect of leukotriene modifier on the transcription regulation by IL-4

Corticosteroids represent one of the most widely used and the most effective anti-inflammatory treatment of immune and anti-inflammatory diseases, including persistent asthma of all degree severity (Boumpas, 1993). Their mechanism of action involves regulation of gene transcription and RNA stability. CysLTs have been identified as an important brochoconstrictor in asthma, particularly in AIA and CysLTR1 selective antagonists could block bronchoconstriction induced by lysine aspirin in some patient with AIA (Dahlen B et al, 1993). To investigate whether MK-571 and dexamethasone possess an inhibitory effect on transcriptional induction of CysLTR1 induced by IL-4, we compared transcription activities of

Fig. 9. Effect of dexamethasone and MK-571 on CysLTR1 promoter activity. Luciferase reporter activities were in A549 cells transfected with CysLTR1 promoter construct (B2400). After total transfection time of 48h, the cells were incubated with various concentrations of dexamethasone and MK-571 for 24hr. The results are presented as the mean ± SD; n=9.

Drug dose 0.0

5.0 10.0 15.0 20.0 25.0 30.0

RLU

Dex (M): - 10^-9 10^-8 10^-7 10^-6 - - - - MK-571 (M): - - - - - 10^-9 10^-8 10^-7 10^-6

WT [C-A-A]

MT [T-C-G]

CysLTR1 promoter gene between major allele ht1 [C-A-A] and minor allele ht2 [T-C-G]. Compare to untreated cells, maximal inhibitions was observed at 10^-8M of dexamethasone and 10^-7M of MK-571 (Fig. 9). We next determined the effects of Th2 cytokine (IL-4), dexamethasone, and CysLTR1 specific antagonist (MK-571) on the CysLTR1 promoter activity in A549 cells. Significant increase of promoter activity was noted in mutant allele ht2 [T-C-G] (p=0.008), not in major allele ht1 [C-A-A], which was inhibited by 10^-7M of MK-571 pretreatments (p=0.004, Fig. 10).

However, dexamethasone pretreatments increased promoter activity in both mutant allele ht2 [T-C-G] and major allele ht1 [C-A-A] (p<0.001).

F. Effect of dexamethasone on CysLTR1 mRNA expression in A549 cells

To investigate whether to assess the effect of dexamethasone on CysLTR1 expression in A549 cells, cells were incubated with fresh medium in presence or absence of various dexamathasone from 10^-9M to10^-6M (Fig. 11). Treatment of A549 cells with various concentration dexamethasone increased CysLTR1 mRNA expression compared to untreated cells. Maximum enhancement was observed between 10^-6M and 10^-5M dexamethasone.

DEX (10^-8M) - + - - + -

IL-4 significantly increased CysLTR1 promoter activity in mutant genotype (p=0.008), which was significantly inhibited by the pretreatment of MK-571 in mutant genotype (p=0.004). In contrast, dexamethasone pretreatment increased promoter activity in both mutant and wild genotype (p< 0.001). ** P<0.001and

*P<0.01.

**

**

Fig. 11. Realtime PCR analysis effects on CysLTR1 mRNA with addition of dexamethasone in A549 cells.A549 cells were further incubated with fresh medium in the presence or absence of dexamethasone (10^-5M~10^-9M) for 6hr. RNA was extracted and realtime-PCR was performed as described methods.

A: A549 cell only; B: A549 cells + IL-4 (10ng/ml, 18hr); C: dexamethasone (10^-9M);

D: dexamethasone (10^-8M); E: dexamethasone (10^-7M); F: dexamethasone (10^-6M);

G: dexamethasone (10^-5M)

IV. DISCUSSION

CysLTs such as LTC₄, LTD₄, and LTE₄ are important proinflammatory lipid mediators in the development of asthma by mediating bronchoconstriction as well as increasing mucus secretion, vascular permeability, and cellular infiltration and airway hyperresponsiveness (Lewis and Soberman, 1990; Henderson WR, 1994).

CysLTs exert their biological action by binding to types of GPCRs such as CysLTR1 and CysLTR2 (Hui Y and Funk, 2002). Because CysLTR1 mRNA is abundantly expressed in inflammatory leukocytes such as eosinophils, monocytes and macrophages, it is thought that CysLTR1 may have an important role in these cells, main effectors in asthma, particularly in AIA. Although the pathogenesis of aspirin intolerance has not understood yet, overproduction of CysLTs and CysLT receptors may play major roles in pathogenesis mechanism of AIA.

In the present study, we are the first to demonstrate allele polymorphisms of CysLTR1 promoter gene in AIA patients. The 3 novel SNPs that we discovered (C-634T, A-475C, and A-336G) were associated with AIA risk in males; males with AIA had significantly higher frequencies of minor alleles (T-C-G) of three SNPs than male control subjects. Moreover, the two common 3-SNP haplotypes were associated with AIA in males. The ht1 [C-A-A] haplotype was associated with decreased disease risk and the ht2 [T-CG] haplotype with increased disease risk. Within the AIA group, the male patients were younger than female patients.

We therefore can speculate that male AIA patients carrying ht2 [T-C-G] may be at greater risk of developing AIA at an earlier age. In female subjects, there were no

significant associations of CysLTR1 genotype or 3-SNP haplotype with AIA risk.

These results suggest that the male patient with mutant allele of CysLTR1 has a higher risk genetic factor in AIA development. In fact, this result was consistent with other previous report that the male subjects showed higher level of CysLTR1 than female subjects in corectral adenocarcinoma (Ohd and Sjolander, 2003).

These results suggest that the observed differences in association between males and females may be related to allele-dependent dysregulation of the CysLTR1 gene in AIA patients.

In females, the CysLTR1 C-634T polymorphism was associated with AIA-related quantitative phenotype, total serum IgE level. Where no association was found in case of males. AIA was not through allergic mechanism but through a pharmacological mechanism, there has been no evidence of an IgE response to aspirin. (Cowburn et al, 1998; Lam and Frank Austen, 2000; Pierzchalska and Szczeklik 2003). In addition, CysLTs promoted generation of Th2 cytokines such as IL-4, IL-5, IL-13 and those cytokines also enhanced generation of CysLTs in marchrophages, eosinophils, and monocytes by inducing the expression of CysLTR1 (Thivierge and Pleszczynski, 2000; Thivierge and Rola-Pleszczynski, 2001; Chibana and Fukuda, 2003; Peters-Golden and Sampson, 2003;). IL-4 may also promote tissue eosinophilia by VCAM-1 (Schleimer et al, 1992), and it is an essential cofactor for IgE swiching in B lymphocyte (Del Prete, et al, 1988). There were extensive interactions between CysLTs and other mediators such as macrophage inflammatory protein (MIP)-1β (Frieri et al., 1999),

TNF-α (Elizabeth AM et al., 2002), NF-kB, and RANTES (Kawano T et al., 2003), which are relevant to asthmatic inflammation via a CysLTR1 mediated mechanism.

These previous reports show that the expression of CysLTR1 mRNA can be increased by Th2 inflammatory cytokines, implying a positive feedback by endogenously produced CysLTs. The role of CysLTR1 in total IgE production has a lack of supporting biological data. However, it may be possible that Th2 cytokine-rich environment can up-regulate the expression of CysLTR1 and therefore production of IgE in human with the C-634T polymorphism in CysLTR1.

To investigate the association of the promoter 3 SNPs with CysLTR1 expression, we performed in vitro functional studies using Jurkat cells, U937 cells,

To investigate the association of the promoter 3 SNPs with CysLTR1 expression, we performed in vitro functional studies using Jurkat cells, U937 cells,