INTRODUCTION

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Chronic obstructive pulmonary disease (COPD), which includes chronic pulmonary emphysema (CPE) and chronic bronchitis (1), is basically a benign disease, but the prognosis is so poor that its mortality rate is similar to that of some malignant disease. It is generally accepted that cigarette smoking is the most important risk factor for COPD. Nevertheless, only 10-15% of smokers develop the severe impairment of pulmonary function associated with COPD (2); moreover, the factors that determine the susceptibility to cigarette smoking and disease progression are poorly understood. Among those factors, the genetic contribution to the disease has been implicated (3). Although the nature of the genetic influences remains undefined, factors that regulate the inflammatory responses to direct exposure to inhaled insults are important in the pathogenesis of COPD (4).

Tumor necrosis factor  (TNF-) is a multifunctional cytokine. TNF- could promote tracheal smooth muscle proliferation (5) and alter smooth muscle function (6). The level of TNF- is elevated in bronchoalveolar lavage fluid (7), bronchial biopsies (8), and induced sputum (9) of patients with COPD. These studies suggest that TNF- may contribute to the airway remodeling and alter smooth muscle cell function in COPD.

A polymorphism at position 308 of the TNF- gene promoter has been described. This is a guanine (G)-to-adenine (A) substitution in the TNF- gene at position 308 in the promoter region (the G allele was denoted as 1, and the A allele as 2). TNF--308*2, the rarer allele, has been associated with higher baseline and induced expression of TNF- (10). Several studies have demonstrated a positive association with the presence of the TNF--308*2 allele in a number of inflammatory diseases, such as asthma (11) and sarcoidosis (14). A prior study showed an association between the TNF--308*2 allele and the risk of development of chronic bronchitis in a male Taiwanese population (15). However, one study showed no association between the TNF--308*2 allele and the severity of smoking-related COPD in a white population (16). The differences in the association between COPD in Asian and white populations may not be explained by differences in TNF--308*2 allele frequency between the populations. There seems to be an ethnic difference in the prevalence, but the lack of association in a white population may not be explained on this basis. In this study, the patient group was restricted only to those with smoking-related COPD to exclude other factors related to diffuse panbronchiolitis that are observed mainly in Japanese and Southeast Asians. The purpose of the present study was to determine whether the TNF--308*1/2 polymorphism was associated with the development of smoking-related COPD.

	METHODS

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Subjects

The patient group consisted of 106 patients with smoking-related COPD recruited from the Respiratory Outpatient Department at Chiba University Hospital and affiliated hospitals (Chiba, Japan). COPD was diagnosed on the basis of past history, physical examination, and spirometric data, according to American Thoracic Society guidelines (1). Pulmonary function tests were performed to determine forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV₁), using a standard spirometer (Fudac-60; Fukuda Denshi, Tokyo, Japan), in a standing position. Chronic airflow obstruction was defined as (1) FEV₁/FVC less than 70% and (2) FEV₁ less than 80% of predicted values. Predicted values were calculated according to the Japanese Thoracic Society statement on standardized lung function testing. Subjects were excluded if they had a history of productive cough for 3 mo in each of two successive years (chronic bronchitis).

Two control groups were used in the study: (1) a smoker control group (n = 110) that included asymptomatic smokers and ex-smokers matched for sex and age with a smoking history of at least 10 pack-years but without COPD and asthma. The control group included subjects who visited the same hospital for a health checkup. They had normal pulmonary function (FEV₁/FVC > 70% and FEV₁ > 80% of predicted values); (2) a population control group (n = 129) of adult Japanese blood donors from Chiba Prefecture, Japan. The study was approved by the Research Ethics Committee of Chiba University School of Medicine, and all subjects gave their informed consent in writing.

Detection of the TNF- Polymorphism

The base composition at position 308 of the TNF- gene was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Genomic DNA was obtained from blood lymphocytes, using a QIAamp DNA blood minikit (Qiagen, Valencia, CA). The 5' region of the TNF- gene (positions 331 to 14) was amplified, and the PCR conditions were similar to those already described (17), with some modifications (15): the 5' primer was 5'-AGGCAATAGGTTTTGAGGGCCAT and the 3'-primer was 5'-GAGCGTCTGCTGGCTGGGTG. PCR conditions: genomic DNA was amplified primers (0.2 µM each), dNTPs (100 µM each), 10 mM Tris, 1.5 mM MgCl₂, 50 mM KCl, and 0.1% Triton X-100. Cycling: 94° C for 1 min, 60° C for 1 min, and 72° C for 1 min for 30 cycles followed by 60° C for 1 min and 72° C for 5 min. The PCR product was ethanol precipitated and digested with NcoI (Nippon Gene, Tokyo, Japan) and analyzed on a 3% NuSieve agarose gel (FMC BioProducts, Rockland, ME). DNA products were visualized by ethidium bromide staining. The TNF1 allele would be digested into two fragments (325 and 20 bp, respectively), while the TNF2 allele would not be digested (345 bp).

Statistical Analysis

The difference in allele distribution and allele frequency among the groups was examined for statistical significance by the ² test for independence, and with the Fisher exact test when appropriate. Age, smoking index expressed as pack-years, and pulmonary function parameters were compared with the Mann-Whitney U test.

	RESULTS

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Seventy-three men and 33 women were recruited into the COPD patient group. The smoker control group consisted of 66 men and 44 women. The population control group was composed of 129 blood donors, aged 22-72 yr. The smoking history of the population control subjects was unknown. Age, smoking history, and pulmonary function data of patients with COPD and smoker control subjects are summarized in Table 1. No significant differences were observed, in age or smoking history, between patients and control groups.

The results of the comparison of the base composition at position 308 of the TNF- gene are summarized in Table 2. The distribution of the genotype was significantly different between patients and smoker control groups, as well as between patients and population control groups. ² analysis showed that the patient group had a significantly higher TNF--308*2 frequency than either the smoker control group or the population control group (p < 0.01). The presence of the TNF--308*2 allele (including both homozygous and heterozygous subjects) was associated with an increased risk for COPD (odds ratio = 2.58; 95% confidence interval = 1.28-5.23) compared with TNF--308*1-homozygous subjects.

	DISCUSSION

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In the present study, we compared the frequency of a polymorphism in the promoter region of the TNF- gene (designated TNF--308*1/2) in Japanese patients with smoking-related COPD and asymptomatic smoker/ex-smoker control subjects matched for age and sex. The results indicate that the TNF--308*2 allele frequency is higher in patients with COPD compared with control subjects, suggesting an association of TNF--308*1/2 alleles with the presence of COPD. It is possible that the TNF2 allele, which is a G-to-A polymorphism at position 308 in the TNF- promoter, increases expression of TNF-, and this may explain these associations. Alternatively, the polymorphism could be in linkage disequilibrium with other causal mutations.

The TNF--308*2 rare allele is associated with higher baseline and inducible TNF- expression in transcription studies in vitro (10), thus, various authors have investigated the possibility of an association between the TNF--308*2 allele and disease manifestation. TNF- is a potent modulator of immune and inflammatory responses and has been implicated in a variety of autoimmune diseases, including asthma. However, a positive association with the TNF--308*2 allele and asthma was not found in all studies (11). These inconsistent results suggest that the association of the TNF--308 polymorphism with the inflammation underlying asthma may not be expressed through the influence of the polymorphism on TNF secretion. The TNF- gene is located within the major histocompatibility complex (MHC) Class III region on chromosome 6p21.3. This is a highly polymorphic region, and some of these polymorphisms form extended haplotypes with the HLA Class I and II alleles. Thus, the TNF--308 polymorphism could be in linkage disequilibrium with HLA variants associated with different inflammatory responses. For example, the TNF--308*2 allele is strongly associated with the HLA-A1, -B8, and -DR3 alleles (18).

The etiology of COPD could be attributable to both environmental and genetic factors. Like asthma, it is probable that COPD is a polygenic disorder with considerable genetic heterogeneity. Among the many gene products involved in the airway inflammation and remodeling, TNF- has been tested as a candidate gene for COPD, because it is a potent proinflammatory cytokine that is found in increased concentration in bronchoalveolar lavage fluid (7) and induced sputum (9). The data from this study suggest that the TNF--308*2 allele is a risk factor for the development of COPD. The result is unlikely to be confounded by population stratification given the homogeneity of the Japanese population. However, there are alternative interpretations of these results. The TNF--308*2 allele may not affect susceptibility to COPD but could be in linkage disequilibrium with the true susceptibility allele in a closely linked gene, because multiple genes may be involved in the development of COPD. In addition, there are no obvious mechanisms for the involvement of TNF- in the pathogenesis of COPD. There may be another functional gene in disequilibrium with the TNF--308*2 allele that could be responsible for an increased risk of COPD.

It is generally accepted that one of the significant risk factors for COPD is male sex. Female sex hormones, such as estrogen and progesterone, may play a protective role against insults that injure lung tissues. It was demonstrated that TNF- production by peripheral blood lymphocytes varied considerably in premenopausal females, while it was more constant in men and postmenopausal females, suggesting that it may be regulated by female sex hormones (19). However, the TNF--308*2 allele was not more frequently detected in male patients with COPD than in female patients in the present study.

The odds ratio of patients with COPD among TNF--308*2 allele carriers is lower than that of patients with chronic bronchitis in the Taiwanese population (15). The frequency of the TNF--308*2 allele seems to be much lower in the Taiwanese population than in the Japanese population. The low allele frequency in the Taiwanese population may account for the higher odds ratio of TNF--308*2 allele carriers among patients with chronic bronchitis compared with that among Japanese patients with COPD.

There is the fact that two studies of Asian subjects have been positive but that studies of white subjects have been negative. The one possible cause may be an ethnic difference affecting prevalence. The TNF--308*2 allele frequency ranged from 8% (20) to 27% (18) in the white population and from 5% (15) to 14% (21) in the Asian population. Another possible cause may be that the allele could be in linkage disequilibrium with a specific HLA haplotype that increases susceptibility to COPD in smokers only in the Asian population.

In conclusion, we found that the TNF--308*¹/₂ polymorphism may play a role in the susceptibility to smoking-related COPD, although the determinants between smokers susceptible to the development of COPD and those resistant to it cannot be explained by a single gene polymorphism.