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(Chest. 2001;119:753-761.)
© 2001 American College of Chest Physicians

The Gene Polymorphism of Tumor Necrosis Factor-ß, But Not That of Tumor Necrosis Factor-{alpha}, Is Associated With the Prognosis of Sarcoidosis*

Etsuro Yamaguchi, MD; Akihide Itoh, MD; Nobuyuki Hizawa, MD and Yoshikazu Kawakami, MD, FCCP

* From the First Department of Medicine, School of Medicine, Hokkaido University.

Correspondence to: Etsuro Yamaguchi, MD, The First Department of Medicine, School of Medicine, Hokkaido University, Kita-15 Nishi-7, Kitaku, Sapporo 060-8638, Japan; e-mail: etsuro{at}med.hokudai.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objectives: Few genetic markers for the prognosis of sarcoidosis have been found. Tumor necrosis factor (TNF)-{alpha} has been implicated in the pathogenesis of sarcoidosis. Induced TNF-{alpha} or TNF-ß levels have been shown to be associated with the polymorphisms of the TNF genes. We investigated the roles of such polymorphisms in the development and prolongation of sarcoidosis.

Subjects and measurements: One hundred ten Japanese patients with sarcoidosis and 161 control subjects were genotyped for three biallelic polymorphisms in the promoter region of TNF-{alpha} gene by direct sequencing of polymerase chain reaction (PCR) products. A polymorphism of the TNF-ß gene (TNFB*1/TNFB*2) was detected by Nco I restriction fragment length polymorphism analysis of PCR products spanning intron 1 and exon 2 of the TNF-ß gene.

Results: None of the polymorphisms conferred susceptibility to sarcoidosis. However, our study identified the allele TNFB*1, detected by the presence of a Nco I restriction site, as a marker of prolonged clinical course, with the resolution of sarcoidosis being defined as the disappearance of all clinical symptoms, physical signs of active lesions, abnormal chest radiograph findings, and abnormal results of pulmonary function and biochemical tests. When the probability of remission in patients homozygous for TNFB*2 was defined as 1.00, it was 0.48 (95% confidence interval, 0.26 to 0.88; p < 0.05) in patients with TNFB*1 (genotypes TNFB*1/1 and TNFB*1/2).

Conclusions: The TNFB*1 allele is a marker for prolonged clinical course in patients with sarcoidosis. Our study is the first to link a cytokine gene polymorphism to the prognosis of sarcoidosis.

Key Words: gene polymorphism • sarcoidosis • tumor necrosis factor-{alpha} • tumor necrosis factor-ß


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sarcoidosis is a systemic granulomatous disease that affects a wide range of organs and has a highly variable prognosis. Most patients have a short favorable clinical course. However, some patients have a prolonged course and require systemic corticosteroid treatment. In their study on a cohort of Scandinavian patients, Berlin et al1 reported that human leukocyte antigen (HLA)-DR17 (one of the serologic specificities of DR3) was a marker for good prognosis. We have recently shown that the deletion allele of angiotensin-converting enzyme (ACE) gene is associated with prolonged clinical course.2 Other studies3 4 5 have established a correlation between the persistence of sarcoidosis and each of several clinical variables, including the absence of erythema nodosum, older age at onset, hyperglobulinemia, decreased FVC, increased proportion of HLA-DR+ T cells in the BAL fluid (BALF), and increased maximum levels of serum ACE during the disease course. Most of these markers are disease activity. Thus, only a few genetic markers of the prognosis for sarcoidosis have been detected.

It has been well established that cytokines play pivotal roles in the development of alveolitis and the formation of granulomas in sarcoidosis.6 7 8 9 10 Among such cytokines, tumor necrosis factor (TNF)-{alpha} has long been thought to play a crucial role in the formation and sustenance of granulomas.7 11 12 In fact, this cytokine has been targeted as a possible focus of granuloma treatment.13 Several polymorphisms of the TNF-{alpha} gene (TNFA) have been identified.14 15 16 Among them, allele 2 at nucleotide position - 308 has been associated with higher inducible levels of gene transcription and TNF-{alpha} protein production.17 18

TNF-ß (lymphotoxin-{alpha}) is a cytokine that orchestrates lymphoid neogenesis and the formation of germinal center reactions.19 20 In the first intron of the TNF-ß gene (TNFB), there is a Nco I polymorphism consisting of the allele TNFB*1 in the presence of the restriction site, and the allele TNFB*2 in its absence.21 TNFB*1 is the less frequent allele in white subjects and is associated with higher TNF-{alpha} and TNF-ß production.22 23

In order to investigate the possible roles of gene polymorphisms in the development and progression of sarcoidosis, we genotyped 110 patients with sarcoidosis and 161 control subjects for both TNFA and TNFB polymorphisms. Our results provide evidence that the TNFB Nco I polymorphism is a novel genetic marker for prognosis of sarcoidosis in Japanese patients.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
One hundred ten patients with sarcoidosis (36 men and 74 women) were included in the present study. All patients were Japanese and living in Hokkaido, Japan. The diagnosis of sarcoidosis was made when patients had biopsy evidence of noncaseating epithelioid cell granuloma in any organ, and when there were compatible clinical imaging of bilateral hilar and/or mediastinal lymph node enlargement with or without lung parenchymal infiltrates or eye or skin lesions without evidence of mycobacterial, fungal, or parasitic infection. None of the patients had a history of exposure to organic or inorganic materials known to cause lung diseases. Their mean age at the initial visit to Hokkaido University Hospital was 39.4 years (95% confidence interval [CI], 36.3 to 42.6 years). Chest radiographic staging at the time of diagnosis showed that 8 patients were in stage 0, 62 patients were in stage I, 30 patients were in stage II, and 10 patients were in stage III. The mean follow-up time of patients was 67.0 months (95% CI, 54.5 to 79.5 months). Four patients were administered systemic corticosteroids during the follow-up period. The research was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association, and the study was approved by the Ethics Committee of Hokkaido University School of Medicine. Written informed consent was obtained from each patient.

Healthy Volunteers
One hundred sixty-one healthy volunteers served as control subjects. They were all residents of Hokkaido and were selected from subjects who received annual health checkups. None had a history of lung disease or showed any symptoms of lung or other disease. All showed normal findings on chest radiography and laboratory examination, which included complete blood counts, urinalysis, and assays for hepatic enzyme activities and BUN levels.

Assessment of Clinical Outcome
The follow-up evaluation of disease activity included an inquiry into symptoms, such as malaise, fever, dyspnea, cough, sputum, wheezing, palpitation, blurring of vision, myodesopsia, arthralgia, pruritus, skin tenderness, and any other symptoms related to involvement of sarcoidosis; a physical examination for skin rash, joint swelling, superficial lymph node enlargement, hepatosplenomegaly, parotid gland swelling, and any other physical manifestations related to sarcoidosis; an ophthalmologic examination by a slit lamp and fundoscopy; plain chest radiographs; pulmonary function tests, including measuring vital capacity, FEV1/FVC, and diffusion capacity of the lung for carbon monoxide corrected for alveolar volume; an ECG; and measurement of hepatic enzyme activities, including alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, serum calcium level, and serum ACE activity. All these evaluations were repeated at 3-month intervals. When all the symptoms and physical and radiographic manifestations had disappeared, and results of pulmonary function, ECG, and biochemical tests were found to be within the normal range, cases were judged to be in remission and the length of time from the onset of sarcoidosis was recorded. Some patients did not show remission during the study. The length of time from the onset of sarcoidosis to the last follow-up month in such patients was recorded and used for analysis under the Cox proportional-hazards regression model.

Our criteria for remission may seem to be superficial, in that they do not include BAL, gallium scintigraphy, or CT scans. However, it is highly difficult to thoroughly assess the clinical activity of sarcoidosis. Because sarcoidosis is a systemic disease, complete evaluation of the presence or absence of active lesions might necessitate biopsy of all frequently affected organs, such as liver, lung, and lymph nodes, in addition to extensive diagnostic imaging. Such exhaustive assessment is not indicated for asymptomatic patients at outpatient clinics. We therefore performed basic work-ups that were both practical and cost-effective.

Determination of the Genotype of the TNF Gene
Genomic DNA was purified from blood leukocytes using an extraction kit (Sepagene; Wako; Tokyo, Japan). The three polymorphisms at - 308, - 244, and - 23814 15 16 in the 5'-region of the TNFA were determined by directly sequencing polymerase chain reaction (PCR) products. Briefly, a 218-base pair (bp) fragment spanning positions - 395 to - 180 of the TNF-{alpha} promoter sequences was amplified with a sense primer (5'-TCCTGCATCCTGTCTGGAAGTTAG) and an antisense primer (5'-GGAAAGTTGGGGACACACAAGC). PCR was carried out in a 20-µL reaction mixture containing 50 ng of genomic DNA, 10 pmol of each primer, 1 U of Taq polymerase (Ampli Taq Gold; PE Applied Biosystems; Foster City, CA), 0.2 mM each 2'-deoxyribonucleoside 5'-triphosphate, and 2 µL of 10 x PCR buffer (PE Applied Biosystems), 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, 500 mM KCl, 0.01% gelatin). The cycling condition consisted of an initial activation of Taq polymerase at 95°C for 10 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. The amplified PCR products were sequenced by the dideoxynucleotide chain termination method using a fluorescent sequencing kit (DNA Sequencing Kit; PE Applied Biosystems) and an automated sequencer (ABI PRISM 310; PE Applied Biosystems) according to the protocol of the manufacturer.

The Nco I polymorphism in intron 1 of the TNFB was determined by the PCR-restriction fragment length polymorphism method. A 289-bp fragment of genomic DNA including the polymorphic site was amplified with a sense primer (5'-TCTGACTCTCCATCTGTCAG) and an antisense primer (5'-AGACGTTCAGGTGGTGTCAT). PCR was carried out in a 20-µL volume containing 50 ng of genomic DNA, 10 pmol of each primer, 1.0 U of Taq DNA polymerase (Ampli Taq Gold; PE Applied Biosystems), 0.25 mM each 2'-deoxyribonucleoside 5'-triphosphate, 10 mM Tris-HCl, pH 8.3, 4.5 mM MgCl2, 50 mM KCl, and 0.001% gelatin. The cycling condition consisted of an initial activation of Taq polymerase at 95°C for 10 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 64°C for 1 min, and extension at 72°C for 1 min. The PCR products were digested with 10 U of Nco I (New England Biolabs; Beverly, NE) at 37°C for 6 h. Genotypes were determined by electrophoresis on 4% gel (NuSieve 3:1 agarose; FMC Bioproducts; Rockland, ME) and staining with ethidium bromide.

Statistical Analysis
Demographic data of patients having different TNF-ß gene polymorphisms were compared using a one-way analysis of variance. Differences in the frequencies of alleles and genotypes between patients and control subjects were tested by the {chi}2 test or, when appropriate, Fisher’s Exact Probability Test. Odds ratios were calculated by logistic regression analysis adjusted for age and sex. The Cox proportional-hazards regression model was used to compare the clinical courses of patients with sarcoidosis in regard to TNF gene polymorphisms and to calculate the relative probability of remission in patients with each genotype of the TNF genes. The genotype distribution according to the activity of sarcoidosis at several time points during the follow-up period was assessed by the {chi}2 test. Statistical analysis was performed using a statistical software package (SPSS version 7.5; SPSS; Chicago, IL). Differences with a p value of < 0.05 were considered significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We sequenced the promoter region spanning positions - 395 to - 180 of the TNFA in all study subjects (Fig 1 ). Very few subjects had variant alleles at either base position of this region (Table {ddagger} 1 ). In particular, we found no subjects with a variant allele at position - 244, whereas Nedwin et al15 reported an A instead of a G. Similarly, the frequencies of a variant allele at position - 308 (TNFA2) were lower than those previously reported for white subjects,14 but comparable to those observed for Japanese subjects.24 There were no significant biases in either genotype distribution or allele frequencies for the three TNFA polymorphisms between the control subjects and patients with sarcoidosis (Table 1) . Odds ratios were calculated by logistic regression analysis after adjusting for sex and age (Table 1) ; however, none were statistically significant.



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  		Figure 1.. Nucleotide sequences showing biallelic polymorphisms in the promoter region of TNFA. Top left, A: homozygous for - -308 (G/G); bottom left, B: heterozygous for - -308 (G/A); top right, C: homozygous for both - 244 and - 238 (G/G); bottom right, D: homozygous for - 244 but heterozygous for - 238 (G/A).

 

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  		Table 1.. Allele and Genotype Frequencies of TNF Gene Polymorphisms*

 
Three genotypes of the TNFB intron 1 polymorphism (TNFB*1/1, homozygous for TNFB*1; TNFB*1/2, heterozygous for TNFB*1 and TNFB*2; TNFB*2/2, homozygous for TNFB*2) were unambiguously identified after amplification of the 289-bp fragments spanning intron 1 and exon 2 and digestion with Nco I (Fig 2 ). The genotype and allele frequencies for the 161 Japanese control subjects were comparable to those observed for white and Japanese control subjects.25 26 27 There was no significant difference in either allele frequency or genotype distribution between patients and control subjects (Table 1) , nor was the age- and sex-adjusted odds ratio statistically significant. Thus, our study did not provide evidence that any TNF gene polymorphism confers susceptibility to sarcoidosis in Japanese subjects. We found no significant association between the TNFB polymorphism and any of the clinical features listed in Table 2 . None of the polymorphisms for TNFA and TNFB in normal control subjects exhibited significant deviation from Hardy-Weinberg expectations by the {chi}2 tests, demonstrating that they were randomly selected.



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  		Figure 2.. An electrophoretic image of the TNFB Nco I polymorphism.

 

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  		Table 2.. Characteristics of Patients With Sarcoidosis at the Initial Visit According to the TNFB Nco I Polymorphism*

 
We next examined the relationship between the genotypes and clinical course of sarcoidosis. The resolution of sarcoidosis was defined as the time point when all the clinical manifestations including symptoms, skin lesions, and ophthalmologic abnormalities other than sequelae of active lesions disappeared, and when results of chest radiography, pulmonary function tests, and biochemical tests were normal. The Cox proportional-hazards regression model was used by replacing a death event with remission of sarcoidosis, and inverting survival curves so that they represented the cumulative probability of remission for each genotype (Figs 3 4 5 ). Similarly, the hazard ratios in this model represented the probability of remission during the follow-up period in patients with a given genotype relative to that in patients with a reference genotype. Although no TNFA polymorphisms were associated with the clinical course of sarcoidosis partly because of a very low frequency of variant alleles (data not shown), the TNFB polymorphism significantly affected the cumulative probability of remission (Fig 3) . Patients with the TNFB*1 allele (genotype TNFB*1/1 or TNFB*1/2) had a more prolonged clinical course than those without the allele (genotype TNFB*2/2), with the probability of remission being 0.48 (95% CI, 0.26 to 0.88; p < 0.05). Thus, the possibility of remission in patients with the TNFB*1 allele was approximately half that in those with the genotype TNFB*2/2.



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  		Figure 3.. Cumulative probability of remission in all patients.

 


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  		Figure 4.. Cumulative probability of remission in patients not treated with systemic corticosteroids.

 


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  		Figure 5.. Cumulative probability of remission in patients followed up for > 60 months.

 
The use of systemic corticosteroids may be a strong confounding factor for the clinical course of sarcoidosis, as has been suggested.28 There were only four patients in our study who had received systemic corticosteroids during the follow-up period. This was principally because of the fact that few patients had severe symptoms or impairment of lung function even in cases with a prolonged course, and partly to the contention that corticosteroids may be deleterious for a subset of patients.28 When analysis was restricted to the subgroup of patients who had not received systemic corticosteroid, differences in the cumulative probability of remission according to the three genotypes became more evident (Fig 4) . Patients with either TNFB*1/1 or TNFB*1/2 individually had a significantly prolonged clinical course compared with those with TNFB*2/2. The probability of remission in patients with TNFB*1/1 or TNFB*1/2, relative to that in patients with TNFB*2/2, were 0.33 (95% CI, 0.11 to 0.97; p < 0.05) or 0.46 (95% CI, 0.23 to 0.91; p < 0.05), respectively.

The clinical course was monitored for > 24 months in 77 patients and for > 60 months in 40 patients. Even for those patient groups, the allele TNFB*1 was a significant prognostic factor (data not shown; Fig 5 ). The probability of remission in patients with the TNFB*1 allele (TNFB*1/1 or TNFB*1/2) who were followed for > 24 months or > 60 months, relative to those with TNFB*2/2, was 0.48 (95% CI, 0.26 to 0.89; p < 0.05) and 0.53 (95% CI, 0.28 to 0.98; p < 0.05), respectively.

The clinical course was also assessed by a cross-sectional analysis (Table 3 ). The {chi}2 test was used to evaluate genotype distribution according to the activity of sarcoidosis at several follow-up time points. The clinical activity was determined by the same criteria as that used for the Cox proportional-hazards model. The frequency of TNFB*1/1 or TNFB*1/2 in active cases was significantly increased compared with that of TNFB*2/2 at 12 months, 24 months, and 48 months, but not at 96 months. Thus, TNF-ß gene polymorphism was a genetic determinant for early-to-intermediate clinical courses of sarcoidosis patients.


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  		Table 3.. Genotype Distribution According to the Activity of Sarcoidosis at Each Time Point

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present report is the first to demonstrate an allelic polymorphism in TNFB as a genetic factor for the prognosis of patients with sarcoidosis. We found that Japanese patients with the allele TNFB*1 had prolonged clinical courses. Our patient population consisted principally of patients who did not require corticosteroid therapy. Such patients apparently represent a subgroup of patients showing mild-to-moderate disease manifestations. Therefore, our results may not be applicable to other ethnic populations, which usually include a substantial proportion of patients who are indicated for systemic corticosteroid therapy. Nevertheless, the present results can be considered valuable for their clarification of the natural course of patients with sarcoidosis not requiring therapeutic intervention. Indeed, when the analysis was restricted to only those patients who had not received steroid therapy, the results were more specific; patients with genotype TNFB*1/1 and those with TNFB*1/2 having significantly prolonged clinical courses compared with those with TNFB*2/2.

Several lines of evidence suggest that TNF-ß may be implicated in the pathogenic mechanism of sarcoidosis. It has been demonstrated that it plays a crucial role in lymphoid organ development.19 Mice deficient in the TNF-ß gene lack mesenteric and peripheral lymph nodes and Peyer’s patches.19 Meanwhile, mice transgenic for the TNF-ß gene exhibit a marked cellular infiltrate at local sites.20 They also have a specialized vascular system characterized by lymph nodes that express a variety of adhesion molecules, such as intracellular adhesion molecule-1, vascular cell adhesion molecule-1, mucosal addressin cell adhesion molecule -1, and peripheral node addressin. Lymph node swelling and the accumulation of abundant lymphocytes are the hallmarks in the pathology of sarcoidosis. In addition, soluble adhesion molecules have been demonstrated in serum and BALF from patients with sarcoidosis.29 30 Because of these partially common features shared by TNF-ß transgenic mice and sarcoidosis, it seems reasonable to speculate that dysregulated expression of TNF-ß could be a factor leading to the development and/or the progression of sarcoidosis. Indeed, it has been shown that TNFB*1 is associated with higher TNF-ß production by peripheral blood mononuclear cells than is TNFB*2.22 In addition, TNFB*1 is linked with an amino-acid substitution at position 26 of TNF-ß.22 This single amino-acid substitution may further lead to altered biological activity of TNF-ß. However, we acknowledge that more direct evidence of the roles of TNF-ß in granuloma formation or in perpetuation of the inflammation awaits further investigation.

TNFB*1 is also associated with higher TNF-{alpha} production by unstimulated lymphoblastoid cell lines.23 TNF-{alpha} is a central mediator of various inflammatory responses,31 and has been shown to be specifically implicated in the mechanisms of granuloma formation in vitro.12 We have previously demonstrated increased TNF-{alpha} production by alveolar macrophages obtained by BAL in patients with sarcoidosis.7 And it has been shown that sarcoid granulomas show increased expression of TNF-{alpha} messenger RNA.8 Thus, the association between the TNFB polymorphism and TNF protein production, and the critical roles of TNF in granuloma formation collectively support the idea that this gene polymorphism affects the process of disease remission in sarcoidosis through altered expression of TNF-{alpha} and/or TNF-ß. There is, however, some controversy as to the association between TNFB polymorphisms and TNF protein expression.32 33 Nevertheless, our observations indicate that the TNFB polymorphism is a novel genetic marker of the prognosis of sarcoidosis in Japanese patients.

None of the allelic base pair substitutions of TNFA were associated with the development of sarcoidosis. Further, the present study did not identify TNFB*1 as a risk factor for sarcoidosis. This is consistent with the results reported by Seitzer et al.26 In contrast, Ishihara et al27 reported a significant association between TNFB*1 and the development of sarcoidosis in Japanese subjects, and attributed this association to linkage disequilibrium to HLA-DR5, HLA-DR6, and HLA-DR8. This apparent discrepancy between their findings and ours may be reasonable in view of the fact that our patients had only a weak association with those HLA-DR antigens (unpublished observation; March 1998). This difference may have been because of population stratification, because in the former study, most patients were recruited at ophthalmic clinics.

Previous studies1 2 27 34 35 36 37 on genetics of sarcoidosis have demonstrated that genetic factors are involved in both susceptibility to and phenotype determination of the disease. The occurrence of sarcoidosis has been reported to be associated with HLA-A1, HLA-B8, HLA-DR3, and HLA-DR17 in white subjects,1 34 and with the HLA-DR3, HLA-DR5, HLA-DR6, and HLA-DR8 group in Japanese subjects,27 which have common amino-acid residues at positions 10 to 12 of the ß chain of HLA-DR molecules. The frequency of the deletion (D) allele of the ACE gene is higher in Japanese female patients and in African-American patients than in control subjects.35 36 With respect to factors accounting for the heterogeneity of phenotypes, significant relationships have been established in white subjects between early onset of the disease and HLA-B13 and HLA-B35, and between radiographic stage I and HLA-A1, HLA-B8, HLA-B27, and HLA-DR3.34 A higher frequency of TNFA2 at position - 308 has been reported for cardiac sarcoidosis in Japan.37 HLA-DR17 has been shown to be overrepresented in patients with an acute onset of the disease.1 This HLA antigen is also associated with the nonchronic form of the disease, whereas the D allele of the ACE gene confers a prolonged clinical course.2 Thus, distinct genetic components seem to variably affect the susceptibility to and phenotypic heterogeneity of sarcoidosis, depending on the subgroup of patients. Our finding is an example of genetic factors that account for the prognostic heterogeneity of sarcoidosis.

The genes for TNF-{alpha} and TNF-ß lie within a 7-kb stretch of DNA in the class III region of the major histocompatibility complex on chromosome 6p21.3.38 They are located 200 kilobase centromeric of HLA-B and 1,000 to 1,600 kilobase telomeric of the class II genes.39 In view of the close physical proximity of the TNF locus to class II genes, it is tempting to conclude that the association found in the present study is because of linkage disequilibrium. In fact, linkage of TNFB*1 with the HLA-A1, HLA-B8, and HLA-DR3 haplotype has been shown in white subjects.40 41 A significant association between HLA-DR17, one of the serologic specificities of HLA-DR3, and good prognosis has been reported for Scandinavian patients with sarcoidosis.1 However, because DR17 has been generally absent in Japanese control subjects and patients with sarcoidosis,27 42 it is not possible to analyze its significance. In addition to the HLA genes, several non-HLA genes relevant to immune responses, such as transporter-associated protein and heat shock protein 70, are also located near TNF genes on the same chromosome. Further studies will be needed to elucidate which of these genes are primarily associated with the prognosis of sarcoidosis.

In conclusion, we have found that TNFB*1 is associated with a prolonged clinical course of sarcoidosis in Japanese patients. Our study suggests that specific cytokine genotypes predispose patients to a worse clinical outcome, possibly by creating a proinflammatory phenotype.


   Footnotes
 
Abbreviations: bp = base pair; ACE = angiotensin-converting enzyme; BALF = BAL fluid; CI = confidence interval; HLA = human leukocyte antigen; PCR = polymerase chain reaction; TNF = tumor necrosis factor

This study was supported by a Grant-in-Aid (10670526) for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

Received for publication September 15, 1999. Accepted for publication September 27, 2000.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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