  308 Promoter Gene
Polymorphism and Increased Tumor Necrosis Factor
Serum Bioactivity in Farmer's Lung Patients
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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Hypersensitivity pneumonitis (HP) represents an immunologic reaction of the pulmonary parenchyma to an inhaled agent. Since tumor necrosis factor (TNF)-
is thought to be involved in the pathogenesis of HP, and polymorphisms in the TNF genes have
been associated with variations in the production of TNF-, we investigated the serum bioactivity and genotype of TNF in HP. TNF
bioactivity was measured after hay dust challenge in eight patients with farmer's lung (Group A) and in 12 healthy, sensitized
(antibody-positive) controls (Group B). Genotyping for the 
308
TNF- promoter polymorphism and the TNF-
intron 1 gene polymorphism was performed in 20 patients with farmer's lung, 25 patients with pigeon breeder's lung, and 216 controls. TNF bioactivity increased in Group A at 4 to 10 h after hay dust challenge, but
not in Group B (p < 0.05). The frequency for the TNFA2 allele, a
genotype associated with high TNF- production in vitro, was significantly higher in farmer's lung patients (frequency [f] = 0.43, p = 0.0012) than in controls (f = 0.19) or patients with pigeon
breeder's lung (f = 0.16). Genotyping for TNF- revealed no significant abnormalities. Thus, increased production of TNF- after
hay contact, and a genetic predisposition to TNF- production, are
implicated in the pathogenesis of alveolitis in farmer's lung.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypersensitivity pneumonitis (HP) is an interstitial lung disease resulting from a reaction to repeated inhalation of certain antigens. The disease is characterized by chronic inflammation, and its development is immunologically mediated, as reflected by detectable precipitating antibodies against specific antigens (1) and antigen-specific activated lymphocytes (2). Alveolitis in HP has been linked to numerous endogenous mediators, of
which the immunoregulatory cytokine tumor necrosis factor
(TNF)- is thought to play a pivotal role. Alveolar macrophages (AM) from patients with farmer's lung show increased
spontaneous and antigen-induced TNF- secretion (3). Conversely, inhibition of TNF- has been shown to restrain the
development of HP in a mouse model (4).
Recent investigations have described the correlation of cytokine gene polymorphisms with varying levels of cytokine
production. Genetic analysis of healthy subjects revealed biallelic polymorphisms in the TNF- gene promoter (TNFA) (5)
and in the first intron of the TNF- gene (TNFB) (6), both of
which were associated with variations in TNF- production
(7). Considering this, and the association of these polymorphisms with severe diseases and inflammatory processes (10),
cytokine gene polymorphism and individual cytokine secretory capacity might be linked to the development of alveolitis.
The present study sought to answer the following questions: (1) Does hay challenge increase the serum bioactivity of
TNF- in farmer's lung patients, and is there a difference from
sensitized (antibody positive) but healthy subjects? (2) Are
the TNF- 308 and TNF- intron 1 gene polymorphisms
linked to the disease? (3) Are there differences in TNF genotypes in different forms of HP (e.g., farmer's lung and pigeon
breeder's lung)?
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects for Hay Dust Challenge Test
Standardized hay dust challenge was done in an exposure chamber (2 × 1.5 × 2.5 m) as described previously (11). Subjects were exposed for 20 min to a mixture of different moldy hay probes from farmers' homes. The following data were recorded before and at 6 to 8 h after exposure: VC, CO transfer factor (TLCO), arterial blood gas values, body temperature, and complete blood count. Patients were questioned about dyspnea and malaise.
When patients developed systemic and pulmonary symptoms at 6 to 8 h after hay dust exposure, a positive reaction was based on the established criteria (11) of: (1) a systemic reaction including a rise in temperature of > 1° C and a rise in the leukocyte count of > 2.5 cells/ nl; and (2) a pulmonary reaction including a decrease in VC of 20% and a decrease in TLCO of 15% or a reduction in arterial oxygen tension (PaO2) of > 15%.
During hay challenge, serum samples for TNF bioactivity analysis were drawn at 0, 1, 2, 3, 4, 6, 8, and 10 h.
Group A
Two study groups were exposed to a hay dust challenge. The first (Group A) consisted of eight patients with farmer's lung (seven male and one female), with a mean age of 52 yr (range: 32 to 61 yr), and consisting entirely of nonsmokers. The diagnosis of farmer's lung was established according to the American guidelines (12). The patients histories indicated that all were acutely symptomatic, with pulmonary and systemic symptoms at 4 to 8 h after working with hay. All patients had significant antibodies (two or more bands) to Micropolyspora faeni, Thermophilus polyspora, or Thermophilus vulgaris as measured by immunodiffusion according to the method of Ouchterlony (13).
Group B
The second study group (Group B) consisted of 12 farmers (nine male and three female), with a mean age of 48 yr (range: 28 to 62 yr), and consisting of eight nonsmokers and four smokers. All 12 subjects in this group were antibody positive to Micropolyspora faeni, Thermophilus polyspora, or Thermophilus vulgaris, but had no pulmonary symptoms. Only five subjects reported occasional mild systemic symptoms after working with hay.
Subjects for Genotyping
Subjects for genotyping consisted of 45 unrelated patients with acute HP. The diagnosis of HP was performed according to the American guidelines (12). Twenty of these patients had a diagnosis of farmer's lung and 25 had a diagnosis of pigeon breeder's lung (pigeon fancier's lung). In the farmer's lung patients, antibodies were detected against M. faeni, T. polyspora or T. vulgaris, and in the patients with pigeon breeder's lung against pigeon droppings and pigeon bloom, as measured with the Ouchterlony immunodiffusion technique (13). According to the patients' histories, all were acutely symptomatic, with pulmonary and inflammatory symptoms of 4 to 8 h after exposure to hay or after contact with pigeons.
A control group consisting of 216 unrelated healthy blood donors
from Lübeck and Kiel, Germany, were used for TNF- and TNF- genotyping.
Detection of TNF Bioactivity in Serum
The biologic activity of TNF in serum samples was determined by its cytotoxic effect on the fibrosarcoma cell line WEHI 164, subclone 13B9. The detection limit of the assay was 2 pg/ml (14).
DNA Isolation
Genomic DNA was isolated from 300 µl of ethylenediamine tetraacetic acid (EDTA)-treated blood samples with the Puregene DNA- extraction kit (Gentra, Minneapolis, MN), according to the manufacturer's instructions.
Genotyping
Analysis of the TNF- 308 promoter gene polymorphism was done
as described by Wilson and colleagues (4), and that for the TNF- intron 1 gene polymorphism as described by Stüber and coworkers (15).
For polymerase chain reaction (PCR) amplification, 100 ng of genomic DNA was added to 50 µl of reaction mixture containing 1 µM
of each primer. The forward and reverse primers for TNF- consisted
of: 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and 5'-TCCTCCCTGCTCCGATTCCG-3', respectively, and those for TNF- consisted of 5'-CCGTGCTTCGTGCTTTGGACTA-3' and 5'-AGAGGGGTGGATGCTTGGGTTC-3'. The remainder of the reaction mixture
consisted of 200 µM of deoxynucleotide triphosphates, 1 U Taq polymerase (Perkin Elmer, Norwalk, CT), PCR reaction buffer (50 mM
KCl; 10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl2). Cycling conditions for
TNF- were 38 cycles of 1 min at 94° C, 1 min at 60° C, and 1.5 min
(+2 s per cycle) at 72° C. The conditions for TNF- were analogous to
those for TNF-, with an annealing temperature of 70° C. Products of
107 bp and 782 bp were generated for TNF- and TNF-, respectively.
The restriction digests with NcoI of the TNF- PCR product yielded
fragments of 87 bp and 20 bp (TNFA1), and 107 bp (TNFA2), and those
for the TNF- PCR product yielded fragments of 586 bp, and 196 bp
(TNFB1), and 782 bp (TNFB2). The analyses for TNFB and TNFA,
respectively, were monitored with electrophoresis on 2% and 4% agarose gels containing ethidium bromide (0.5 mg/ml).
Statistical Analysis
Differences between challenge-positive and challenge-negative patients were compared through the Mann-Whitney U test. The chi-square test with Yates' correction was used to determine the significance of differences in genotype distribution between patient groups and controls. A value of p < 0.05 was considered significant.
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RESULTS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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At 6 to 8 h after hay dust challenge, all patients in Group A developed a systemic and pulmonary reaction with fever (mean: 1.96° C; range: 1.7 to 3.1° C) and leukocytosis (mean: 6.73 cells/nl; range: 3 to 11.3 cells/nl), as well as more than a 20% decrease in FVC and more than a 15% decrease in the diffusing capacity of carbon monoxide. All patients reported dyspnea and malaise. According to the criteria stated previously (11), all subjects had a positive challenge test.
At 6 to 8 h after exposure, two subjects in Group B developed a systemic reaction with fever (> +1° C), leukocytosis (> 2.5 cells/nl), and malaise. No changes in temperature or leukocytes were seen in 10 subjects. No patients developed a pulmonary reaction (i.e., dyspnea or change in lung function test results). All tests were judged negative.
TNF Bioactivity During Hay Challenge Test
As shown in Figure 1, TNF bioactivity increased from 5 ± 5 pg/ml (mean ± SD) to 58 ± 15 pg/ml at 4 h after allergen challenge in Group A. The increase peaked at 8 h (79 ± 31 pg/ml), followed by a decrease to 50 ± 42 pg/ml at 10 h. In Group B, (healthy, antibody positive) no change in TNF bioactivity was seen. The differences between Group A and Group B at 4, 6, 8, and 10 h were statistically significant (p < 0.05).
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Cytokine Genotype
The results of the genotyping analysis for the TNF- -308 promoter and TNF- Intron 1 are shown in Figures 2A and 2B.
The frequency (f) of the TNFA2 allele was higher in patients
with HP than in healthy controls (f = 0.28 versus f = 0.19), although this difference did not reach statistical significance. In
patients with farmer's lung, a subgroup of those with HP, the
frequency of the TNFA2 allele was significantly higher than in
healthy controls (f = 0.43 versus f = 0.19; p = 0.0012) or in patients with pigeon breeder's lung (f = 0.43 versus f = 0.16; p = 0.0012). Seventy-five percent of farmer's lung patients had the
TNFA2, as compared with 34% of the control group and 26%
of patients with pigeon breeder's lung.
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No significant differences in allele frequencies were seen
for the TNF- intron 1 gene polymorphisms.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In our study, we found an increase in TNF serum bioactivity
after hay challenge in all farmer's lung patients, but not in antibody-positive asymptomatic controls. We also found that
farmer's lung was associated with the TNFA2 polymorphism
in the TNF- gene promoter and with high TNF- production
in response to a hay challenge (8).
Stimulation of the production of proinflammatory cytokines by allergens is thought to play an important role in pulmonary inflammation (16). M. faeni, the main antigen in
farmer's lung, stimulates AM and blood monocytes from nonfarmer volunteers to produce TNF-, interleukin (IL)-1, and
IL-1 (17). Our study showed increased TNF bioactivity in serum after hay dust challenge, in association with a pulmonary
and a systemic reaction in all tested farmer's lung patients. No
TNF increase and no change in lung function testing were
seen in sensitized (antibody positive) but asymptomatic farmers (Figure 1). Two otherwise asymptomatic farmers developed fever and malaise upon challenge with hay dust, most
likely due to nonspecific irritants in moldy hay. These subjects did not show an increase in TNF serum bioactivity. Therefore, the detection of increased circulating TNF after antigen exposure seems to be of diagnostic value for farmer's lung.
Release of TNF from AM in response to M. faeni is known to
cause a pyrogenic reaction (18). Pulmonary inflammation in HP and granuloma formation in different lung diseases have been
linked to TNF (4, 19). In a mouse model of M. faeni-induced
HP, Denis and colleagues were able to completely abrogate
the development of alveolitis by using a polyclonal antiserum
against TNF- (4). Therefore, we conclude that endogenous
TNF production plays a critical role in the development of
farmer's lung.
Considering that 20% to 30% of farmers produce precipitating antibodies but that only 1% to 8% develop HP (20), cofactors must be present for the development of alveolitis.
Since the incidence of HP in relatives of patients with farmer's
lung is double that in the general population, genetic cofactors
are postulated for this observation (21). The degree of production of TNF- in response to a stimulus is known to be linked
to a biallelic polymorphism in the TNF- gene: it has been
shown that individuals homozygous for the TNFA2 allele at
position 308 in the TNF- gene promoter produce more
TNF- upon stimulation than do individuals homozygous for
the TNFA1 allele, whereas heterozygous individuals produce intermediate amounts of this cytokine (8, 9). In our analysis, the rare TNFA2 allele was represented significantly more often in farmer's lung patients than in the control group (Figure
2A), offering a possible explanation for the TNF increase after
hay dust challenge in farmer's lung patients. A significantly
greater prevalence of the less common TNFA2 allele in 75%
of the farmer's lung patients (TNFA2 homozygous or heterozygous) with a possibly concomitantly greater capacity for
TNF- production, implies a genetic predisposition to greater
TNF- production as an important factor in the manifestation
of farmer's lung. Löfgren's syndrome, the acute form of sarcoidosis, and coal miners pneumoconiosis, representing pulmonary diseases also linked to TNF- release, have previously
been shown to be associated with TNFA2 (22, 23). In contrast,
the patients with pigeon breeder's lung did not show an association with TNFA2, suggesting that besides being precipitated
by different antigens, subtypes of HP are a divergent group of
diseases. TNF- production might not be a key factor for the
development of alveolitis in pigeon breeder's lung.
In conclusion, our data demonstrate an increased production of TNF after hay dust challenge in farmer's lung patients
but not in sensitized asymptomatic controls. In addition, genotyping revealed a higher frequency of the TNFA2 allele in
farmer's lung patients, indicating a genetic predisposition for
increased TNF- production in the pathogenesis of alveolitis
in farmer's lung. Since farmer's lung patients usually do not
develop pulmonary fibrosis, the TNFA2 genotype at position
308 2 of the TNF- promoter, and high TNF- secretion,
might predispose to a better outcome in alveolitis. Future
studies should investigate the prognostic role of TNF genotypes in the course of HP, focusing especially on the development of progressive interstitial lung disease.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. med. Bernhard Schaaf, Medizinische Klinik II, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail: bernhard.schaaf{at}medinf.mu-luebeck.de
(Received in original form February 14, 2000 and in revised form August 30, 2000).
Acknowledgments: Beside the blood samples that we took, blood samples of patients with HP were received from the following members of the German Alveolitis Study Group: T. Schaberg (Rothenburg Wümme), G. Liebetrau (Lostau), K. Bergmann (Bad Lippspringe), J. Sennekamp (Bonn), and D. Müller-Wening (Zusmarshausen). The authors greatly appreciate the dedicated and expert technical assistance of Ms. Simone Ross and Ms. Margrit Hahn.
Supported by grant DFG SFB367/C1 from the Deutsche Forschungsgemeinschaft.
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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