 Is Associated with the
 308 TNF- Promoter Polymorphism and with Clinical
Severity in Chronic Beryllium Disease
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Beryllium (Be)-antigen stimulates tumor necrosis factor-alpha (TNF-
)
from bronchoalveolar lavage (BAL) cells in chronic beryllium disease (CBD). This study tested the hypothesis that high concentrations of Be-stimulated TNF- are related to polymorphisms in the
TNF- promoter and clinical markers of disease severity in CBD.
Demographic and clinical information was obtained from patients
with CBD (n = 20). TNF- concentrations were measured in BAL
cell culture supernatant by ELISA. A priori, we categorized CBD
subjects as either high or low TNF- producers using a cutoff of
1,500 pg/ml. The TNF- promoter sequence, +64 to 
1045, was
determined by direct sequencing. Human leukocyte-associated antigen (HLA)-DPB1 and -DRB1 genotyping was determined by polymerase chain reaction (PCR). High Be-stimulated TNF- was associated with TNF2 alleles, Hispanic ethnicity, presence of HLA-DPB1
Glu69, and absence of HLA-DR4. Be-stimulated TNF- concentrations correlated with markers of disease severity, including chest
radiograph, beryllium lymphocyte proliferation, and spirometry.
We found no novel TNF- promoter polymorphisms. These data
suggest that the TNF2 A allele at 308 in the TNF- promoter region is a functional polymorphism, associated with a high level of
Be-antigen-stimulated TNF- and that these high TNF- levels indicate disease severity in CBD.
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Keywords: chronic beryllium disease; TNF-; genetic polymorphisms;
functional genetics; genetic susceptibility
Tumor necrosis factor-alpha (TNF-) has a broad range of biologic effects, from proinflammatory activities stimulating production of other cytokines to inhibitory effects on cells containing parasites (1). The TNF- gene is located in the Class
III region of the major histocompatibility complex (MHC),
between Class I and Class II loci. Because of its biologic activities and location, early studies explored associations between
human leukocyte-associated antigen (HLA) genotypes and
TNF- concentrations in diseases with apparent immune regulation, such as systemic lupus erythematosus (2). These studies suggested that Class II genes may modulate TNF- production, as HLA-DRB1, DR3, and DR4 were associated with higher mitogen-stimulated TNF- levels, whereas HLA-DR2
was associated with lower TNF- production (2). More recent studies have associated a TNF- promoter polymorphism
with a G to A transition at the 308 nucleotide position with
greater mitogen-stimulated or serum TNF- production, suggesting that the HLA-DRB1 associations might be due to
linkage disequilibrium with the TNF gene (5). There is a
paucity of data relating antigen-stimulated or disease site-specific TNF- production to either the -308 TNF promoter polymorphism or HLA Class II genotypes. The study of beryllium
(Be)-antigen-stimulated TNF- production in chronic beryllium disease (CBD), a granulomatous lung disease, offers the
opportunity to examine this relationship in light of evidence
that Be induces a cellular immune response to Be salts with
concomitant in vitro bronchoalveolar lavage (BAL) cell production of TNF- (8, 9).
Be is used in a variety of manufacturing processes, including those associated with the ceramics, telecommunications, automotive, computer, and defense industries (10). After exposure to Be in the workplace, as much as 20% of workers become sensitized to Be (BeS) (11), with many eventually developing CBD (10). Individuals with BeS demonstrate a specific immune response to Be-antigen, as evidenced by positive peripheral blood Be lymphocyte proliferation tests (BeLPT) (12, 13). These individuals do not have any evidence of pulmonary pathology on lung biopsy or physiologic abnormalities. BeS progresses to CBD at a rate of approximately 10% per year (14). Individuals with CBD have granulomatous inflammation as evidenced by noncaseating pulmonary granulomas and mononuclear cell infiltrates on lung biopsy. Both their peripheral blood and BAL lymphocytes demonstrate a Be-antigen-specific response in the BeLPT (12, 13). Previous studies have shown that the Be-antigen-stimulated T-cell proliferation is MHC Class II restricted (15). Moreover, epidemiologic studies involving subjects with CBD show an increased risk of disease in those with an HLA-DPB1 containing a glutamic acid substitution at position 69 (Glu69) (16). However, it is likely that CBD is a multigenetic disease and that other susceptibility factors contribute to the immune response to Be in CBD.
From the previously cited studies, we hypothesized that either novel or known polymorphisms within the TNF- promoter region (19) might contribute to the magnitude of the
Be-stimulated CBD BAL cell TNF- response. We further
hypothesized that if there were an association between high
concentrations of Be-stimulated CBD BAL cell TNF- and
TNF- promoter polymorphisms, then high TNF- concentrations might also be associated with more severe disease in
CBD. Because HLA Class II influences T-cell proliferation, we also investigated whether Be-stimulated TNF- production was associated with HLA-DRB1 and DPB1 alleles. In
this study, we found that subjects with CBD whose BAL cells
express high TNF- concentrations in response to Be stimulation were likely to possess the TNF2 A allele (a G to A transition at position 308). We also found that the absence of the
HLA-DR4 allele and the presence of the HLA-DPB1 Glu69
allele was associated with higher Be-antigen-stimulated BAL
TNF- concentrations in CBD. High levels of Be-stimulated CBD BAL cell TNF- were associated with clinical parameters of disease severity in CBD.
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Study Design
From previous studies, we observed that some CBD individuals produce low levels of Be-stimulated TNF-, whereas others produced high TNF- (8, 20). Based on these data, a priori, we classified individuals as "high" TNF- producers if Be stimulated > 1,500 pg/ml
from BAL cells, whereas "low" TNF- producers were those who
produced < 1,500 pg/ml Be-stimulated TNF-, using this value to approximate the median production. A random group of 10 high and 10 low TNF- producers was selected for participation in this study from
those previously studied (8, 20). All study subjects provided informed
consent, according to the protocol approved by the National Jewish
Medical and Research Center's Human Subjects Review Board.
CBD study subjects. All 20 subjects with CBD underwent clinical evaluation consisting of a chest radiograph, pulmonary function testing, exercise testing, venipuncture, bronchoscopy with BAL, and peripheral blood and BAL LPT as described previously (12, 21). Those who participated in our study met the following case definition: (1) history of occupational or environmental Be exposure; (2) histologic evidence of noncaseating granulomas on transbronchial or open lung biopsy; (3) Be-stimulated blood or BAL lymphocyte proliferation, or both (12, 13). Demographic and work history information was obtained using a modified version of the American Thoracic Society (ATS) respiratory questionnaire (22) as described previously (21).
BAL and peripheral blood mononuclear cells (PBMCs). BAL was performed by standard methods reported previously (21, 23). Using density-gradient purification (Ficoll-Hypaque), PBMCs were separated from peripheral blood (20).
TNF- Protein and Gene Analysis
Culture of BAL cells. Freshly isolated CBD BAL cells were suspended at 1.0 × 106 cells per ml and cultured alone, or in the presence of 100 µM BeSO4 (20). Cell supernatants were collected at zero time, 24, 48, and 72 h after stimulation.
Determination of TNF- protein levels. We measured TNF- protein in CBD BAL cell culture supernatants using ELISA kits purchased from R&D Systems (Minneapolis, MN) as previously described (20). The peak production of TNF- was chosen as the highest
level of TNF- at or after an interval of 24 h (8, 20).
Amplification and sequencing of the TNF- promoter. Using polymerase chain reaction (PCR), we amplified and sequenced the TNF-
promoter, +64 to 1045, from PBMCs. Genomic DNA was prepared from 5 × 106 cells using a Wizard Genomic Purification Kit (Promega,
Madison, WI). Sequences were amplified in 100-µl reactions containing approximately 1 µg of genomic DNA, 1 µM of each primer, 2 mM
MgCl2, 200 µM deoxyribonucleoside triphosphates (dNTPs), and 2 U
of Taq Gold polymerase (Perkin Elmer, Boston, MA). The DNA was
amplified using the flanking TNF- 5'-CAA AGG AGA AGC TGA
GAA GAT G- and TNF- 3'-CAG TTG CTT CTC TCC CTC TTA
G-; heated at 95° C for 10 min; amplified for 14 cycles at 96° C/20 s, 72° C/
45 s decreasing the subsequent cycles by 1° C per cycle, 72° C/2 min
then 25 cycles at 96° C/20 s, 58° C/45 s, and 72° C/2 min followed by a final cycle at 72° C for 7 min. PCR products were directly purified using
the Promega Wizard PCR Purification System (Madison, WI). The
TNF- promoter region was sequenced by Davis Sequencing (Davis,
CA) using sequencing primers and an automated ABI Prism 377 DNA
Sequencer (Applied Biosystems, Perkin Elmer, Foster City, CA).
Polymerase chain reaction-sequence-specific primer (PCR-SSP)
analysis of HLA-DPB1 and DRB1. HLA typing was performed with blinding to the subject's disease and TNF- status as previously described by Bunce and coworkers and Gilchrist and coworkers (24, 25).
Statistical analysis. An odds ratio (OR) with a 95% confidence interval (CI) was calculated to evaluate the degree of association between the high and low TNF--producing groups and the TNF- and
other genotypes. For purposes of this analysis, TNF1 G homozygotes were compared with TNF2 A heterozygotes combined with homozygotes. For HLA genotyping with multiple genotypes, comparisons
were made between the presence of the allele of interest in high and
low TNF- groups. Comparisons were made between TNF- supernatant levels, TNF- genotype, and clinical parameters of disease severity, as described subsequently. Continuous variables were compared using Wilcoxon's rank sum test, Kruskal-Wallis test, and Student's t
test when appropriate. Categorical variables were compared using chi-square or Fisher exact test. Spearman's rank correlation coefficient (
) was used to evaluate the relationship between the levels of Be-stimulated CBD BAL cell TNF- and other continuous variables. To assess the contribution of different genotypes and demographic variables on TNF- concentrations, we used multiple linear regression. Those variables significantly associated with TNF- production in univariate analysis were entered into the model using a stepwise method. The log of TNF- was used in the analysis to approximate a
normal distribution. All statistical analyses were performed using
JMP-SAS or SAS (SAS Institute, Cary, NC). All tests were two-sided,
and a p value of < 0.05 was used as a level of statistical significance.
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Be-Stimulated CBD BAL Cell TNF-
For our 20 subjects with CBD, Be stimulated (100 µM BeSO4)
a median production of 1,998 pg/ml of TNF- (interquartile
range [IQR] 25%/75%; 488 pg/ml, 7,591 pg/ml) by CBD BAL
cells. The high TNF- group (n = 10) had a median of 7,365 pg/ml TNF- (IQR 5,298 pg/ml, 9,568 pg/ml, minimum = 2,846 pg/ml, maximum = 30,000 pg/ml). In comparison, the low
TNF- group (n = 10) had a median of 501 pg/ml TNF-
(IQR 226 pg/ml, 1,106 pg/ml, minimum = 178 pg/ml, maximum = 1,149 pg/ml) (p < 0.05) as shown in Figure 1. The
above-mentioned results reflect our a priori study design.
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CBD Subject Demographics
We compared the demographics, smoking, corticosteroid therapy status, Be exposure, and the time from first Be exposure
by TNF- group (high or low) in these CBD subjects (Table
1). The mean age, sex, and race of our study population reflect
the Be workforce in Colorado (26). Overall, 40% (8 of 20) of
our study participants were of Hispanic ethnicity, higher than
that usually seen in our work-based studies (26, 27). We found
a significantly higher frequency of Hispanics among our high
TNF- producers (70% versus 10% in the low TNF--producing group, p = 0.02). Among the high TNF- producers, 50%
of the subjects were current steroid users, and of these, two
were prescribed methotrexate plus prednisone. Seventy percent of both high and low TNF- producers were ceramics workers. When we compared ethnicity by category of Be exposure, we found no association (p > 0.05).
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Be-antigen-stimulated Cell Proliferation
Results of BAL cell counts and differentials for all 20 subjects
showed the typical elevations in BAL WBC number and lymphocyte percentages seen in CBD (26, 28). We observed no
significant differences in the percentages of leukocyte classes
between low and high TNF- producers. The low TNF- producers had 41 ± 26 × 104 white blood cells (WBC)/ml of BAL
fluid (BALF) with 61 ± 23% macrophages and 38 ± 22%
lymphocytes. The high TNF- producers had 47 ± 28 × 104
WBC/ml of BALF with 43 ± 17% macrophages and 55 ± 18% lymphocytes. Consistent with previous studies using the
BeLPT (12, 13), CBD BAL cells proliferated in response to
Be-antigen stimulation. The median peak BAL stimulation index (SI) for the low TNF- producers was 3.4 (IQR 1.75, 10.75), whereas the median peak BAL SI for the high TNF-
producers was 74.2 (IQR 43.4, 176.8) (p < 0.01, Figure 2). The
PBMC median peak SI did not differ between groups (low
TNF- producers, median = 5.4, IQR 1.2, 20.3 versus high TNF- producers, median 7.4, IQR 3.5, 11.1, p > 0.05).
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Sequence Analysis of the TNF- Promoter Region from
Patients with CBD
We tested the hypothesis that high Be-stimulated TNF- concentrations might be associated with TNF- promoter polymorphisms, including the two most well known polymorphisms at the 238 or the 308 nucleotide positions. We
observed polymorphic variability at the 308 nucleotide position (Table 2), with a TNF1 G allele frequency of 77.5% (31 of
40) and a TNF2 A allele frequency of 22.5% (9 of 40) in our
subjects with CBD. The genotypes and the gene frequencies
were in Hardy-Weinberg equilibrium. We observed a trend
between the high TNF- group and the TNF2 A heterozygous or homozygous genotype (OR of 13.5, 95% CI 1.00 to 687.9, p = 0.057). In addition to the 308 nucleotide position, we
found polymorphic variation at the 856 nucleotide position
(29). Specifically, 20% of the alleles in our CBD subjects contained a C to T transition at 856. Among the high TNF- producers, 10% were heterozygous at the 856 position. Although
there was a higher frequency of this allele among the low
TNF- producers, the frequency did not differ significantly
(p > 0.05) from the high TNF- producers. Our sequence
analysis revealed no novel sequence changes in the TNF-
promoter between low and high TNF--producing groups, or as compared with the consensus TNF- promoter sequence
from +64 to 1045 (30). There were no polymorphic changes
in the nucleotide sequence at 238, 574, or 862 in any (20 of 20) of the CBD subjects' TNF- promoters.
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High TNF- Levels are Associated with CBD Disease Severity
We compared the concentration of Be-stimulated CBD BAL
cell TNF- with clinical parameters, testing the hypothesis
that higher TNF- concentrations are reflective of more severe disease in CBD. Figure 2 shows a significant (median,
IQR, and 10%/90% limit) difference between the values for
high versus low TNF- producers' chest radiographic score or
profusion rank (p = 0.02), the peak SI (lavage peak SI) in the
BAL BeLPT (p 
0.01), and spirometric measurement of
FEV1/FVC ratio (p = 0.03). Specifically, the higher TNF-
group had worse lung infiltrates on chest radiograph, a higher
BAL BeLPT, and airflow obstruction. Although not statistically significant, there were notable trends between high and
low TNF- producers' exercise alveolar-arterial (A-a) gradient
at baseline (high TNF- producers median = 10.5, IQR 6.75, 18.75 compared with the low TNF- producers median = 7, IQR 0.55, 9.2, p = 0.07), percent predicted diffusing lung capacity (DLCO) (high TNF- producers median = 83, IQR 67.5, 96.25 compared with the low TNF- producers median = 92.5, IQR 83.5, 108.75, p = 0.07), and the percentage of lymphocytes in the BAL cell population (high TNF- producers median = 50, IQR 45.5, 65.25 compared with the low TNF- producers median = 33, IQR 18.75, 65.25, p = 0.12).
We observed no statistically significant association between
corticosteroid use and Be-stimulated TNF- production (steroid users median = 4,597.5 pg/ml, IQR 514.8, 12,084.8 compared with the nonsteroid users median = 1,142.5 pg/ml, IQR
289.3, 7,942.8, p > 0.05). TNF- concentrations did not differ
by smoking status (p > 0.05). However, we did find a significant association between Hispanic ethnicity and Be-stimulated TNF- production, with the Hispanic individuals producing a median of 8,068.5 pg/ml (IQR 6,313, 11,762.6) compared
with the non-Hispanics with a lower median of 983 pg/ml (IQR
348.3, 2,421.8, p < 0.01).
TNF Promoter Genotype and Disease Severity in CBD
The foregoing data reveal an association between Be-stimulated CBD BAL cell TNF- concentrations and measures of
disease severity in CBD. Because we also found an association
between TNF- genotypes and high and low TNF- producers, we tested the hypothesis that the TNF- genotype might
also be associated with high TNF- concentrations and CBD
disease severity. A comparison of TNF- levels by TNF promoter genotype showed no significant difference (p = 0.10). However, the median TNF- concentrations were lower in the
TNF1 G homozygous patients with CBD (median 1,096 pg/ml,
IQR 391, 5,054) compared with the TNF2 A homozygous and
heterozygous patients with CBD (median 6,113 pg/ml, IQR
2,846, 8,471). We dropped one subject from this portion of our
analysis, because he was an outlier with significantly higher
TNF- concentrations (30,000 pg/ml) and the longest Be latency. Comparing TNF- levels by TNF promoter genotype
excluding this individual, we found that CBD patients with the
TNF2 A homozygous or heterozygous promoter genotype
produced significantly higher concentrations of Be-stimulated
CBD BAL cell TNF- (p = 0.04) (Figure 3). Evaluation of the
clinical parameters, excluding this one individual, revealed
that patients with CBD with the TNF2 A allele had a more
lymphocytic-predominant alveolitis (Table 3). In addition, we
found no association between the TNF2 A genotype and demographic variables such as race, sex, ethnicity, steroid use,
Be material exposure, or latency (p > 0.05).
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TNF- Promoter, HLA-DPB1, and DRB1 Genotypes and
Associations with TNF- Production
Previous studies have found associations between TNF- concentrations and HLA-DRB1. HLA-DPB1 is a known susceptibility factor for CBD, likely functioning in Be-antigen presentation. Therefore, we determined the HLA-DRB1 and
HLA-DPB1 genotypes in our subjects with CBD. Because of
limited DNA, we were able to obtain typing results on 18 of
our subjects with CBD. The results are presented in Table 4
for nine high and nine low TNF- producers.
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As has been shown previously (16), the majority of the
CBD individuals had at least one Glu69-containing HLA-DPB1 allele (15/18 or 83%). The frequency of non-Glu69-containing alleles, with *0401 and *0402 predominant, did not differ between the high and low TNF- groups (data not shown,
p > 0.05). The frequency of carrying a DPB1 Glu69 allele was
more prevalent in the high TNF- group (p = 0.02, OR = 5.5, 95% CI 1.06-31.5). Those individuals with a Glu69 and those
homozygous for Glu69 (33%) produced higher Be-antigen-stimulated TNF- concentrations (Table 5). Interestingly, all
seven of the Hispanic subjects and TNF2 A carrying subjects had at least one Glu69 allele, although this did not achieve statistical significance (p = 0.25 for Glu69 versus TNF genotype
and Hispanic ethnicity).
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Only one of our 18 CBD subjects in whom we performed
HLA typing had a HLA-DRB1*0301 allele (Table 4). The
high TNF- group had a lower frequency of the HLA-DRB1
*04 allele, although this was not statistically significant (6%
versus 28% in the low TNF- group, p = 0.18). Interestingly,
the subjects with the HLA-DRB1 *04 allele produced significantly lower Be-stimulated BAL cell TNF- concentrations
(p = 0.02, Table 5). Evaluating the association between Hispanic ethnicity and HLA-DR4, we observed that only one of
our seven Hispanic subjects carried a HLA-DR4 allele (p > 0.05). This individual had one of the lowest Be-stimulated
TNF- concentrations (185 pg/ml) and was the only TNF2 A
subject who also had a HLA-DR4 allele. Of those CBD subjects with a Glu69 allelic substitution, only 33% (5 of 15, p > 0.05) had a HLA-DR4 allele. Of note, four of the highest six
TNF- producers carried a TNF2 A and Glu69 but did not
have a HLA-DR4 allele.
We evaluated TNF- levels by the TNF- 856 T promoter
polymorphism (Table 5). Although there were no statistically
significant differences in TNF- concentrations, the subjects
with the 856 polymorphism tended to have lower Be-stimulated CBD cell TNF- levels. All of the five CBD subjects
who carried the C to T transition at 856 also carried the
TNF1 G genotype, whereas only one subject carried the HLA-DRB1 *04 allele.
To assess the contribution of the TNF- genotype, when controlling for other variables associated with TNF- production,
such as HLA-DR4 and Glu69 genotype, we employed linear regression (Table 5). We developed a model to predict TNF-
concentrations, Y, based on the TNF2 A genotype, X1, Hispanic
ethnicity, X2, and DPB1 *04, X3: Y = 3.02 + 0.38X1 + 0.51X2 0.52X3 (adjusted R2 = 0.48, p < 0.01). The HLA-DPB1
Glu69 genotype was not found to be predictive and thus was not
included in our model (p = 0.64). We did not attempt to include
any interaction terms because of our small sample size.
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In this study, we found an association between the TNF2 A allele and high Be-antigen BAL cell TNF- production in individuals with the granulomatous lung disease CBD. Although
we did not find novel mutations in the TNF- promoter between nucleotides +64 and 1045, based on direct sequencing
we identified a known polymorphism at the 856 nucleotide
position. The BAL TNF- concentrations correlated with profusion abnormalities on chest radiograph, Be-antigen-stimulated proliferation of BAL cells, and airway obstruction, reflecting more severe disease in those individuals with higher
TNF- production by lung cells. The TNF2 genotype may explain why some individuals with CBD progress to a more
chronic severe form of the disease. We found a protective effect of the HLA-DR4 allele on Be-stimulated TNF- production. In addition to the TNF- gene, this suggests that other
genetic loci may also influence antigen-stimulated TNF- production. To our knowledge, this is the first evidence that the
TNF2 A genotype is associated with specific antigen-stimulated TNF- production at the site of disease pathology.
Previous studies have shown associations between unstimulated TNF- concentrations and disease severity in diverse
disease processes, including sarcoidosis, malaria, and systemic
lupus erythematosus (2, 5, 31). A few of these studies associated the TNF 308 genotype with disease severity (2, 5, 34). A limited number of studies evaluated the impact of the TNF- 308 polymorphism on TNF- levels from a functional standpoint (35). However, the mechanism by which TNF- induces or affects disease severity is still not well understood.
TNF- is a key cytokine in the early stages of granuloma formation and granuloma maintenance. It stimulates an amplification loop wherein TNF- increases the accumulation of
macrophages, stimulates proliferation of activated T cells, induces its own production, and promotes the differentiation of
noncaseating granulomas (1, 38). As markers of pathologic
granuloma formation in the alveolar interstitium, we found
more gas exchange and chest radiographic abnormalities and
higher BAL cell proliferation in our high TNF- producers. This suggests that the high TNF- production in CBD may result in more significant granulomatous inflammation. In addition, TNF- may propagate granulomatous inflammation because of its proinflammatory effects (8, 39). In CBD, these effects
may be more pronounced in the setting of persistent antigen,
ongoing exposure, and resultant chronic antigen presentation
in lung tissue.
Based on these previous studies and our current data, we
propose a model by which high TNF- levels may promote a
chronic severe form of CBD. First, those individuals with the
TNF2 A genotype, when exposed to Be in an antigenic form,
produce higher concentrations of TNF- in the lungs. This sets
the stage for the accumulation of lymphocytes (40) and the
proliferation of activated T cells (15, 28). A local cytokine amplification loop perpetuates this cycle and contributes to the
formation of additional noncaseating granulomas (8, 9, 41,
42). This scenario may then be maintained by the persistence
of Be-antigen in tissue and result in a severe, disabling form of
CBD characterized by higher Be-stimulated BAL cell TNF-
levels; ongoing T-cell proliferation; lymphocytic alveolitis; and
spirometric, gas exchange, and chest radiographic abnormalities, all of which were demonstrated in the present study.
Our data are consistent with published evidence that the
TNF- 308 polymorphism may dictate the magnitude of in
vitro TNF- response to inflammatory stimuli. Several studies
show that the 308 polymorphism may enhance transcription
by affecting an activator protein-2 (AP-2) binding site, provide
a binding site for a novel transcription factor, and increase levels of TNF- transcription (35). We believe this enhanced
transcription promotes higher Be-antigen-stimulated BAL cell
TNF- concentrations in CBD. In contrast to the 308 polymorphism, Uglialoro and coworkers (29) found no effect of
856 polymorphism on TNF- expression. Thus, the apparent
trend of lower TNF- concentrations in our subjects with the
TNF- 856 T polymorphism was likely a result of the subjects'
TNF1 G genotype.
Factors other than TNF2 A genotype may regulate TNF-
production and granulomatous inflammation. We found associations between the HLA-DPB1 Glu69 genotype, the absence of HLA-DRB1 DR4, Hispanic ethnicity, and inducible
TNF- concentrations. No previous studies have found an association between Glu69 and TNF- levels. Actually, because
such a large number of our subjects with CBD carry at least
one Glu69 allele, this genotype was found to be the least predictive in our multiple linear regression model. In CBD,
Glu69 might not directly affect TNF- concentrations but promote optimal antigen presentation and subsequent higher
TNF- levels in those individuals with the TNF2 A genotype.
In our CBD subjects, DR4 was a protective marker associated
with lower TNF- concentrations. Although not statistically
significant, all but one of our TNF2 A subjects were HLA-DR4-negative. Conversely, the low TNF- group had a higher
frequency of HLA-DR4. This suggests that HLA-DR4 may
favor lower Be-stimulated TNF- production. This finding
stands in contrast to previous studies of malaria, lupus, and
inflammatory bowel disease showing HLA-DR4 association
with higher TNF- concentrations. No association between
the TNF2 A genotype and HLA-DR4 has been substantiated
to date (2, 6, 43). In contrast to previous studies, we did not
find a link between the TNF2 A genotype and HLA-DR3 (6,
44). McGuire and coworkers also found the TNF2 A allele
was independent of HLA Class II variation in cerebral malaria (5). A larger number of cases and control subjects will be
needed to substantiate the findings in our study.
A striking association was found between high TNF- concentrations and self-reported Hispanic ethnicity in our multiple regression model. In this study, the majority of our Hispanic
subjects were TNF2 A-positive (4 of 7, 57%), DR4-negative
(6 of 7, 86%), and Glu69-positive (7 of 7, 100%). Thus, in our
multiple linear regression model, Hispanic ethnicity may have
effectively acted as an interaction term between all three of
our relevant genotypes and as a surrogate for the genotypes
themselves. It is also possible that Hispanic ethnicity is a surrogate for another, as yet undefined gene important in the regulation of TNF- production. We do not currently know the
frequency of the TNF2 A allele in a control or large CBD or Hispanic population, as compared with a group of Be-exposed
workers containing Hispanics. Our study does not suggest that
Hispanics are more susceptible to CBD, because we do not
know the prevalence of the TNF2 A genotype in a Be-exposed
nondiseased population of Hispanics. Recent studies have not
found an increased rate of the TNF2 A allele in Mexican Hispanics (45). Previous studies suggest Glu69 subjects were as
likely to be Hispanic as the Glu69-negative subjects (34% versus 29%) (17). Population data on TNF2 A and HLA-DPB1
frequencies are limited for ethnic groups and will be addressed
in a future case control study in CBD.
We believe that genetic and environmental interactions are
important in understanding and defining who will or will not
develop CBD. In our study, we found no association between
Hispanic ethnicity or TNF- concentrations and work with Be
ceramic or metal. Our results were also not confounded by
steroid exposure or smoking status, both of which are known
to reduce TNF- levels (46, 47). Previous studies suggest that
Be acts as an adjuvant in immune regulation. We believe it is
unlikely that TNF- production results from Be-adjuvant effects because Be is unable to stimulate TNF- from BeS or
normal individuals' BAL cells (8, 48).
Based on these data, we believe that the TNF2 A genotype
is likely the first identified gene marker of Be-antigen-stimulated TNF- production and disease severity in CBD. At this
time, it is unclear whether TNF- is a marker of CBD susceptibility, compared with a Be-exposed worker population, similar to the HLA-DPB1 Glu69. It is also unknown whether this
genetic marker may be important in the progression from BeS
to CBD. Further larger studies will be required to address
these two questions. However, our current study suggests that
TNF2 A may be an important prognostic indicator, which might
find application in the risk counseling of BeS and CBD patients
with further study and substantiation of our current results.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Lisa A. Maier, National Jewish Medical and Research Center, 1400 Jackson Street, Room M210, Denver, CO 80206. E-mail: MaierL{at}njc.org
(Received in original form December 28, 2000 and accepted in revised form May 31, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: The authors would like to thank Dr. Lawrence Abraham for his review of this manuscript and helpful suggestions. They thank Mary Solida, RN, for her patient care. They thank Eric Wilcox for technical assistance and Heather Davis, Kieran Nelson, and Malkah Tannenbaum for expert secretarial support. They would also like to acknowledge those patients who make this and other Be-related research possible.
Supported by K08 HL03887, R01 ES06358-06, and M01 RR00051 from the National Institutes of Health.
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References |
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REFERENCES
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1. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol 1992; 10: 411-452 [Medline].
2.
Jacob CO,
Fronek Z,
Lewis GD,
Koo M,
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