 , Interleukin-6, and Their Soluble
Receptors in Chronic Beryllium Disease
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ABSTRACT
INTRODUCTION
METHODS
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Chronic beryllium disease (CBD) provides a model system in which to evaluate the antigen-stimulated, cell-mediated, immune response that leads to granulomatous lung disease. We hypothesized that beryllium salts would stimulate bronchoalveolar lavage (BAL) cell release of tumor necrosis factor-
(TNF-) and interleukin-6 (IL-6), and their soluble receptors, soluble TNF receptor I (sTNF RI),
sTNF RII, and sIL-6R and that chronic exposure to antigen would increase production of soluble receptors in the serum and BAL fluid (BALF) of beryllium-sensitized and CBD patients. We have demonstrated (1) similar constitutive TNF-, IL-6, and soluble receptor production by control subjects and
CBD patients, (2) a BeSO4-stimulated increase in TNF- and IL-6 production by CBD-derived BAL cells,
and (3) a BeSO4-induced decrease in sTNF RII production by BAL cells from control subjects. We measured increased serum sTNF RI and serum and BALF sIL-6R in beryllium-sensitized subjects and increased sTNF RI and RII in serum and sIL-6R and sTNF RII and BALF in CBD patients. These changes
correlated with pulmonary lymphocytosis and clinical measures of disease severity, indicating that
soluble receptors may reflect disease status.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Granulomatous diseases, such as sarcoidosis, schistosomiasis, and chronic beryllium disease (CBD) develop in response to different antigens but share a similar pathologic outcome (1, 2). Cytokines play a pivotal role in the antigen-stimulated, cell-mediated, immune response and in the development of noncaseating granulomas (1, 3). The cellular response to cytokines is modulated, in part, by the interplay between the cytokine ligand, cell surface receptor, and corresponding soluble receptor. Cytokines and their soluble cytokine receptors act as both agonist and antagonists and are important in understanding the behavior of cells in the inflammatory response (4).
CBD is an occupationally acquired lung disease that begins
as a cell-medicated immune response to beryllium particulates that, over time, results in the development of noncaseating
granulomas. Our central hypothesis states that dysregulation
of the pro-inflammatory cytokine network underlies the progression from a protective immune response to disease. Studies from our laboratory and others have demonstrated that in
CBD, beryllium salts stimulate release of interleukin-2 (IL-2),
the soluble alpha subunit of the IL-2 receptor (-sIL-2R), and
interferon-
(IFN-) from bronchoalveolar lavage (BAL) cells
from CBD patients (5, 6), as well as T-lymphocyte proliferation (7, 8). Additional studies have shown that freshly isolated
alveolar macrophages from CBD patients express increased
levels of tumor necrosis factor- (TNF-) and IL-6 mRNA (5).
Research in other immunologic diseases also demonstrates
that in response to antigenic stimulation, the levels of TNF-, IL-6, and their soluble receptors rise at the site of inflammation (6, 9) and reflect disease status (10, 11). However, the interplay of cytokine ligand and sR in response to pathogenic antigen has not been studied in human lung disease. We hypothesized that beryllium salts would stimulate CBD-derived
BAL cells to produce TNF- and IL-6, as well as their soluble
receptors sTNF RI, sTNF RII, and sIL-6R. Furthermore, we
hypothesized that the levels of sTNF RI, sTNF RII, and sIL-6R would be elevated in the serum and BAL fluid (BALF) of
beryllium-sensitized and CBD patients and would serve as
molecular markers of disease severity.
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INTRODUCTION
METHODS
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Study Design
We evaluated the in vitro time course for TNF-, sTNF RI, sTNF RII,
IL-6, and sIL-6R production in unstimulated and beryllium salt, tetanus toxin-, lipopolysaccharide (LPS)-, and phytohemagglutinin (PHA)-
stimulated BAL cell supernatants. The clinical relevance of the soluble receptors as biomarkers of granulomatous disease was determined
by comparing their levels in serum and BALF samples with physiologic and radiographic measures of disease severity obtained at the
time of BAL.
Study Populations
CBD subjects. We evaluated 23 patients who met the following case definition of CBD: (1) history of occupational or environmental beryllium exposure, (2) histologic evidence of noncaseating granulomas on lung biopsy, and (3) BeSO4-stimulated blood and/or BAL lymphocyte proliferation. One patient had been on corticosteroids for 4 yr at the time of BAL.
Beryllium-sensitized subjects. The 16 beryllium-sensitized patients had (1) history of occupational or environmental beryllium exposure, (2) no histologic evidence of granulomas on biopsy, (3) at least two positive BeSO4-stimulated blood lymphocyte proliferation tests (LPT), (4) a negative BeSO4-stimulated BAL LPT, and (5) normal chest radiographs and spirometry at the time of testing. One patient was on a tapering dose of steroids for a skin rash at the time of BAL.
Control subjects. The 14 control subjects had no known exposure to beryllium, were free of respiratory symptoms and lung disease, and had normal chest radiographs and spirometry at the time of testing.
We obtained informed consent from all participants, according to the protocol approved by our institution's Human Subject's Review Board. Demographics and smoking status for the subset of subjects providing serum and BALF are detailed in Table 1. Differences in sex and smoking status did not confound our statistical analysis. The number of patients providing cells for each experiment is indicated in the test and figure legends.
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Sample Collection
BAL. Lavage was performed by standard methods reported previously (8). Briefly, we instilled four 60-ml aliquots of room temperature normal saline and harvested the fluid by gentle suction on the instilling syringe. The fractions were pooled, centrifuged, aliquotted,
and stored at 
20° C. Cell viability, evaluated by trypan blue exclusion, was 90% or higher. Cell counts were reported as total white
blood cells per milliliter of returned BALF (WBC/ml BALF). Cell
differential counts included macrophages, lymphocytes, eosinophils,
and neutrophils and excluded ciliated epithelial cells and erythrocytes,
reported as both percentage and as total number of cell type (Table 2).
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Serum. Venous blood was collected at the time of BAL in serum
separator tubes, aliquotted, and stored at 20° C.
Clinical Measurements
At the time of BAL, all beryllium-sensitized and CBD patients underwent clinical evaluations consisting of chest radiograph and measurements of pulmonary physiology (Table 2).
Radiography. A certified B-reader scored the severity of parenchymal infiltrates on chest radiographs using standard posteroanterior chest films according to the International Labour Organization classification system for radiographs of pneumoconiosis (12). Data were analyzed on a 0 to 10-point scale in which profusion ranks 0/- and 0/0 were combined.
Pulmonary physiology. We measured FVC and FEV1 with a recording spirometer or a pneumotachograph, reporting maximal values obtained from three satisfactory maneuvers. Normal predicted values were derived from the work of Morris and coworkers (13).
We determined the diffusing capacity for carbon monoxide by the single-breath method and calculated the results as the percentage of predicted normal values of Crapo and Morris (14). We evaluated the maximal exercise capacity and gas exchange during exercise using a Seimens Elema 380B cycle ergonometer, with continuous monitoring of cardiac rhythm and oxygen saturation (Hewlett-Packard, Waltham, MA). Mass spectrometry (1100 medical gas analyzer; Perkin-Elmer Medical Instruments, Pomona, CA) measured inspired air and expired oxygen and carbon dioxide concentrations. Using an ABL-2 blood gas analyzer (Radiometer, Copenhagen, Denmark), we measured arterial blood gases through an indwelling arterial line at rest and after each minute of graded exercise and reported gas exchange data as the alveolar-arterial oxygen gradient (A-a gradient).
Culture of BAL Cells
Fresh BAL cells were cultured in RPMI 1640 (Irvine Scientific, Santa
Ana, CA) containing 25 mM HEPES, 2 mM L-glutamine, 10% (vol/
vol) fetal bovine serum (Gibco BRL, Gaithersburg, MD), 100 U/ml
penicillin, and 100 mg/ml streptomycin at 1 × 106 cells/ml under standard mammalian tissue culture conditions. Cells were cultured in the
presence or absence of 100 µM BeSO4 (8) and in the presence or absence of 10 µg/ml final concentration PHA (Sigma Chemical Co., St.
Louis, MO), 1 µg/ml LPS, or 2 LFU/ml tetanus toxin (Wyeth Ayerst
Laboratories, Philadelphia, PA) as positive controls. At 0.25, 3, 6, 24, 72, and 120 h supernatants were harvested, centrifuged at 1,500 rpm
for 5 min, aliquotted, and stored at 20° C.
Quantification of Cytokines and Soluble Receptors
Cytokine and soluble receptor concentrations were measured with
commercially available solid-phase, two-site enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN). The minimum sensitivities reported for the TNF- and IL-6 ELISAs were 4.4 and 3.5 pg/ml, respectively. The minimum sensitivity for 10-fold dilutions of serum was 25 pg/ml for sTNF RI and 5 pg/ml for sTNF RII.
For measurements using cell culture supernatants, those values are 1.0 and 0.5 pg/ml, respectively. The minimum sensitivity for sIL-6R is 140 pg/ml in serum diluted 40-fold and 3.5 pg/ml in cell supernatants.
ELISA data are reported as the mean of duplicates and, in the statistical analysis, results for BALF were normalized against the number of
milliliters of fluid recovered.
Statistical Analysis
Time-response curves for BAL cells from control and CBD subjects, cultured in the presence and absence of BeSO4, were compared using repeated-measures analysis of variance models (15). To stabilize the variance estimates and to ensure more normally distributed residuals, data were log-transformed before analysis. When necessary, one was added to all data points before log transformation to avoid taking the log of zero. When the differences across time or the interaction between condition and time were significant, pairwise contrasts were used to determine which pairs of means differed significantly. For analyses presented on a log scale, the mean and SE of the original data are included. We tested for differences in cytokine and soluble receptor levels in serum and BALF between subject groups using Dunn's nonparametric multiple-comparisons procedure. We tested associations between clinical parameters and cytokine or soluble receptor concentrations by Spearman's correlation coefficient (rho). Differences between physiologic parameters for sensitized and CBD patients were evaluated by the Wilcoxin rank sum test and differences in BAL cellularity between normal, sensitized, and CBD subjects were tested by the Kruskal-Wallis method. Statistical significance was defined as p < 0.05. All tests were two-sided.
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RESULTS |
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INTRODUCTION
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Beryllium Sulfate Increased Release of TNF- and
IL-6 from BAL Cells of CBD Patients
To determine if beryllium salts stimulate increased release of
TNF- and IL-6 in CBD, we evaluated BAL cells from five
control and seven CBD subjects under different conditions of
stimulation and at selected points over a 120-h time course.
Cytokine measurements were transformed using natural logs,
and the differences between means across time and the interactions between group, time, and stimulating condition were
evaluated. We determined that the constitutively low levels of
TNF- and IL-6 measured in supernatants from unstimulated cells of control subjects were not altered by the addition of BeSO4. At 24 h, the median concentrations of TNF- in supernatants from unstimulated and beryllium-stimulated control
cells were 0.441 ng/ml (interquartile range, twenty-fifth and
seventy-fifth percentiles [IQR]: 0.054 ng/ml, 1.36 ng/ml) and
0.394 ng/ml (IQR: 0.106 ng/ml, 1.33 ng/ml) (p = 0.99), respectively. The 24 h median concentrations of IL-6 produced by
unstimulated and beryllium-stimulated control cells were
0.800 ng/ml (IQR: 0.462 ng/ml, 2.68 ng/ml) and 1.05 ng/ml (IQR:
0.237) ng/ml, 2.57 ng/ml) (p = 0.89), respectively.
In contrast, BeSO4-stimulated BAL cells from CBD patients produced statistically higher levels of TNF- and IL-6
(Figure 1). At 24 h, the median IL-6 level in unstimulated cell
supernatants was 0.321 ng/ml (IQR: 0.209 ng/ml, 0.756 ng/ml)
compared with 2.14 ng/ml (IQR: 1.82 ng/ml, 2.38 ng/ml) in supernatants from beryllium-stimulated cells (p = 0.0005). The
TNF- concentration increased from 0.209 ng/ml (IQR: 0.117 ng/ml, 0.416 ng/ml) to 3.48 ng/ml (IQR: 2.86 ng/ml, 5.06 ng/ml)
(p = 0.0001). Furthermore, the constitutive levels of TNF-
and IL-6 measured in unstimulated CBD-derived BAL cells
did not differ from those of control subjects, except for IL-6 at
24 h (control subject median: 0.800 ng/ml [IQR: 0.462 ng/ml,
2.68 ng/ml]; CBD median: 0.321 ng/ml [IQR: 0.209 ng/ml,
0.756 ng/ml] [p = 0.045]).
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Beryllium Sulfate Decreased sTNF RII Production from BAL Cells of Control Subjects
We also evaluated the release of sTNF RI and RII and sIL-6R in the BAL cell supernatants described above. For control subjects, the concentrations of sTNF RI and RII and sIL-6R in supernatants from unstimulated cells increased across the 120-h time course (p < 0.01), with median concentrations of sTNF RI, sTNF RII, and sIL-6R at 24 h measuring 68.2 ng/ml (IQR: 53.3 ng/ml, 92.0 ng/ml), 141.6 ng/ml (IQR: 113.5 ng/ml, 204.2 ng/ml), and 65.6 ng/ml (IQR: 33.4 ng/ml, 107 ng/ml), respectively. Soluble receptor levels in BeSO4-stimulated cell supernatants were 47.6 ng/ml (IQR: 43.1 ng/ml, 77.4 ng/ml) for sTNF RI, 86.0 ng/ml (IQR: 56.4 ng/ml, 92.8 ng/ml) for sTNF RII, and 52.1 ng/ml (IQR: 15.0 ng/ml, 88.2 ng/ml) for sIL-6R. Beryllium salts decreased significantly the concentration of sTNF RII (at 24 h, p = 0.014), whereas LPS stimulated an increase (at 24 h, 389.8 ng/ml [IQR: 161.6 ng/ml, 537.6 ng/ml]; p = 0.006). The concentrations of all three soluble receptors also increased across the 120-h time course in unstimulated BAL cell supernatants for CBD patients and were unaffected by beryllium stimulation (p > 0.05) (Figure 2). The median concentrations of sTNF RI, sTNF RII, and sIL-6R at 24 h were 55.8 ng/ml (IQR: 39.4 ng/ml, 70.6 ng/ml), 148.3 ng/ml (IQR: 98.9 ng/ml, 188.2 ng/ml), and 45.2 ng/ml (IQR: 28.6 ng/ ml, 65.6 ng/ml), respectively, for unstimulated conditions and 58.0 ng/ml (IQR: 41.9 ng/ml, 86.0 ng/ml), 159.0 ng/ml (IQR: 82.7 ng/ml, 196.6 ng/ml), and 45.0 ng/ml (IQR: 20.9 ng/ml, 66.0 ng/ml) in BeSO4-stimulated cell supernatants. Tetanus toxin and PHA induced small but statistically significant increases in the concentration of sTNF RI over unstimulated levels (76.8 ng/ml [IQR: 65.5 ng/ml, 88.1 ng/ml], p = 0.032 and 93.7 ng/ml [IQR: 66.9 ng/ml, 108.8 ng/ml], p = 0.002, respectively), whereas LPS stimulated a large increase in sTNF-RII (471.3 ng/ml [IQR: 228.5 ng/ml, 641.2 ng/ml], p = 0.0005).
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LPS, tetanus toxin, the PHA did not alter the constitutively produced concentrations sIL-6R in BAL cell supernatants for either CBD or control subjects. Furthermore, comparison of the log-transformed medians for each soluble receptor demonstrated no statistical difference between the CBD and control groups for unstimulated cell supernatants and for LPS-stimulated supernatants (at 24 h, p > 0.05).
Soluble Receptor Levels are Elevated in the Serum and BALF of Beryllium-sensitized and CBD Patients
Previous studies have shown that the severity of many immunologically mediated disorders correlates with elevations in
TNF- and IL-6 levels in serum and the disease organ (1, 11).
To determine if TNF- and IL-6 were elevated in our study
population, we measured by ELISA the concentration of
these cytokines in serum and BALF obtained from 12 control
subjects, 15 beryllium-sensitized patients, and 23 CBD patients. We did not detect measurable amounts of TNF- or
IL-6 in serum or BALF for control subjects or for most sensitized (n = 11) and CBD (n = 19) patients. The subset of patients with measurable cytokine levels had 4 to 7 pg/ml serum or BALF.
We did observe significant differences in soluble receptor concentrations in serum and BALF obtained from 14 control subjects, 16 beryllium-sensitized patients, and 23 CBD patients. We measured elevated concentrations of sIL-6R in the serum and BALF for beryllium-sensitized patients (median: 47.08 ng/ml; IQR: 38.48 ng/ml, 59.36 ng/ml) and CBD patients (median: 0.59 ng/ml; IQR: 0.36 ng/ml, 0.96 ng/ml) and in the BALF from CBD subjects (median: 0.86 ng/ml; IQR: 0.54 ng/ ml, 2.23 ng/ml) (Figure 3, Table 3). No statistical differences in sTNF RI concentration were observed in BALF; however, increased serum levels were measured for both beryllium-sensitized patients (median: 1.13 ng/ml; IQR: 1.06 ng/ml, 1.39 ng/ ml) and CBD patients (median: 1.17 ng/ml; IQR: 0.87 ng/ml, 1.42 ng/ml). sTNF RII levels were significantly higher in the serum (median: 2.26 ng/ml; IQR: 1.84 ng/ml, 3.11 ng/ml) and BALF (median: 0.76 ng/ml; IQR: 0.54 ng/ml, 2.23 ng/ml) of CBD patients.
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Correlation of Soluble Receptor Levels with Measures of CBD Severity
To determine if changes in the soluble receptor levels reflect
the severity of CBD, we correlated these measurements with the BAL cellular profile and with the clinical status of each subject. We observed significant associations between the number of WBC per milliliter of BALF and sIL-6R (rho = 0.50, p < 0.02) and sTNF RII (rho = 0.77, p < 0.01), as well as an association between the number of lymphocytes in the BALF and
the BALF concentration of sIL-6R (rho = 0.49, p < 0.02) and
sTNF RII (rho = 0.80, p < 0.01) (Table 4). The A-a gradient
was positively associated with serum sIL-6R (rho = 0.47, p < 0.50) and negatively correlated with BAL sTNF RII (rho = 0.59, p < 0.01). sTNF RII concentration in BALF was associated also with the profusion of small opacities on chest radiograph (rho = 0.52, p < 0.02). Serum sTNF RI levels correlated negatively with restrictive lung physiology for CBD patients, as reflected by FVC (rho = 0.45, p < 0.03) and no
other clinical parameters of disease. Additionally, no correlation was observed between the peak stimulation index determined from the beryllium-stimulated peripheral blood LPT
and the serum soluble receptor levels for either the beryllium-sensitized or CBD subjects (p > 0.05), consistent with previous soluble receptor studies using mitogen stimulation of peripheral blood mononuclear cells (PBMC) (15).
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This study demonstrates that TNF- and IL-6 participate in
the initiation of the CBD-derived BAL cell-mediated response to beryllium salts in vitro. TNF- and IL-6 have a central role in initiating the cell-mediated immune response to
antigen and in other granulomatous lung diseases (3). IL-6 is
involved in T-lymphocyte activation, probably controlling
early steps in T-lymphocyte activation and, in combination
with IL-1, induces T-lymphocyte production of IL-2 and IL-2R
(16). TNF- is a key mediator of inflammation and triggers
the release of cytokines that amplify and extend the inflammatory response (17). Furthermore, the effects of IL-6 are synergistic with TNF- (16). Our in vitro findings are also consistent
with previous observations that TNF- and IL-6 mRNA expression is increased in freshly isolated alveolar macrophages
from the beryllium-containing lungs of CBD patients (5), and
the absence of TNF- and IL-6 in BALF may be due to the dilutional effect of the lavage procedure.
In contrast to our findings for TNF- and IL-6, we measured no BeSO4-stimulated increase in sIL-6R, sTNF RI, and
sTNF RII from CBD-derived BAL cells and a BeSO4-induced
decrease in sTNF RII in supernatants from control subjects.
Constitutive soluble receptor release from unstimulated BAL
cells from both study groups is similar and measured approximately 0.1 to 0.4 ng soluble receptors/ml supernatant. These
concentrations of soluble receptors are similar to constitutive
levels reported by other investigators for T-lymphocyte cell
lines but significantly lower than the 0.5 to 10.0 ng soluble receptors/ml measured in supernatants from unstimulated PBMC
and the human monocytic cell line THP-1 (18). LPS stimulated significant increases in sTNF RII levels from CBD and
control BAL cells, and tetanus toxin stimulated a modest increase in sTNF RI levels in CBD patients. Others have shown
that PHA- and tetanus toxin-activated T-cell lines shed sTNF
RI and RII within 24 to 48 h of stimulation (20). None of the
stimulating conditions employed in this study increased either
control or CBD-derived sIL-6R levels, and no other studies
have reported an antigen-induced decrease in soluble receptor
levels. In combination, these data indicate that antigen-stimulated BAL cells respond differently, or on a different time
course, than T-lymphocyte cell lines and PBMC. It is also possible that beryllium salts affect directly cytokine and cytokine
receptor expression BAL cells. Biochemical research on the
cellular effects of beryllium has shown that beryllium inactivates enzymes by binding irreversibly to serine or threonine in
the active site (21, 22) or alters gene transcription by binding
to acidic nuclear proteins (23, 24). Further studies will be necessary to distinguish between the immunologic effects of beryllium as a hapten and its direct biochemical effects as a divalent cation.
An imbalance between cytokines that initiate the antigen-stimulated, cell-mediated immune response and the inhibitory
molecules that attenuate the response is thought to underlie
many disease processes, including CBD (6, 9, 25). Research
has demonstrated that sIL-6R, sTNF RI, and sTNF RII can act
as both agonists and antagonists. The sIL-6R (the gp80 subunit) enhances the response to antigen two ways: (1) by stabilizing the Il-6 molecule, thus extending its half-life, and (2) by
permitting trans-signaling in which the IL-6-sIL-6R complex
binds membrane-bound gp130, the IL-6R signal transducing
subunit (26). The gp130 subunit can be found on cells that do
not synthesize the gp80 subunit, thus extending the effects of
IL-6 to cells previously unable to respond. Other studies have
identified a soluble form of gp130 that is capable of binding to
the soluble IL-6-sIL-6R complex and neutralizing IL-6-sIL-6R
potentiation of the cell-mediated immune response (27). Data
from several laboratories have demonstrated that TNF- increases sTNF receptor levels (28) and that the agonist/antagonist effects of the sTNF receptors are concentration dependent
(28, 29). At high concentrations, sTNF RI and RII inhibit
TNF- activity and at low concentrations enhance bioavailability. In our BAL cell system, BeSO4 stimulated high levels
of TNF- and IL-6 but did not increase the concentration of
the corresponding soluble receptors that modulate the TNF-
and IL-6 response. Previous work in our laboratory documented beryllium salt-stimulated release of IFN- in vitro
(25). IFN- is a potent stimulus for soluble TNF receptor
shedding (30) and for alveolar macrophage release of sTNF RII,
in particular (31). We also demonstrated increased production
of -sIL-2R within 72 h of beryllium salt stimulation (25).
We evaluated the soluble receptor levels in the serum and BALF of three populations: control, beryllium sensitized, and CBD. Consistent with other reports, the control group had measurable levels of all three soluble receptors in serum and BALF, and these levels were the lowest recorded in our study (10, 32). Beryllium-sensitized patients manifest higher soluble receptor levels that are statistically significant for sIL-6R in BALF and sIL-6R and sTNF RI in serum. Although the sensitized population displays normal pulmonary physiology, elevated soluble receptor levels may indicate a subclinical, inflammatory response to beryllium antigen. Except for serum sIL-6R, the highest concentrations of soluble receptors were measured in the CBD group. Elevated sIL-6R and sTNF RII levels in BALF were correlated with cellularity and lymphocytosis, whereas serum sIL-6R and sTNF RII in BALF were associated with measures of gas exchange. These clinical parameters were previously reported to reflect disease severity and compartmentation of the beryllium-stimulated immune response to the lung (33). The moderate degree of association between soluble receptor levels and several clinical parameters observed in our study population may be due to a high degree of measurement variability or the limited range of observed values for these clinical parameters.
In addition to changes in the concentration of cytokine and soluble receptor, the pattern of their release may provide additional information about disease status (Table 5) (34). For example, sIL-6R levels are elevated in serum and BALF in the sensitized population but only in BALF in the CBD population. sTNF RI is increased in the serum of both sensitized and CBD subjects, but serum sTNF RII is elevated in CBD subjects only. It is an intriguing possibility that patients with an abnormal beryllium-stimulated peripheral blood LPT and elevated serum sIL-6R are more likely to be beryllium sensitized and those with increased serum sTNF RII are more likely to have noncaseating granulomas on biopsy.
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Further studies will be necessary to address the apparent incongruency between our BAL cell data and our serum and BALF data. It is possible that elevated soluble receptor levels in serum and BALF are indicators of chronic inflammation and alveolar lymphocytosis, whereas the in vitro BAL cell data reflect initiating events in the beryllium-stimulated, cell-mediated immune response. Continued research will also determine if soluble cytokine receptor levels indicate changes in the cell-mediated immune response that accompany progression from a protective response to a pathologic mechanism and if these proteins will serve as clinical markers of disease progression in CBD and other granulomatous lung diseases.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Sally S. Tinkle, Ph.D., National Institute for Occupational Safety and Health, Health Effects Laboratory Division, 1095 Willowdale Rd., Morgantown, W V 26505.
(Received in original form October 16, 1996 and in revised form July 29, 1997).
Acknowledgments: The authors wish to thank the patients for their participation in this research. They also thank Becki Bucher Bartelson, Ph.D., and Elaine Daniloff, M.S.P.H., for assistance with the statistical analysis; Lori A. Kittle, Elizabeth A. Barker, and Margaret M. Mroz, M.S.P.H., for helpful discussions; and Nina Rice for secretarial assistance.
Supported by FIRST Award ES-04843 (to L.S.N.), NHLBI Specialized Center of Research (SCOR) Grant HL-27353 (to L.S.N.), General Clinical Research Center Grant MO1 RR00051, a Clinical Research Grant from the National Jewish Center for Immunology and Respiratory Medicine Clinical Investigation Committee, U.S. Public Health Services Training Grant 5-T32-HL07085-20 (to S.S.T.), and National Research Service Award (NRSA) I-F32-Al/HL09448-01 (to S.S.T.).
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References |
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REFERENCES
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1. Chensue, S. W., K. S. Warmington, J. H. Ruth, P. Lincoln, and S. L. Kunkel. 1995. Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. J. Immunol 154: 5969-5976 [Abstract].
2. Newman, L. S.. 1995. Beryllium disease and sarcoidosis: clinical and laboratory links. Sarcoidosis 12: 7-19 [Medline].
3. Kunkel, S. L., S. W. Chensue, R. M. Strieter, J. P. Lynch, and D. G. Remick. 1989. Cellular and molecular aspects of granulomatous inflammation. Am. J. Respir. Cell Mol. Biol 1: 439-447 .
4. Howarth, P. H., P. Bradding, D. Quint, A. E. Redington, and S. T. Holgate. 1994. Cytokines and airway inflammation. Ann. N.Y. Acad. Sci 725: 69-82 [Medline].
5.
Bost, T. W.,
D. W. H. Riches,
B. Schumacher,
P. C. Carré,
T. Z. Khan,
J. A. Martinez, and
L. S. Newman.
1994.
Alveolar macrophages from
patients with beryllium disease and sarcoidosis express increased levels of mRNA for TNF- and IL-6 but not IL-1.
Am. J. Respir. Cell
Mol. Biol
10:
506-513
[Abstract].
6. Rydberg, J., H. Miörner, A. Chandramuki, and M. Lantz. 1995. Assessment of a possible imbalance between tumor necrosis factor (TNF) and soluble TNF receptor forms in tuberculous infection of the central nervous system. J. Infect. Dis 172: 301-304 [Medline].
7. Rossman, M. D., J. A. Kern, J. A. Elias, M. R. Cullen, P. E. Epstein, O. P. Preuss, T. N. Markham, and R. P. Daniele. 1988. Proliferative response of bronchoalveolar lymphocytes to beryllium: a test for chronic beryllium disease. Ann. Intern. Med 108: 687-693 .
8. Mroz, M. M., K. Kreiss, D. C. Lezotte, P. A. Campbell, and L. S. Newman. 1991. Reexamination of the blood lymphocyte transformation test in the diagnosis of chronic beryllium disease. J. Allergy Clin. Immunol 88: 54-60 [Medline].
9. Brennan, F. M., D. I. Gibbons, A. P. Cope, P. Katsikis, R. N. Maini, and M. Feldmann. 1995. TNF inhibitors are produced spontaneously by rheumatoid and osteoarthritic synovial joint cell cultures: evidence of feedback control of TNF action. Scand. J. Immunol 42: 158-165 [Medline].
10. Gaillard, J.-P., R. Bataille, H. Brailly, C. Zuber, K. Yasukawa, M. Attal, N. Maruo, T. Taga, T. Kishimoto, and B. Klein. 1993. Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy. Eur. J. Immunol 23: 820-824 [Medline].
11. van Deuren, M., J. van der Ven-Jongekrijg, A. K. M. Bartelink, R. van Dalen, R. W. Sauerwein, and J. W. M. van der Meer. 1995. Correlation between proinflammatory cytokines and anti-inflammatory mediators and the severity of disease in meningococcal infections. J. Infect. Dis 172: 433-439 [Medline].
12. International Labour Organization. 1980. Guidelines for the Use of ILO International Classification of Radiographs of Pneumoconioses. International Labour Office (Occupational Safety and Health Series, No. 22 [REV]), Geneva, Switzerland.
13. Morris, A. H., R. E. Kanner, R. O. Crapo, and R. M. Gardner. 1984. Clinical Pulmonary Function Testing: A Manual of Uniform Laboratory Procedures, 2nd ed. Intermountain Thoracic Society, Salt Lake City. 154-155.
14. Crapo, R. O., and A. H. Morris. 1981. Standardized single breath normal values for carbon monoxide diffusing capacity. Am. Rev. Respir. Dis 123: 185-189 [Medline].
15. Snedecor, W. G., and W. G. Cochran. 1989. Statistical Methods, 8th ed. Iowa State University, Ames, IA. 142-144, 193-194.
16. Hirano, T. 1994. Interleukin-6. In A. W. Thompson, editor. The Cytokine Handbook, 2nd ed. Academic Press, New York. 145-168.
17. Tracey, K. J. 1994. Tumour necrosis factor-alpha. In A. W. Thompson, editor. The Cytokine Handbook, 2nd ed. Acadmic Press, New York. 289-304.
18. Hwang, C., M. Gatanaga, G. A. Granger, and T. Gatanaga. 1993. Mechanisms of release of soluble forms of tumor necrosis factor/lymphotoxin receptors by phorbol myristate acetate-stimulated human THP-I cells in vitro. J. Immunol 151: 5631-5638 [Abstract].
19. Leeuwenberg, J. F., T. M. Jeunhomme, and W. A. Buurman. 1994. Slow release of soluble TNF receptors by monocytes in vitro. J. Immunol 152: 4036-4043 [Abstract].
20. Cope, A. P., D. Aderka, D. Wallach, M. Kahan, N. R. Chu, F. M. Brennan, and M. Feldmann. 1995. Soluble TNF receptor production by activated T lymphocytes: differential effects of acute and chronic exposure to TNF. Immunology 84: 21-30 [Medline].
21. Thomas, M., and W. N. Aldridge. 1966. The inhibition of enzymes by beryllium. Biochem. J 98: 94-99 [Medline].
22. Reiner, E. 1971. Binding of beryllium of proteins. In W. N. Aldridge, editor. Symposium on Mechanisms of Toxicity. St. Martin's Press, New York. 111-125.
23.
Sirover, M. A., and
L. A. Loeb.
1976.
Metal-induced infidelity during
DNA synthesis.
Proc. Natl. Acad. Sci. U.S.A
73:
2331-2335
24. Parker, V. H., and C. Stevens. 1979. Binding of beryllium to nuclear acidic proteins. Chem-Biol. Interact 26: 167-177 [Medline].
25.
Tinkle, S. S.,
L. A. Kittle,
B. A. Schumacher, and
L. S. Newman.
1997.
Beryllium induces IL-2 and IFN- in berylliosis.
J. Immunol
158:
518-526
[Abstract].
26. Rose-John, S., H. Schooltink, D. Lenz, E. Hipp, G. Dufhues, H. Schmitz, X. Schiel, T. Hirano, T. Kishimoto, and P. C. Heinrich. 1990. Studies on the structure and regulation of the human hepatic interleukin-6 receptor. Eur. J. Biochem 190: 79-83 [Medline].
27. Stoyan, T., U. Michaelis, H. Schooltink, M. Van Dam, R. Rudolph, P. C. Heinrich, and S. Rose-John. 1993. Recombinant soluble human interleukin-6 receptor: expression in Escherichia coli, renaturation, and purification. Eur. J. Biochem 216: 239-245 [Medline].
28. Winzen, R., D. Wallach, O. Kemper, K. Resch, and H. Holtmann. 1993. Selective up-regulation of the 75-kDa tumor necrosis factor (TNF) receptor and its mRNA by TNF and IL-1. J. Immunol 150: 4346-4353 [Abstract].
29.
Aderka, D.,
H. Engelmann,
Y. Maor,
C. Brakebusch, and
D. Wallach.
1992.
Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors.
J. Exp. Med
175:
323-329
30. Nicod, L. P., B. Galve-De-Rochemonteix, and J. M. Dayer. 1994. Modulation of IL-1 receptor antagonist and TNF-soluble receptors produced by alveolar macrophages and blood monocytes. Ann. N.Y. Acad. Sci 725: 323-345 [Medline].
31.
Galve-de-Rochemonteix, B.,
L. P. Nicod, and
J.-M. Dayer.
1996.
Tumor necrosis factor soluble receptor 75: the principal receptor form released by human alveolar macrophages and monocytes in the presence of interferon-.
Am. J. Respir. Cell Mol. Biol
14:
279-287
[Abstract].
32. Shapiro, L., B. D. Clark, S. F. Orencole, D. D. Poutsiaka, E. V. Granowitz, and C. A. Dinarello. 1993. Detection of tumor necrosis factor soluble receptor p55 in blood samples from healthy and endotoxemic humans. J. Infect. Dis 167: 1344-1350 [Medline].
33. Newman, L. S., C. Bobka, B. Schumacher, E. Daniloff, B. Zhen, M. M. Mroz, and T. E. King Jr.. 1994. Compartmentalized immune response reflects clinical severity of beryllium disease. Am. J. Respir. Crit. Care Med 150: 135-142 [Abstract].
34. Frieling, J. T. M., M. van Deuren, J. Wijdenes, J. W. M. van der Meer, C. Clement, C. J. van der Linden, and R. W. Sauerwein. 1995. Circulating interleukin-6 receptor in patients with sepsis syndrome. J. Infect. Dis 171: 469-472 [Medline].
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