Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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To characterize the time course and localize the
production of proinflammatory cytokines after inhalation of corn dust,
we exposed mice (C3H/HeBFeJ) by inhalation challenge to sterile corn dust extract (CDE) and contrasted this response to inhalation of
Escherichia coli 0111:B4
lipopolysaccharide (LPS) or pyrogen-free saline. After both CDE and LPS
exposure, an increase in the concentration of bronchoalveolar lavage
neutrophils was detected 1 h postinhalation and persisted for 48 h.
Significant increases in the bronchoalveolar lavage concentration of
tumor necrosis factor (TNF)-, interleukin (IL)-1
, and macrophage
inflammatory protein (MIP)-2 resulted after inhalation of either CDE or
LPS. Although the time courses of these cytokines were distinct, a
similar pattern of release was observed after both CDE and LPS
exposure. A single inhalation exposure of either CDE or LPS resulted in
enhanced expression of mRNA for TNF-
, IL-1
, and MIP-2 that was
evident and most pronounced within 1 h of the inhalation challenge.
Although enhanced expression of mRNA for TNF-
was detectable 12 h
after completion of the inhalation challenge, IL-1
and MIP-2 mRNA
expression remained elevated through the 24-h time point. TNF-
,
IL-1
, and MIP-2 expression was localized by in situ hybridization to
inflammatory cells in the airways and alveoli from 1 to 24 h in both
CDE- and LPS-exposed lungs. Interestingly, there was no convincing
evidence that MIP-2 was substantially produced by airway epithelial
cells. The pattern, timing, and location of expression of TNF-
,
IL-1
, and MIP-2 mRNA after a single inhalation exposure of CDE in
comparison with LPS is similar, supporting a common etiology and
mechanism of inflammation in the lower respiratory tract. Moreover, our findings indicate that inhalation of corn dust or LPS results in an
acute inflammatory process that is primarily mediated by inflammatory
cells and appears to be self-limited.
endotoxin; lipopolysaccharide; kinetics; bronchoalveolar lavage
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INTRODUCTION |
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OCCUPATIONAL and environmental exposure to grain dust
can cause a spectrum of clinical syndromes including asthma, acute and chronic changes in airway reactivity, bronchitis, and chronic obstructive lung disease (4). Among grain handlers, the prevalence of
work shift changes in forced expiratory volume in 1 s
(FEV1; 10% decline) varies
between 3.9 and 11% (5, 10, 13). Exposure to grain dust causes
seasonal decrements in airflow (23, 24, 28) and an accelerated
longitudinal decline in FEV1 (6,
19, 29, 33, 41). Chronic bronchitis occurs in a high prevalence (23-37%) of grain handlers (14, 15, 19, 29). Moreover, acute
changes in airflow across either the work shift or workweek are
consistently associated with accelerated longitudinal declines in lung
function (2, 6, 23, 33, 41). These observations indicate that grain
dust-induced airway disease is a common occupational and environmental
problem and that understanding the inflammatory response associated
with acute inhalation may provide necessary information to prevent the
chronic manifestations of grain dust-induced airway disease.
Although the pathogenesis of grain dust-induced airway disease is not
entirely understood, the acute inflammatory response to inhaled grain
dust involves recruitment of inflammatory cells, shedding of airway
epithelial cells, and release of specific cytokines (8). After
inhalation of grain dust, healthy, nonasthmatic, nonatopic study
subjects without airway hyperreactivity develop significant declines in
airflow (FEV1 decreases by
10-40%) that are associated with a neutrophilic alveolitis and an
increase in the concentration of specific proinflammatory cytokines
[tumor necrosis factor (TNF)-, interleukin (IL)-1
, IL-6,
and IL-8] (8, 22). Inhalation studies in mice (11, 21, 37) have shown that after a single exposure to grain dust, neutrophils are
rapidly recruited to the lung, and these cells produce and release
proinflammatory cytokines (TNF-
, IL-1
, and IL-6) and chemokines
[macrophage inflammatory protein (MIP)-2]. However, the
specific source of these mediators of inflammation are not known nor is
the temporal pattern of production well defined.
These previous studies led us to hypothesize that acute injury to the
airway epithelium and the associated airflow obstruction induced by
grain dust is mediated by proinflammatory cytokines and chemokines that
are produced and released from alveolar and airway macrophages,
recruited polymorphonuclear neutrophils (PMNs), and airway epithelial
cells. Furthermore, we speculate that the inflammatory response to
grain dust is compartmentalized, with TNF- and IL-1 (
and
)
being primarily expressed by inflammatory cells (macrophages and PMNs),
whereas MIP-2 [the murine homologue of IL-8 (40)] is
produced by both inflammatory cells and the airway epithelium. The
purpose of this study was to examine the kinetics and localization of
proinflammatory cytokines (TNF-
and IL-1
) and a chemokine (MIP-2)
that appear to be important in mediating the acute inflammatory
response to inhaled corn dust in mice. This study represents an
important step in determining whether these cytokines and chemokines
play a role in grain dust-induced airway disease. Our results indicate
that a single exposure to corn dust produces inflammation of the lower
respiratory tract that is mediated by macrophages and PMNs, may persist
for several days, and is very similar to the inflammatory response
induced by similar doses of inhaled lipopolysaccharide (LPS).
Additionally, although inflammatory cells in the lung actively produce
TNF-
, IL-1
, and MIP-2, the airway epithelium does not appear to
be involved in the production of these cytokines and chemokines.
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METHODS |
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General experimental protocol. Endotoxin-sensitive (C3H/HeBFeJ) mice were exposed for 4 h to either an aerosol of corn dust extract (CDE), LPS, or sterile saline in a 75-liter glass exposure chamber. After exposure, the mice were euthanized at selected time points and underwent whole lung lavage. The end of the exposure was designated as time 0. Lavage fluid was collected and analyzed for its cellularity and protein concentration. Whole lungs from animals not lavaged were dissected and either quick-frozen for RNA extraction or fixed in 4% paraformaldehyde followed by 20 and 30% sucrose and quick-frozen in liquid nitrogen for in situ hybridization.
Animals. Endotoxin-sensitive (C3H/HeBFeJ) male mice (Jackson Laboratories, Bar Harbor, ME) were obtained at 6 wk of age and used within 2 wk of their arrival. The mice were maintained in an approved animal care facility. All animal care and housing requirements set forth by the National Institutes of Health Committee on Care and Use of Laboratory Animal Resources were followed. Animal protocols were approved by the Institutional Animal Care and Use Committee.
Chemicals. LPS was purchased as lyophilized purified Escherichia coli 0111:B4 (Sigma, St. Louis, MO). The corn dust used in these experiments was obtained from the air filtration systems at an eastern Iowa grain-handling facility. Sterile pyrogen-free saline was obtained from Baxter Medical Laboratories (Deerfield, IL).
CDE preparation. We prepared the
extracts with standard methods (21) by adding 3.0 g of dust to 30.0 ml
of Hanks' balanced salt solution, vortexing for 2 min, and shaking for
1 h at 4°C. Insoluble particles were removed from the suspension by
centrifugation at 800 g for 20 min.
CDE was passed through a sterile 0.22-µm low-protein-binding
nonpyrogenic polyvinylidene difluoride filter (Acrocap, Gelman
Sciences, Ann Arbor, MI) to yield sterile CDE. The resulting filtrate
was adjusted to pH 7.0, tested for sterility, and frozen at
70°C.
Endotoxin assay. The endotoxin concentrations of CDE solution, LPS solution, saline, and resulting aerosols were assayed with the chromogenic Limulus amoebocyte lysate assay (QCL-1000, BioWhittaker, Walkersville, MD) with sterile pyrogen-free labware and a temperature-controlled microplate block and microplate reader (405 nm). The extracts and LPS solutions were serially diluted in pyrogen-free water (pfw) and assayed. The airborne concentration of endotoxin was assessed by sampling 0.30 m3 of air drawn from the exposure chamber through 47-mm binder-free glass microfiber filters (EPM-2000, Whatman, Maidstone, UK) held within a 47-mm stainless in-line air-sampling filter holder (Gelman Sciences). Air-sampling filters were extracted with 10 ml of pfw at room temperature with gentle shaking for 1 h. The extracts were then serially diluted with pfw and assayed for endotoxin. Four to six air samples were assayed for each exposure. All standard curves achieved a linear regression coefficient exceeding r = 0.995. Our laboratory routinely runs spiked samples and filter blanks and participates in interlaboratory validation studies. The concentration of endotoxin was 0 µg/m3 in the saline aerosol, 8.3 µg/m3 for the CDE aerosol, and 7.5 µg/m3 for the LPS aerosol.
Exposure and monitoring apparatus. CDE
and LPS aerosols were generated and directed into a glass 75-liter
exposure chamber by use of a PITT no. 1 nebulizer supplied with extract
by a syringe pump. Liquid feed rates ranged from 0.0027 to 0.21 ml/min.
High-efficiency particle apparatus-filtered air was supplied to the
nebulizer at flow rates of 10-17 ml/min. Mixing within the chamber
was aided by a magnetically coupled rotor. The chamber atmosphere was
exchanged at a rate of one change per minute. Aerosol concentrations
were determined with an aerodynamic particle sizer (TSI, St. Paul, MN)
and gravimetrically with a Marple personal cascade impactor sampling
within the chamber. Count median aerodynamic diameter for the aerosols
was 1.3-1.4 µm for all three exposures. Mass median
aerodynamic diameters determined with an eight-stage cascade impact
were 1.2 µm for each of the three exposures. The mass
median diameter (g) for the
mass distributions were between 1.4 and 1.7. Thus 80% of
the mass for the aerosol exposures was represented by particles with
<2-µm aerodynamic diameter.
Measurement of cytokines in lavage
fluid. Standard, commercially available ELISA kits were
used for measurements of murine TNF- (R&D Systems, Minneapolis, MN),
IL-1
(Endogen, Boston, MA), and MIP-2 (R&D Systems). In all cases, a
monoclonal antibody was used as a capture reagent in a standard
sandwich ELISA. The assays were performed according to the
manufacturer's instructions, and standard curves were derived with
known concentrations of the recombinant specific cytokine supplied by
the manufacturer. All assays were performed with the standard diluent
supplied in the ELISA kit.
Preparation of RNA and multiprobe RNase protection
assay. We extracted the total RNA from lung specimens
with the single-step method (7, 25), lysing flash-frozen lungs in RNA
Stat-60 (Tel-Test "B," Friendswood, TX). The composition of RNA
Stat-60 includes phenol and guanidinium thiocyanate in a monophase
solution. The lung parenchyma was homogenized in the RNA Stat-60 with a polytron homogenizer. Chloroform was added, the total RNA was precipitated from the aqueous phase by addition of isopropanol, and the
total RNA was washed with ethanol and solubilized in water. After
drying the pellet in a vacuum desiccator, we quantitated the yield and
purity of RNA by measuring the ratio of absorbances at 260 and 280 nm.
Minigel electrophoresis was used to confirm the integrity of the 28S
and 18S rRNA bands. Gene transcripts were detected with the RNase
protection assay and probes as previously described (18). Five
micrograms of total RNA were hybridized with a
32P-labeled antisense cRNA probe
in a solution of hybridization buffer for 14 h at 56°C. The
nonhybridized single-stranded RNA was digested with a mixture of RNases
A and T1. The remaining protected
RNA fragment was extracted with phenol-chloroform-isoamyl alcohol
(25:24:1) and precipitated with ethanol. The protected hybridization
products were separated on a 5% acrylamide-8 M urea gel. The gel was
dried on a vacuum gel dryer at 80°C, wrapped in plastic wrap, and
exposed to X-ray film at 70°C. Densitometry was performed to
control for the concentration of RNA in each lane with Sigma gel
(Jandel Scientific Software, San Rafael, CA).
In situ hybridization.
Eight-micrometer-thick paraformaldehyde-fixed sucrose-embedded sections
of mouse lung were cut at 20°C and mounted onto Superfrost
Plus glass slides. Slides were fixed for 10 min in 4%
paraformaldehyde, rinsed in 2× saline-sodium citrate (SSC), and
digested with 1 µg/ml of proteinase K. To acetylate amino groups in
the tissue and reduce the electrostatic nonspecific binding of the
probe, we incubated the slides in triethanolamine buffer containing
acetic anhydride (0.25%). Sections were rinsed with 2× SSC and
dehydrated with ethanol. Slides were hybridized overnight at 50°C
in a hybridization buffer containing a 12.0 × 103
counts · min
1 · µl
labeled probe
1 (42). After
hybridization, the slides were rinsed in 4× SSC containing 10 mM
1,4-dithiothreitol at 50°C until the coverslips fell off. The
slides were then rinsed in an RNase solution, dehydrated through an
ethanol series, and air-dried. Autoradiography was performed with NTB-2
liquid emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 with
distilled water at 42°C. Slides were coated with emulsion, allowed
to dry, packed in a lighttight slide box containing desiccant, and
stored at 4°C. Slides were developed in D19 developer for 3 min,
rinsed in distilled water for 30 s, and fixed for 3 min. Slides were
counterstained in hematoxylin and dehydrated through an ethanol series.
The following cDNA clones were used to generate
35S-labeled sense and antisense
cRNA probes for the in situ studies: murine (mu) TNF-
(pMuTNF,
American Type Culture Collection, Mannasas, VA), muIL-1
(pMuIL1
cDNA, American Type Culture Collection), and muMIP-2 (a kind
gift from Dr. Barbara Sherry, Laboratory of Medical
Biochemistry, Rockefeller University, New York, NY). All cDNA clones were cloned into a pBluescript II SK vector
(Stratagene, La Jolla, CA).
Statistical analysis. Comparisons were performed to investigate the kinetics of the inflammatory response to inhaled CDE and LPS. At each time point, for each response, the concentrations of lavage cells and cytokines were compared with those of saline-exposed mice. In addition, the relative effect of CDE and LPS was evaluated by directly comparing the inflammatory response of CDE and LPS at each time point after the inhalation challenge. The Mann-Whitney U-test was used to assess significance of these comparisons (35).
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RESULTS |
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Similar cellular inflammatory changes in the lower respiratory tract
were observed after exposure to CDE and LPS (Fig.
1). Moreover, the kinetic profile of
cellular changes in the lavage fluid was remarkably similar after
exposure to either CDE or LPS. A marked increase in the concentration
of PMNs was noted in the lavage fluid after exposure to either of these
agents. The concentration of PMNs peaked at 1 h after exposure to CDE
and 4 h after inhalation of LPS. Elevations in lavage concentration of
PMNs persisted for 48 h. A small but significant elevation in lavage
macrophage concentration was present 48 h after exposure to CDE but not
to LPS. In addition, a small but significant increase was observed in
the concentration of lymphocytes after inhalation of either CDE
(4-48 h) or LPS (24-48 h). There were no significant changes
in the concentration of eosinophils in the lavage fluid at any time
point (data not shown). No significant differences were observed in the
cellular inflammatory responses between CDE and LPS at any of the time points postexposure. At 96 h after inhalation of either CDE or LPS, the
concentrations of PMNs, macrophages, and lymphocytes were similar to
baseline or preexposure levels.
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Inhalation of CDE and LPS resulted in significant increases in the
concentration of proinflammatory cytokines in the lavage fluid of mice
(Fig. 2). Again, the kinetic profile of
cytokine inflammatory changes in the lavage fluid was similar after
exposure to either CDE or LPS. Concentrations of inflammatory mediators TNF-, IL-1
, and MIP-2 were significantly increased in the initial postexposure lavage fluid for both CDE and LPS. Lavage fluid TNF-
concentrations peaked at 1 h for CDE inhalation and 4 h for LPS inhalation and remained significantly elevated for 48 h after inhalation of either CDE or LPS. Lavage IL-1
and MIP-2
concentrations peaked at 1 h after inhalation of either CDE or LPS.
IL-1
levels returned to baseline at 96 h after the inhalation
challenge. For CDE inhalation, MIP-2 levels returned to baseline at 48 h, and for LPS, MIP-2 levels returned to baseline at 24 h.
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Similar patterns of mRNA cytokine expression for TNF-, IL-1
, and
MIP-2 were observed in the lungs after inhalation of CDE and LPS at
each of the time points (Fig. 3). TNF-
mRNA was expressed in CDE- and LPS-exposed mouse lungs at 1 h (the
earliest time point) and returned to near baseline at 24 h. IL-1
and
MIP-2 mRNA expression peaked at 1 h and continued to be expressed
through 24 h after exposure to either CDE or LPS. No cytokine mRNA
expression was observed in the lungs of saline-exposed mice at any time
point postinhalation. Comparing densitometry measures and controlling for the total RNA in each sample, we observed no substantial difference in the induction or kinetics of mRNA for specific cytokines after inhalation of CDE or LPS (densitometry values not presented).
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TNF-, IL-1
, and MIP-2 mRNAs were detectable in CDE- and
LPS-exposed mouse lungs with the use of
35S-labeled antisense cRNA probes
for in situ hybridization (Figs. 4-6,
respectively). TNF-
and IL-1
mRNAs were present in inflammatory cells scattered throughout the airways and alveoli from 1 through 12 h.
This was observed for samples obtained after inhalation of either CDE
or LPS but was not observed after inhalation of saline. In situ
hybridization of lavage cytospins confirmed expression of TNF-
and
IL-1
mRNAs by macrophages and PMNs. Occasional hybridization was
seen at 24 h for TNF-
and IL-1
but at a much lower level; the
persistence of mRNA for TNF-
and IL-1
as assessed by in situ
hybridization (in comparison with the results from the RNase protection
assay) is most likely due to the increased sensitivity of in situ
hybridization. MIP-2 was present in inflammatory cells throughout the
airway from 1 through 24 h in lung tissue in both CDE- and LPS-exposed
samples. Interestingly, the airway epithelium does not convincingly
express mRNA for MIP-2 after inhalation of either CDE or LPS (Fig.
7). When tissue from CDE-, LPS-, and saline-exposed lungs was hybridized with
35S-labeled TNF-
, IL-1
, or
MIP-2 sense cRNA probes, no message was detected at any time point
(Figs. 4-6). Also, there was no expression of mRNA for TNF-
,
IL-1
, or MIP-2 in lungs obtained from mice exposed to saline.
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DISCUSSION |
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The results from our study indicate that inhaled corn dust causes an
inflammatory response in the lower respiratory tract of mice that is
similar to that with inhaled LPS. These inflammatory responses persist
for several days after the inhalation challenge, are largely mediated
by airway inflammatory cells, and are ultimately self-limited. Although
airway inflammatory cells produce and release TNF-, IL-1
, or
MIP-2 into the air spaces, the airway epithelium does not appear to be
involved in the production or release of any of these proinflammatory proteins.
The results presented in this study demonstrate that the kinetics and pattern of cell recruitment and cytokine release and/or expression are remarkably similar in mice after inhalation of corn dust or LPS. These findings further support a growing body of literature that indicates that endotoxin is one of the primary agents in grain dust that causes airway inflammation and airflow obstruction. Previously, our laboratory (38) has shown that the concentration of endotoxin in the bioaerosol appears to be the most important occupational exposure associated with the development of airway disease in grain handlers. Physiologically, inhaled endotoxin (3, 17, 30, 31, 36) or grain dust (8, 9, 12) can cause airflow obstruction in naive or previously unexposed subjects. Our laboratory's previous exposure-response studies have shown that inhaled grain dust and endotoxin produce similar physiological and biological effects (8, 22), the concentration of endotoxin appears to play an important role in the acute biological response to grain dust (21, 22, 37), a competitive antagonist for LPS (Rhodobacter sphaeroides diphosphoryl lipid A) reduces the inflammatory response to inhaled grain dust (20), and genetic or acquired hyporesponsiveness to endotoxin substantially reduces the biological response to grain dust (37). In aggregate, these findings, in conjunction with those presented with this study, clearly indicate that endotoxin is one of the principal agents in grain dust that causes the acute biological and physiological responses that characterize this disease.
Because inflammatory cells in the airway appear to play a major role in
mediating the inflammatory response to inhaled grain dust, we speculate
that macrophages and PMNs are essential to the release of these
cytokines. This hypothesis is supported by findings by Xing and
colleagues who observed that PMNs and macrophages are the primary
source of TNF- (44) and MIP-2 (43) in a rat model of acute
inflammation induced by intratracheal instillation of LPS. Furthermore,
our results indicate that the airway epithelium is not involved in the
production of MIP-2. Importantly, epithelial cells (in vitro) respond
poorly to LPS and appear to require a specific host-derived signal
(TNF-
or IL-1) for induction of IL-8 (1, 27, 32, 34, 39, 40), the
human chemokine equivalent of MIP-2 (21). The inability to respond to
LPS has been demonstrated in vitro in A549 cells (39) and bronchial epithelial cells (27, 32); however, if these cells are exposed directly
to either TNF-
or IL-1, they are able to produce and release IL-8.
In a baboon model of sepsis, pretreatment with anti-TNF-
antibody
significantly reduced the circulating concentration of IL-8 (42),
suggesting that TNF-
and/or IL-1 is needed to stimulate other cells
to release IL-8 and promote neutrophil chemotaxis. However, other
causes of lung inflammation, such as IL-1
(45) and
-quartz (16),
have been shown to directly induce airway epithelia to upregulate the
expression of MIP-2 mRNA. Our findings suggest that inhaled organic
dust initiates a complex interaction between macrophages and other
inflammatory cells (primarily PMNs) mediated by specific
proinflammatory cytokines released by these cells. Because MIP-2 is not
produced by the airway epithelium, other factors such as adhesion
proteins must function to localize the inflammatory cells to the
airway. Alternatively, the airway epithelium may produce MIP-2 at a
much earlier time point. Because our inhalation protocol takes 4 h and
the initial specimens are obtained 1 h after the exposure ends, it is
conceivable that MIP-2 (or any of the other cytokines) was produced by
the airway epithelium before the sample period.
A key, unanswered question is the relationship between the acute reversible inflammatory and physiological response to inhaled grain dust and the development of chronic airway disease (4). Our model of grain dust-induced airway disease represents an excellent opportunity to study this relationship for the following reasons. First, we have developed a reliable and valid model of the acute physiological and biological response to inhaled grain dust in humans (8, 11, 22) and mice (11, 20, 21, 37). Second, our laboratory (11) has shown that the acute physiological and inflammatory response to inhaled grain dust is self-limited, with airflow obstruction persisting for 48 h, and the inflammatory response, most prominent within hours of exposure, ultimately resolves. These findings indicate that a single inhalation challenge of grain dust results in airflow obstruction and lower respiratory tract inflammation that may last for several days but is self-limited, completely resolving 1 wk after challenge. Third, our laboratory (11) demonstrated similar inflammatory responses in mice and humans after inhalation of grain dust, suggesting that the mouse model provides an opportunity to study the pathogenesis of grain dust-induced airway disease in humans. Fourth, as reviewed above, endotoxin appears to be one of the primary agents in grain dust, causing airflow obstruction and airway inflammation. On the basis of this finding, one could develop specific interventions directed at either endotoxin or the cytokines involved in the inflammatory response to endotoxin to downregulate the inflammatory response to inhaled grain dust. Fifth, chronic exposure to grain dust causes accelerated longitudinal declines in FEV1 (6, 19, 29, 41), is associated with a high prevalence of chronic bronchitis handlers (14, 15, 19, 29), and causes irreversible and progressive airway disease (26). Finally, because acute changes in airflow across either the work shift or workweek are consistently associated with accelerated longitudinal declines in lung function (6, 23, 41), modifying the acute response to inhaled grain dust is likely to alter the risk of developing chronic grain dust-induced airway disease.
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ACKNOWLEDGEMENTS |
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We appreciate and acknowledge the support of Dr. Peter Thorne and the Inhalation Toxicology Facility in the Environmental Health Science Research Center.
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FOOTNOTES |
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This study was supported by National Institute of Environmental Health Sciences Grants ES-06537, ES-07498, ES-05605, and ES-97004; National Heart, Lung, and Blood Institute Grant HL-62628; and the Department of Veterans Affairs (Merit Review).
Address for reprint requests and other correspondence: D. A. Schwartz, Pulmonary Diseases, Critical Care, and Occupational Medicine Div., Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242-1081 (E-mail: david-schwartz{at}uiowa.edu).
Received 18 November 1997; accepted in final form 19 January 1999.
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