1 Division of Pulmonary
Biology, Transforming growth factor-
Teflon; ultrafine particulates; macrophage inflammatory protein-2; interleukin-6
TRANSFORMING GROWTH FACTOR- In response to acute injury, TGF- The precise physiological actions of TGF- In the present study, we examined the effects of TGF- Experimental design. To induce acute
lung injury, mice were exposed to PTFE with the method described by
Oberdorster et al. (25). To determine whether TGF- Transgenic mice and Southern blot
analysis. Methods for generating transgenic mice
expressing hTGF- Aerosol generation. Air-generated PTFE
(Fluon, ICI Chemical) exposures were conducted with the method
described by Oberdorster et al. (25). Briefly, PTFE (0.75 g) was heated
(420-430°C; Lindberg Hevi-Duty Furnace, Sola Basics
Industries, Watertown, WI) to produce a mixture of ultrafine particles
(count median diameter 18 nm) and hydrogen fluorocarbon gases (flow
rate 5 l/min) (26). This mixture was combined with air (30 l/min) and
introduced into a 43-liter Plexiglas inhalation chamber. Before
exposure, PTFE was heated for >20 h to achieve a particle
concentration of 107
particles/cm3 (electric aerosol
size analyzer model 3030, Thermo-Systems, St. Paul, MN). In rats,
Johnston et al. (12) reported that this exposure produces pulmonary
edema, inflammation, and increased transcript levels of inflammatory
cytokine and antioxidant enzymes, consistent with oxidant-induced acute
lung injury.
Pulmonary histology. To assess lung
injury after PTFE exposure, mice were killed with
intraperitoneal pentobarbital sodium, the tracheae were cannulated, and
the lungs were inflated (1 min, 25 cmH2O) with 4% paraformaldehyde
in phosphate-buffered saline (PBS), pH 7.2. The lungs were washed in
PBS, dehydrated in graded alcohol and xylene, and embedded in paraffin
(60°C). Paraffin-embedded lungs were sectioned (5 µm) and placed
on Polysine glass slides (Erie Scientific, Portsmouth, NH). The slides
were deparaffinized with xylene and rehydrated with graded alcohol
washes and PBS. The sections were stained with hematoxylin and eosin.
Pulmonary function. Pulmonary function
was assessed in conscious mice by whole body plethysmography (model PLY
3115, Buxco Electronics, Troy, NY) as previously described (10, 18).
Chamber pressure was measured with a transducer (model TRD 5700)
connected to preamplifier modules (model CHA 0150) and analyzed by the
XA software of the system (model SFT 1810; all from Buxco Electronics). The chamber pressure was used as a measure of the difference between thoracic expansion (or contraction) and air volume removed from (or
added to) the chamber during inspiration (or expiration) and was
differentiated to produce a pseudoflow that was proportional to the
difference between the rate of change of the thoracic volume and nasal
flow. The mice were placed in each chamber until stable breathing
patterns were observed (2-6 min). Continuous measurements including respiratory frequency, tidal volume, expiratory time (TE), inspiratory time,
relaxation time (RT), peak expiratory flow (PEF), and peak inspiratory
flow (PIF) were obtained. Airflow obstruction was assessed by measuring
the enhanced pause (Penh) with
the manufacturer's recommended formula:
Penh = [(TE/0.3RT) BAL fluid cell differential and
protein. After death, the tracheae were cannulated, and
the lungs were lavaged three times with 1 ml of Hanks' balanced salt
solution (137 mM NaCl, 5.4 mM KCl, 0.44 mM
KH2PO4,
0.34 mM
Na2HPO4,
4.2 mM NaHCO3, and 5.6 mM glucose). BAL fluid was pooled and immediately cooled to 4°C. Differential cell counts were performed on Diff-Quick-stained (Baxter
Diagnostics, McGaw Park, IL) cytospin (Cytospin 3, Shandon Scientific)
slides of cells from 200 µl of BAL fluid. Two hundred cells per slide
were counted. Total BAL protein was measured with the bicinchoninic
acid method as previously described (20), with bovine serum albumin for
the standard.
Cytokine production. After death, the
lungs were homogenized in 2 ml of PBS, pH 7.2 and centrifuged at 1,500 g, and the supernatant was stored at
Statistics. Statistics for survival
time were based on Kaplan-Meier product limit survival curves, and
differences in survival time between groups was tested by log rank
test. For all other parameters, differences were compared with paired
and unpaired t-tests. A
P value < 0.05 was used as the level
of significance.
Survival. Average survival time for
the four transgenic lines and the wild-type control mice are presented
in Fig. 1. All TGF-
ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
) is produced in the lung in experimental and human lung
diseases; however, its physiological actions after lung injury are not
understood. To determine the influence of TGF-
on acute lung injury,
transgenic mouse lines expressing differing levels of human TGF-
in
distal pulmonary epithelial cells under control of the surfactant
protein C gene promoter were generated. TGF-
transgenic and
nontransgenic control mice were exposed to polytetrafluoroethylene
(PTFE; Teflon) fumes to induce acute lung injury. Length of survival of
four separate TGF-
transgenic mouse lines was significantly longer
than that of nontransgenic control mice, and survival correlated with
the levels of TGF-
expression in the lung. The transgenic line
expressing the highest level of TGF-
(line 28) and nontransgenic
control mice were then compared at time intervals of 2, 4, and 6 h of PTFE exposure for differences in pulmonary function, lung histology, bronchoalveolar lavage fluid protein and cell
differential, and lung homogenate proinflammatory cytokines. Line 28 TGF-
transgenic mice demonstrated reduced histological changes,
decreased bronchoalveolar lavage fluid total protein and neutrophils,
and delayed alterations in pulmonary function measures of airway
obstruction compared with those in nontransgenic control mice. Both
line 28 and nontransgenic control mice had similar increases in
interleukin-1
protein levels in lung homogenates. In contrast,
interleukin-6 and macrophage inflammatory protein-2 levels were
significantly reduced in line 28 transgenic mice compared with those in
nontransgenic control mice. In the transgenic mouse model, TGF-
protects against PTFE-induced acute lung injury, at least in part, by
attenuating the inflammatory response.
INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
) is a
polypeptide member of the epidermal growth factor (EGF) family that
binds and activates the EGF receptor (6). TGF-
is produced by most
epithelial cells and is mitogenic for both epithelial cells and
fibroblasts in many organs (2, 3, 23, 24, 29).
and EGF are produced in the lung.
Rodent lungs injured with intratracheal administration of asbestos,
naphthalene, or bleomycin produce increased TGF-
mRNA transcripts or
protein in the airway and alveolar epithelia as well as in the
interstitium (19, 21, 32). In humans, elevated TGF-
protein levels
are found in acute and chronic lung injury. TGF-
levels from
bronchoalveolar lavage (BAL) fluid increase in patients with
acute respiratory distress syndrome (22). Infants dying
from bronchopulmonary dysplasia have increased immunohistochemical staining for EGF and TGF-
in alveolar and airway epithelial cells and macrophages (30, 31). Lung biopsies from patients with idiopathic
pulmonary fibrosis and cystic fibrosis have increased immunohistochemical TGF-
staining in airway epithelial cells, peribronchial submucosal cells, and macrophages compared with that in
nondiseased control subjects (4, 9).
after lung injury is not
completely understood. In the lungs of rodents exposed to asbestos or
naphthalene, increased levels of TGF-
protein were detected in foci
of cellular proliferation (19, 32). An increased number of cells
expressing the nuclear proliferation antigen Ki67 and TGF-
was
detected in diseased submucosal areas of cystic fibrosis lungs,
suggesting that TGF-
may promote proliferation of various cells in
response to injury (9). In addition, TGF-
promotes wound healing in
the skin, gut, and lungs by augmenting cell motility and spreading (8,
13-16, 28). Finally, a recent study (1) supports a role for
TGF-
in inducing cellular resistance to the antiproliferative
activity of tumor necrosis factor-
(TNF-
), suggesting that
TGF-
may alter cellular signaling in inflammation.
expression in
acute lung injury produced by the inhalation of polytetrafluoroethylene (PTFE; Teflon) fumes. Lung injury from these fumes is caused primarily by ultrafine particulates generated from the heating of PTFE powder (25). We chose to expose mice to PTFE because this is a
well-characterized model of acute lung injury in rodents (12). A
previous study in rats (12) demonstrated that inhalation of PTFE caused
acute pulmonary hemorrhage and inflammation. In this model, acute
injury was associated with the recruitment of neutrophils and the
production of proinflammatory cytokines [TNF-
, interleukin
(IL)-6, and IL-1
], chemokines [macrophage inflammatory
protein-2 (MIP-2)], and antioxidants. To study the in vivo
effects of TGF-
on the pulmonary toxicity of PTFE, we used
previously generated transgenic mice expressing human TGF-
(hTGF-
) in distal respiratory epithelial cells under control of the
human surfactant protein C (SP-C) promoter (17). We exposed four
separate transgenic lines expressing different levels of TGF-
and
their nontransgenic controls to PTFE fumes and monitored survival time.
Survival was increased in all transgenic lines compared with the age-
and strain-matched nontransgenic control lines. Alterations in lung
morphology, BAL fluid protein and neutrophils, pulmonary function, and
proinflammatory cytokines and chemokines were markedly less in the
highest-expressing TGF-
transgenic mice (line 28) compared with
those in nontransgenic control mice. Taken together, these data suggest
that TGF-
reduces the extent of PTFE-induced acute lung injury by
modifying the inflammatory response.
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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protects against
acute lung injury, four transgenic and one nontransgenic mouse lines
were continuously exposed to PTFE for up to 50 h, and survival time was
recorded. The mouse lines were selected because each expresses different levels of TGF-
in the lung. To examine PTFE-induced lung
injury, pulmonary histology, physiology, and BAL fluid protein concentrations were compared between mice with the highest TGF-
expression (line 28) and age- and strain-matched (FVB/N) nontransgenic mice at time 0 and 2, 4, and 6 h of
exposure. To assess lung inflammation, BAL fluid cell differentials and
lung homogenate cytokine levels (TNF-
, IL-1
, IL-4, IL-6, and
MIP-2) were also determined in line 28 and nontransgenic mice.
under control of the human SP-C 3.7-kb
promoter/enhancer sequences were previously described (17). Mice from
four founder lines were studied. Line 28 mice express hTGF-
at high
levels, with average lung homogenate concentrations of 570 pg/ml by
ELISA. Line 28 mice develop a consistent pattern of lung pathology,
with emphysema and pleural and peribronchial fibrosis, but have no
differences in life span compared with nontransgenic littermate control
mice (10, 17). Line 2 mice express 540 pg/ml of hTGF-
(95% of line
28) and have less extensive emphysema with no fibrosis. Line 4 mice
express 230 pg/ml of hTGF-
(40% of line 28) and have minimal air
space enlargement and no fibrosis. Transgenic line 6108 mice express
120 pg/ml of hTGF-
(21% of line 28) and have no gross pathological
abnormalities. Mice were maintained in a virus-free environment and
handled in accordance with the Institutional Animal Care and Use
Committee of the Children's Hospital Research Foundation and the
University of Cincinnati Medical Center (Cincinnati, OH). Transgenic
mice were identified by a diagnostic 1.4-kb band on genomic Southern
blots of Pst I-digested genomic tail
DNA as previously described (17).
1] × (2PEF/3PIF). This measurement was calculated from the
mean data of repetitive samples obtained over 2 min.
70°C. TNF-
, IL-1
, IL-6, IL-4, and MIP-2 were determined with a quantitative murine sandwich enzyme-linked
immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the
manufacturer's directions. All plates were read on a microplate reader
(Molecular Devices, Menlo Park, CA) and analyzed with the use of a
computer-assisted analysis program (Softmax, Molecular Devices). Only
assays having standard curves with a calculated regression line value
>0.95 were accepted for analysis.
RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
transgenic mice
survived significantly longer than strain-matched nontransgenic mice.
Mouse lines with >500 pg/ml of transgenic TGF-
survived the
longest, with all line 2 and five of nine line 28 mice alive at 50 h of
exposure.
View larger version (28K):
[in a new window]
Fig. 1.
Survival time of nontransgenic and transforming growth factor (TGF)-
transgenic mice exposed to polytetrafluoroethylene (PTFE; Teflon)
ultrafine particles. Values are means ± SE;
n = 13 FVB/N nontransgenic mice, 5 TGF-
line 6108 mice, 5 line 4 mice, 4 line 2 mice, and 9 line 28 mice. * P < 0.01 compared with
FVB/N nontransgenic mice.
Histology. Two hours into PTFE
exposure, all nontransgenic mice demonstrated infrequent areas of
alveolar hemorrhage as well as evidence of early pulmonary edema
signified by perivascular swelling (Fig. 2,
arrow). By 4 h, extensive pulmonary edema was noted, with pronounced
perivascular swelling and interstitial thickening (Fig. 2, arrow).
Alveolar hemorrhage was extensive, and interstitial and alveolar
macrophages and neutrophils were increased. At 6 h, pathological
changes were similar to those at 4 h. Line 28 transgenic mice did not
exhibit significant histological changes at any point during PTFE
exposure compared with time 0 except
for small, infrequent foci of alveolar hemorrhage seen at 4 and 6 h.
|
In line 2 mice, the histological changes were similar to those described in line 28 mice. Small foci of alveolar hemorrhage were occasionally detected after 4 or 6 h of exposure. However, at 6 h, mild perivascular swelling was detected in two of five mice.
Physiological comparisons between nontransgenic and
transgenic mice. At time
0, no significant differences in pulmonary function were found between nontransgenic control and line 28 mice (Table 1). After PTFE exposure, PIF, PEF, and
respiratory frequency decreased for both nontransgenic and line 28 mice. These changes were accompanied by increases in inspiratory time,
TE, and
Penh. In line 28 mice, all
post-PTFE exposure changes in pulmonary function were attenuated
compared with those in nontransgenic mice, with significant differences
in respiratory frequency and TE
at 2 and 4 h and Penh at 2 h. Data
for pulmonary function were obtained over three separate experiments
and were reproducible.
|
BAL fluid protein and cellular
content. At time 0,
there were no differences in total protein recovered in BAL fluid from nontransgenic and line 28 transgenic mice. Increased BAL fluid protein
was observed in nontransgenic and line 28 mice after PTFE exposure;
however, this increase was attenuated in transgenic mice compared with
that in nontransgenic mice (Fig.
3).
|
The percent neutrophils in BAL fluid in line 28 mice after PTFE exposure did not change during the experimental period, with a mean percent neutrophils of 2.8 ± 0.2% at time 0, increasing to 3.7 ± 1.8% at 6 h. In contrast, the percent neutrophils increased >50-fold in nontransgenic mice, from 0.3 ± 0.2% at time 0 to 16.1 ± 7.7% at 6 h.
Cytokines. After PTFE exposure,
IL-1 levels increased equally in both line 28 transgenic and
nontransgenic mice (Fig.
4A). TNF-
and IL-4 levels at all time points increased only minimally from time 0 for both groups of mice
(data not shown). IL-6 and MIP-2 levels increased significantly in both
groups of mice from time 0 after PTFE
exposure; however, the increases were significantly less in line 28 mice. IL-6 levels were significantly decreased in line 28 transgenic
mice compared with those in nontransgenic mice at 4 and 6 h (Fig.
4B). MIP-2 levels were significantly
decreased in line 28 transgenic mice compared with those in
nontransgenic mice at each time point (Fig.
4C).
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DISCUSSION |
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The present work demonstrates the differences between TGF-
transgenic and nontransgenic mice in the development of acute lung
injury after PTFE exposure. All four TGF-
transgenic mouse lines
survived significantly longer than the nontransgenic strain-matched mice. Inflammation and airway obstruction were attenuated in the higher
TGF-
-expressing transgenic lines compared with those in nontransgenic mice. These findings suggest that hTGF-
expression protects the lungs from oxidant-induced injury by attenuating the
inflammatory response.
Increased survival was associated with higher TGF--expressing
transgenic lines and resembled a dose-response curve. Among the two
highest expressing lines, line 2 mice survived longer than the
higher-expressing line 28 mice. Previous characterizations (10, 17) of
line 28 mice demonstrated significant lung remodeling, with
emphysematous alveoli and pleural fibrosis associated with physiological abnormalities of increased compliance and airflow obstruction. In contrast, line 2 mice have less emphysema and no
baseline pulmonary function abnormalities. The lower survival among
line 28 mice may reflect their increased lung remodeling and
physiological abnormalities. Thus these findings implicate increasing
TGF-
gene dosage with increased survival.
The amount of PTFE particulate deposited into the lungs and alveoli of nontransgenic and transgenic mice was not quantified. We, therefore, cannot dismiss the possibility that lung remodeling of higher-expressing transgenic lines influenced particle deposition in the lungs. However, increased survival was demonstrated in transgenic lines where no lung remodeling was detected, and thus no change in PTFE distribution would be expected. In addition, previous studies (5, 11, 27) in animal models of emphysema have demonstrated that emphysematous lungs are generally no more or less susceptible to injury by gaseous and water-soluble irritants than normal lungs. Furthermore, because of the small size of the singlet ultrafine PTFE particle (10-20 nm), deposition would be more similar to an insoluble gas rather than to a longer diameter particle. Such particles may diffuse equally in emphysematous and nonemphysematous lungs.
The characterization of the inflammatory response in nontransgenic mice
after PTFE exposure is similar to the response in rats described by
Johnston et al. (12). In both Johnston et al.'s study and ours, an
increase in BAL fluid protein and neutrophils as well as a marked
increase in proinflammatory chemokines and cytokines, including MIP-2,
IL-6, and IL-1, was present. In TGF-
transgenic mice, this
inflammatory response was attenuated, suggesting that TGF-
may
possess anti-inflammatory properties. A direct effect of TGF-
on
chemokine and cytokine levels has not been described. However, TGF-
has been reported to decrease some of the biological activities of
TNF-
. Aggarwal et al. (1) demonstrated that either transfection or
exogenous addition of TGF-
to cells in vitro decreased the
antiproliferative effects of TNF-
. The mechanism of this protection
is unknown; however, the proliferative effects of TGF-
in cells are
mediated through tyrosine kinase protein phosphorylation, whereas the
antiproliferative effects of TNF-
are mediated through
dephosphorylation (1). Thus TGF-
may inactivate the protein tyrosine
phosphatase pathway, resulting in cellular resistance to the effects of
TNF-
.
In the present study, levels of TNF- and IL-4 were only minimally
increased and no differences between transgenic and nontransgenic mice
were found. However, IL-6 and MIP-2 were significantly lower in TGF-
transgenic mice compared with nontransgenic control mice after PTFE
exposure. MIP-2 is a known chemoattractant for neutrophils (7). In
TGF-
mice, neutrophil counts were unchanged, in association with
only modest increases in MIP-2 levels after exposure. In contrast, in
nontransgenic control mice, neutrophil counts increased 50-fold 6 h
into PTFE exposure in association with marked increases in MIP-2
levels. Thus TGF-
may inhibit MIP-2 release after PTFE exposure,
thereby reducing neutrophil migration to the lung and preventing
neutrophil-mediated lung injury. The effects of TGF-
on the levels
of inflammatory mediators were specific to MIP-2 and IL-6 because
IL-1
levels were equally increased in both transgenic and
nontransgenic mice. TGF-
-mediated protection of the lung may be due
to selective inhibition of inflammatory cytokines and chemokines.
In summary, transgenic mice overexpressing TGF- demonstrated
increased survival and reduced inflammation after acute lung injury
caused by exposure to PTFE. Transgenic mice had reduced BAL fluid
neutrophils and little evidence of lung injury at the same study points
where nontransgenic mice demonstrated hemorrhagic inflammation with
increased neutrophil influx. There was a marked difference in IL-6 and
MIP-2 levels in lung homogenates between transgenic and nontransgenic
mice, whereas TNF-
, IL-4, and IL-1
levels were similar. These
findings suggest that TGF-
protects against acute lung injury by
altering the extent of pulmonary inflammation.
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ACKNOWLEDGEMENTS |
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We thank Kim Kurak and Jaymi Semona for assistance with mouse husbandry, Patricia Carrigan for assistance with pulmonary function testing and graphs, and Jennifer Westrich and Ann Maher for assistance with manuscript preparation.
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FOOTNOTES |
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This work was supported in part by a Trustee's Grant from the Children's Hospital Research Foundation (Cincinnati, OH); grants from the Center for Environmental Genetics (Cincinnati, OH); National Institute of Environmental Health Sciences Grant ES-06096; National Heart, Lung, and Blood Institute Grant HL-58275; and the Health Effects Institute (Cambridge, MA).
This work was presented in part at the American Thoracic Society meeting in Chicago, IL, in April 1998.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. D. Hardie, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: bill.hardie{at}chmcc.org).
Received 29 January 1999; accepted in final form 1 July 1999.
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