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From the Department of Pathology, College of Medicine, University of Vermont, Burlington, Vermont
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.2 mg/m3
air) inhalation exposures to
crocidolite or chrysotile asbestos fibers induce transient inflammation
in bronchoalveolar lavage (BAL) samples and reversible inflammatory
lesions in lung typified by focal aggregations of fiber-laden alveolar
macrophages and maintenance of a normal lung
architecture.1,2
In contrast, high-dose (
7.0
mg/m3
air) exposures result in a more intense and
protracted inflammation as demonstrated by an increased proportion of
neutrophils in BAL and cellular foci along terminal bronchiolar and
alveolar ductal regions.2,3
Rapid and reversible
proliferation of bronchiolar and alveolar type II epithelial cells, as
indicated by enhanced incorporation of 5-bromo-2'-deoxyuridine (BrdU),
is observed within days of high-dose inhalation exposures to
asbestos.3
Proliferation within pulmonary interstitial
cells, increases in lung hydroxyproline content, a biomarker of
collagen synthesis, and morphological evidence of pulmonary fibrosis
become apparent with prolonged high-dose exposure (14 days) to
crocidolite asbestos.1 Recent evidence indicates that asbestos can stimulate gene expression in a variety of cell types in vitro via downstream intracellular signaling cascades.4 These signaling cascades include the mitogen-activated protein kinases (MAPK) which can lead to increased transcriptional activation of genes intrinsic to cell proliferation, apoptosis and inflammation. The MAPK cascade is characterized by a sequential series of phosphorylation events catalyzed by the extracellular signal-regulated kinases (ERK), c-jun NH2-terminal kinases (JNK) or stress-activated protein kinases (SAPK), and p38.5 In vitro exposure to asbestos fibers selectively induces ERK phosphorylation and activity in rat mesothelial cells, leading to apoptosis and increases in BrdU incorporation.6,7 Furthermore, preferential activation of ERK by asbestos in rat mesothelial cells can be mediated through the phosphorylation of the Epidermal Growth Factor receptor.6 Ultimately, asbestos-induced MAPK signaling events may be critical factors in gene transcription governing phenotypic outcomes of lung injury.
In the present study, we used a murine inhalation model of chrysotile asbestos exposure to enhance our understanding of the ERK signaling cascade in relationship to the development of asbestosis. Our primary objective was to demonstrate the spatial patterns of ERK phosphorylation during the development of epithelial cell proliferation, inflammation and asbestosis. Our findings support the hypothesis that MAPK signaling cascades are activated during the development of fibroproliferative lung diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two- to three-month-old male C57Bl/6 mice were obtained from the National Institutes of Health (Bethesda, MD). Animals were housed and allowed to acclimate for 1 week in suspended cages under controlled conditions of temperature, humidity, and light and provided food and water ad libitum before the initiation of inhalation exposures. Animal and exposure facilities are approved by the American Association for Accreditation of Laboratory Animal Care and operated under the supervision of the Institutional Animal Care and Use Committee of the University of Vermont.
Experimental Protocol and Inhalation Exposures
The experimental protocol consisted of two experiments as depicted
in Figure 1
. In the first experiment 4
animals per group were exposed to either ambient air or NIEHS reference
samples of chrysotile asbestos for 6 hours per day, 5 days a week for a
total of 10 or 20 days. These animals were assigned for bronchoalveolar
lavage and lung procollagen mRNA analyses. In the second experiment, 5
animals per group were exposed as above for a total of 4, 14, or 30
days and an additional group was exposed for 30 days and allowed to
recover with exposure to ambient air for an additional 28 days. These
animals were analyzed for lung hydroxyproline content, histopathology
and by immunohistochemistry.
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Bronchoalveolar Lavage (BAL)
Following asbestos exposure for 10 and 20 days, 4 mice from each exposure group received a lethal dose of pentobarbital. Chest cavities were opened and then lungs were cannulated via the trachea with polyethylene tubing. Lungs were then lavaged in situ 6 times with sterile Ca2+- and Mg2+-free phosphate-buffered saline (PBS) at a volume of 1 ml for each lavage. One lung lobe was excised following lavages and flash-frozen in liquid nitrogen and stored at -70°C for Northern blot analysis. BAL fluids were centrifuged to obtain a cell pellet for total and differential cell counts and a cell-free supernatant for total protein determination. Cell numbers and differentials were determined from hemacytometer counting and cytocentrifuge preparation of resuspended cells, respectively. Cytocentrifuge preparations were stained with Giemsa and May-günwald stains and 500 cells were counted in each preparation. Total protein content was determined in cell-free BAL fluid supernatants stored at -20°C with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Northern Blot Analysis
Total RNA was extracted from lung lobes taken from 20 day sham-
and chrysotile-exposed animals and prepared for Northern blotting as
previously described.10
Briefly, 15 µg of RNA was
denatured and fractionated by electrophoresis on a 1.0%
agarose-formaldehyde gel and transferred onto nitrocellulose filters. A
murine procollagen type I cDNA probe (obtained from Dr. David Rowe,
University of Connecticut Health Center, Farmington, CT) was labeled
with 
[32P]-dCTP (New England Nuclear,
Cambridge, MA) by using a Prime-a-Gene labeling system (Promega,
Madison, WI). Hybridizations were performed on duplicate RNA samples
and signals were quantitated by PhosphorImage analysis (Bio-Rad
Laboratories).
Histopathology
Following asbestos exposures of 4, 14, 30, and 30 + 28 days, 5 mice from each exposure group also received a lethal dose of pentobarbital and chest cavities were opened. The lungs were then perfused with PBS via the right ventricle and the right lung lobes were clamped off at the main bronchi and removed for hydroxyproline analysis as previously described.2,11 The left lung lobes were then fixed by intratracheal instillation of 4% paraformaldehyde at a constant pressure of 14 cmH2O. The lungs were then immersed in fixative overnight at 4°C before embedding tissue blocks in paraffin. Lung sections were cut 3 µm thick for immunohistochemistry, described below, or stained using the Masson’s trichrome technique for detection of collagen.
Immunohistochemistry for BrdU and Phosphorylated ERK
To measure cell proliferation, all animals (n = 40; 5/group/time period) in the second experiment were intraperitoneally administered BrdU (100 mg/kg; Boehringer Mannheim, Indianapolis, IN) 48 and 24 hours before sacrifice.12 Lung sections from sham- and chrysotile-exposed animals were deparaffinized in xylene for 5 minutes three times, rehydrated through graded ethanols and equilibrated in PBS. BrdU immunoreactivity was detected with the Zymed streptavidin-biotin based BrdU staining kit (Zymed Laboratories, San Francisco, CA) and 3,3'-diaminobenzidine (DAB) as a chromogen (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Following color development, sections were rinsed in distilled water, counterstained with hematoxylin, dehydrated through graded ethanols, cleared with xylene, and mounted with Histomount (Zymed Laboratories) before microscopy. BrdU incorporation by cells undergoing DNA synthesis was quantitated with image analysis (BioQuant, Nashville, TN). The number of BrdU immunoreactive epithelial cells were determined in 10 bronchial and/or bronchiolar airways for duplicate lung sections per animal and normalized to airway epithelial length. The number of BrdU immunoreactive alveolar cells were also determined in 20 random 400x field areas (15,700 µm2) for duplicate lung sections per animal.
Phosphorylation (activation) of ERK was also determined by immunohistochemistry in lung sections from all animals in the second experiment. Lung sections were deparaffinized, rehydrated, equilibrated, as described above, permeabilized with 100% methanol at -20°C for 10 minutes and washed with 0.1% Triton X-100 in PBS (Tr-PBS). Endogenous peroxidase activity was dampened by treatment with 3% hydrogen peroxide in methanol for 10 minutes, followed by a 10-minute wash in PBS. Sections were encircled with a hydrophobic film (PAP PEN, Electron Microscopy Sciences, Ft. Washington, PA) and nonspecific protein binding was obstructed with a 200-µl overlay of 2% normal goat serum (Jackson ImmunoResearch Labs, West Grove, PA) in Tr-PBS for 20 minutes 3 times. Excess buffer was absorbed before overlaying with 200 µl of polyclonal rabbit anti-phosphorylated-p44/42 MAPK (phosphorylated ERK; New England Biolabs, Beverly, MA) 1:250 in Tr-PBS containing 2% normal goat serum overnight at 4°C in a humidor. Specificity of the phosphorylated ERK primary antibody was confirmed by Western blot analysis of proteins isolated from mouse lung homogenates and from murine alveolar type II epithelial cells stimulated with epidermal growth factor or hydrogen peroxide (data not shown). Mouse intestinal sections were treated identically as positive controls and negative controls consisted of lung and intestinal sections incubated with a rabbit IgG type-matched monoclonal antibody (Zymed Laboratories) in place of the phosphorylated-ERK primary antibody. Immunoreactivity was then detected using the anti-rabbit-IgG Vectastain ABC Elite kit (Vector Laboratories) and DAB as a chromogen according to the manufacturer’s protocols. Following color development, sections were rinsed in distilled water, counterstained with hematoxylin, dehydrated, cleared, and mounted with VectaMount (Vector Laboratories).
Light Microscopic Image Capture and Processing
Masson’s trichrome-stained and immunostained lung sections were imaged using Kodak T160 color slide film and an Olympus PM-30 photomicrograph system attached to an Olympus BX-50 microscope. Color slide images (35 mm) were scanned at a resolution of 320 pixels per inch into .tif format using a Nikon LS 2000 slide scanner and imported into Adobe Photoshop version 5.0 for cropping, assembly, and labeling. Finally, image montages were printed with a Fujix Pictrography 3000 video printer.
Statistical Analysis
All data are presented as means ± SE. Differences between
means for group data were tested for significance by analysis of
variance. A Student-Newman-Keuls test was used to identify significant
differences between chrysotile asbestos-exposed and sham-exposed groups
at each time point. All statistical analyses were performed blindly by
Dr. Pamela M. Vacek in the Department of Biostatistics at the
University of Vermont, and P values 0.05 were
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In comparison to sham controls, chrysotile inhalation resulted in
pulmonary inflammation after 10 and 20 days of exposure, as indicated
by elevations in the percentages of neutrophils in BAL and a
concomitant decrease in percentages of alveolar macrophages (Figure 2)
. A slight humoral inflammatory
response was suggested by a lymphocytic infiltration after 20 days of
exposure, as compared to control animals (Figure 2B)
. However, the
increased presence of inflammatory cells were not accompanied by a
change in the total number of leukocytes present in the BAL fluid (data
not shown). Evidence of pulmonary inflammation after 10 and 20 days of
asbestos exposure was supported by increases in total protein levels in
BAL (Figure 2D)
, indicative of plasma extravasation into luminal
spaces.
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, the development of
focal pulmonary fibrotic lesions became apparent after 30 days of
exposure (Figure 3B)
, but were not associated with increases in lung
hydroxyproline content (Figure 4)
or
elevations in steady-state procollagen type I mRNA levels (data not
shown). Masson’s trichrome staining revealed increases in
peribronchiolar cellularity and collagen deposition, whereas larger
airways appeared unaffected. Developing fibrotic lesions were
consistently confined to bronchiolar and to bronchiolar-alveolar ductal
bifurcations. The severity and extent of lung injury and pulmonary
fibrosis at 30 days did not appear to progress during a 4-week recovery
period (Figure 3C)
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In comparison to lung tissue sections from control mice (Figure 3D)
, increased numbers of cells undergoing DNA synthesis, as indicated
by BrdU incorporation, were seen in asbestos-exposed lungs. BrdU
immunoreactivity (DAB-positive) was primarily localized to bronchiolar
epithelial (Clara cells) and alveolar ductal cells. Focal alveolar
staining was observed within and adjacent to developing fibrotic
lesions after 30 days of chrysotile inhalation (Figure 3E, 3F)
.
Quantitation of BrdU immunoreactivity revealed increased cellular
proliferation in the alveolar regions of animals after 4, 14, and 30
days of chrysotile exposure (Figure 5A)
.
In contrast, elevations in proliferating bronchiolar epithelial cells
were not statistically significant until 30 days of exposure (Figure 5B)
. The numbers of proliferating cells decreased substantially but
remained elevated, in comparison to control animals, in bronchiolar
epithelium and alveoli during the 4-week recovery period after 30 days
of chrysotile inhalation.
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MAPK activation following asbestos exposure was assessed by
immunostaining for phosphorylated ERK in lung sections from sham- and
chrysotile-exposed animals. Negative control tissues, which were
incubated with a rabbit IgG type-matched monoclonal antibody in place
of the phosphorylated-ERK specific primary antibody, did not reveal any
immunoreactivity (Figure 6A)
. In
contrast, phosphorylated ERK was detected in clusters of epithelial
cells in the crypts of intestinal mucosa (positive control; Figure 6B
).
Increases in ERK phosphorylation were not noted in the lungs of
sham-exposed mice, with the exception of an occasional interstitial
cell (Figure 6C)
. However, sporadic clustering of cells immunoreactive
for phosphorylated ERK were noted within bronchiolar bifurcations and
epithelium after 14 and 30 days of chrysotile exposure (Figure 6D, 6E)
.
Focal ERK phosphorylation also was apparent within perivascular and
peribronchiolar fibrotic lesions after 30 days of chrysotile inhalation
(Figure 6F)
. As with BrdU incorporation, ERK phosphorylation after 30
days of exposure increased within bronchiolar epithelium and developing
fibrotic lesions, primarily confined to alveolar ductal regions (Figure 6G, 6H)
. ERK phosphorylation was comparable to levels of
immunoreactivity in sham-exposed animals after a 4-week recovery period
(observations not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The characterization of inflammation, cell proliferation, and fibrotic
endpoints now allows the utilization of molecular strategies in
transgenic mice to elucidate the critical signaling cascades that are
activated following asbestos exposure. For example, epidermal growth
factor (EGF), a known inducer of ERK, causes proliferation of
epithelial cells in a number of models.21,22
Increased EGF
receptor (EGF-R) expression is observed in asbestos
patients23
and in human mesothelial cells exposed to
asbestos.20
Following inhalation of asbestos by rats,
transforming growth factor- (TGF-), which can bind EGF-R, is also
up-regulated with corresponding increases in cellular proliferation
within developing asbestotic lesions.24
Furthermore,
in vitro studies have demonstrated that asbestos and TGF-
exposure can preferentially induce activation of ERK, as opposed to
JNK, in pleural mesothelial cells.6
Activation of ERK
signaling was subsequently observed to be mediated through
phosphorylation of EGF-R and linked to apoptosis and/or cell
proliferation.6,7,25,26
Our observation that ERK
phosphorylation is focally induced in developing fibrotic lesions after
14 and 30 days of asbestos inhalation provides further evidence that
ERK signaling cascades may be key mediators in the development of
asbestos-associated lung disease through regulation of cell
proliferation or apoptosis.
The induction of ERK phosphorylation at sites of developing fibrotic lesions at peribronchiolar and at bronchiolar-alveolar junctions are consistent with the deposition patterns of inhaled asbestos in acute inhalation models. After brief inhalation of chrysotile asbestos, fibers are primarily deposited on alveolar duct bifurcations in both rats and mice.27,28 It is believed that the deposition patterns of inhaled asbestos dictate the sites of pulmonary inflammatory and early cell proliferation in asbestosis.
Proliferation of type II and bronchiolar epithelial cells is a
prominent feature of lung injury, including pulmonary
fibrosis.29
Our work and that of others show bronchiolar
and alveolar epithelial cell proliferation, as measured by increased
numbers of cells incorporating BrdU,
3H-thymidine, and proliferating cell nuclear
antigen (PCNA) immunoreactivity at sites of developing fibrotic lesions
following inhalation of chrysotile asbestos.2,3,16,19,24
The onset of increased cell proliferation could possibly be mediated by
a number of proinflammatory or growth factor molecules. Many members of
the cytokine, chemokine, and growth factor families are candidates for
the critical mediators of asbestos-associated lung diseases including
pulmonary fibrosis.19,23,24,30,31
This mediators include
platelet-derived growth factors (PDGF),32
TGF-24
and
-ß1,16,33
and tumor necrosis
factor- (TNF-)16,21
which are up-regulated or
activated at the bronchiolar-alveolar sites of chrysotile-induced
fibrotic lesions in rats and mice. However, a number of overlapping
intracellular signaling cascades shared by these and other
extracellular stimuli6,34-36
may be inducible agents of
ERK activation in lung cells. Thus, ERK phosphorylation and subsequent
downstream signaling events may represent a common pathway for
protection and/or repair after lung injury.
There is increasing evidence that intracellular MAPK signaling pathways may be critical determinants of the phenotypic outcome of lung diseases.37-40 Patterns of focal ERK phosphorylation at sites of chrysotile-induced cell proliferation and fibrosis may reflect paracrine signaling by the aforementioned cytokines and growth factors at sites of fiber deposition and subsequent lung injury. A similar pattern of focal ERK activation by autocrine/paracrine signaling growth factors has been postulated to occur in low grade gliomas.41 In addition, ERK activation appears to be an important signaling pathway in a murine model of urethane-induced lung injury and carcinogenesis.38 It might be that ERK-mediated signaling is a shared pathway by which inflammatory mediators and growth factors form an autocrine/paracrine system to repair or compensate for injured lung tissue. However, persistent activation of this pathway may override cell growth checkpoints, eventually leading to the development of pulmonary fibrosis and carcinogenesis.
Generation of transgenic mouse models is expanding the knowledge of intracellular signaling events from cellular to physiological phenotypes of disease. Our murine model of asbestosis with quantifiable outcomes of inflammation and cell proliferation will be invaluable in determining whether MAPK signaling is directly related to the development of these endpoints and their relationship to fibrogenesis. Ongoing experiments are using this inhalation model with transgenic mice expressing a mutant form of EGF-R42 or dominant-negative ERK kinase (MEK-1) in bronchiolar epithelial cells to further delineate the signaling cascades involved in the development of asbestosis.
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Footnotes |
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Supported by National Institutes of Health grants T32ES07122, RO1ES/HL09213, RO1HL39469, and RO1ES06499.
Accepted for publication December 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and transforming growth factor-ß1 expression in the lungs of inbred mice that fail to develop fibroproliferative lesions consequent to asbestos exposure. Am J Pathol 1999, 154:853-862 receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. Am J Pathol 1998, 153:1839-1847 in the bronchiolar-alveolar duct regions of asbestos-exposed rats. Am J Pathol 1996, 149:205-217[Abstract]
mice by expression of mutant epidermal growth factor receptor. Am J Respir Cell Mol Biol 1996, 15:499-508[Abstract]
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