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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
From the Lung Biology Program,*
Department of Pathology,
and Department of Medicine,
Tulane University
Medical Center, New Orleans, Louisiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and transforming growth
factor (TGF)-ß mRNA and protein expression and the degree of
fibroproliferative response to inhaled asbestos fibers are clearly
reduced in the 129 inbred mouse strain as compared with typical
fibrogenesis observed in the C57BL/6 inbred strain. The C57BL/6 mice
showed prominent lesions at bronchiolar-alveolar duct (BAD) junctions
where asbestos fibers deposit and responding macrophages accumulate.
The 129 mice, however, were generally indistinguishable
from controls even though the numbers of asbestos fibers deposited in
the lungs of all exposed animals were the same. Quantitative
morphometry of H&E-stained lung sections comparing the C57BL/6 and 129
mice showed significantly less mean cross-sectional area of the BAD
junctions in the 129 animals, apparent at both 48 hours and 4
weeks after exposure. In addition, fewer macrophages had
accumulated at these sites in the 129 mice. Nuclear bromodeoxyuridine
immunostaining demonstrated that the number of proliferating cells at
first alveolar duct bifurcations and in adjacent terminal bronchioles
was significantly reduced in the 129 strain compared with C57BL/6 mice
at 48 hours after exposure (P < 0.01). TNF- and
TGF-ß1 gene expression, as measured by in
situ hybridization, was reduced in the 129 mice at 48
hours after exposure, and expression of TNF- and
TGF-ß1 protein, as measured by
immunohistochemistry, was similarly reduced or absent in the
129 animals. We postulate that the protection afforded the 129 mice is
related to reduction of growth factor expression by the
bronchiolar-alveolar epithelium and lung macrophages.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental evidence is mounting to support the view that peptide
growth factors elaborated at sites of lung injury mediate the cell
proliferation and matrix production that are hallmarks of pulmonary
fibrosis.4
For example, instillation of platelet-derived
growth factor (PDGF) causes airway fibrosis in rats;5
both
isoforms of PDGF are up-regulated rapidly in response to a brief
asbestos exposure;6
transforming growth factor (TGF)-ß is
up-regulated at sites of fiber deposition in rats,7
and
antibodies against tumor necrosis factor (TNF)- block the
development of silicosis.8
These growth factors are among a
large array of cytokines that have been demonstrated as products of
lung cells under a variety of conditions.9
A major problem
facing investigators today is to establish which of these many factors
is playing a role in disease development.
We use well characterized murine models of asbestos-induced lung
fibrosis to test the hypothesis that growth factors such as PDGF,
TGF-, TGF-ß1, and TNF- are involved in the
evolution of early fibroproliferative lesions at the sites of asbestos
fiber deposition.10-13
As transgenic technology is
increasingly applied to investigations of this
nature,14-19
it becomes important to understand the basis
for variations manifested among commonly used inbred mouse strains. In
the work presented here, we have compared the fibroproliferative
process and growth factor expression consequent to inhaled asbestos
fibers in two strains of mice used routinely in making transgenic and
knockout model systems. We demonstrate here that compared with C57BL/6
mice, the 129 strain exhibits 1) reduced growth factor expression and
cell proliferation at sites of fiber deposition and 2) failure to
develop fibrogenic lesions at these same sites. These findings support
the postulate that certain peptide growth factors contribute to
fibroproliferative lung disease, and investigators using the 129 strain
in studies of fibrogenic pulmonary disease should be cautious when
interpreting their results.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific-pathogen-free 6-week-old male C57BL/6 and 129 mice (Jackson Laboratories, Bar Harbor, ME) weighing 25 to 30 g were exposed to room air for 5 hours in exposure chambers or to an aerosol of chrysotile asbestos (~10 mg/m3 respirable mass) in a 39-L inner aluminum chamber containing the exposure atmosphere within a 1.5-m3 stainless steel Rochester outer chamber. Asbestos aerosol was generated from California chrysotile20 and passed through a vertical elutriator to allow only particles less than 10 µm aerodynamic equivalent diameter to enter the chamber. Mice were exposed via the nose only. Dust concentrations in the exposure chamber were measured by sampling onto 37-mm PVC membrane filters placed in unused animal ports followed by gravimetric analysis of the samples. Five animals per group were sacrificed at 0 hour, 48 hours, 1 week, 2 weeks, and 4 weeks after the exposure by intraperitoneal injection of tribromoethanol at 200 mg/kg of body weight followed by exsanguination through the renal artery. Eight hours before sacrifice, each animal received an intraperitoneal injection of 5'-bromodeoxyuridine (BrdU) in PBS at 40 mg/kg of body weight. Lungs were perfused through the trachea with 10% neutral buffered formalin (Sigma Chemical Co., St. Louis, MO) at a pressure of 15 to 20 cm H2O for 30 minutes. After perfusion, the trachea was clamped and the lungs were removed from the chest cavity and placed in fresh fixative for 16 hours at 4°C. After fixation, the lungs were embedded in paraffin and 5-µm-thick sections were cut onto positively charged slides (SuperfrostR, CMS, Houston, TX) for immunohistochemistry and in situ hybridization. Small intestine was excised from each animal and similarly fixed as a positive control for BrdU immunostaining.
Morphometry
Hematoxylin and eosin (H&E)-stained sections from exposed and control animals from the 48-hour and 4-week time points were prepared, and the identity of each was masked. The slides were then reordered randomly and renumbered before measurements were made. Bifurcation area was measured using an Olympus microscope with a video camera interfacing with a PC using V150 imaging software.21 The total area of each of five first alveolar duct bifurcations was measured as previously described.7 The base of the bifurcation was defined to be delineated by the first lateral alveolar wall to transect the axis of the bifurcation. The resulting measurements from each animal were averaged to give a mean bifurcation size per animal.
Fiber Counts
Lung samples were ashed for 4 hours in an LTA plasma asher. After ashing, samples were washed from the ashing boats into glass vials with 20 ml of filtered bleach. Samples were incubated overnight in filtered bleach before being neutralized with sodium bisulfide and resuspended by sonication. The resulting solution was then filtered onto a 25-mm, 0.2-µm, mixed cellulose ester filter (SKC, Eighty Four, PA), and the filter was then dried in a desiccator for a minimum of 2 days. Sections of the filters were then placed on glass slides and cleared with acetone vapor before being coverslipped. Individual fibers were counted under phase contrast light microscopy according to National Institute of Occupational Safety and Health method 7400.22
Macrophage Counts at Duct Bifurcations
One H&E-stained histological section randomly selected from each animal was used to count by light microscopy alveolar macrophages that had migrated to first alveolar duct bifurcations. An average of six bifurcations were counted per animal. The mean numbers of cells/bifurcation were then calculated and recorded in StatView 4.02 (Abacus Concepts, Berkeley, CA) and analyzed by analysis of variance.
Immunohistochemistry
Immunohistochemistry for TGF-ß1 and TNF- protein
was performed using the immunoperoxidase technique as previously
described.23
For TGF-ß1 immunohistochemistry,
the Santa Cruz rabbit polyclonal anti-TGF-ß1 antibody
(catalog item SC-146) was used at a final concentration of 0.1 µg/ml.
For TNF- immunohistochemistry, a protein-A-purified rabbit
polyclonal anti-TNF- antiserum (the generous gift of Dr. Steve
Kunkel) was used at a dilution of 1:2000.
BrdU immunohistochemistry using a mouse monoclonal anti-BrdU antibody at a dilution of 1:100 (Beckton Dickinson, San Jose, CA) was as described with the following modifications. Instead of PBS, 0.05% Tween 20 in PBS (PBST) was used in all cases where PBS was called for. After deparaffinization and rehydration, sections were incubated in 2 N HCl for 20 minutes followed by two washes of 5 minutes each in PBST. After washing, sections were incubated for 6 minutes in 0.05 mol/L Tris-Cl, pH 7.8/0.01% Trypsin/0.1% CaCl2 at 37°C, followed by PBST washes. The secondary antibody was biotinylated goat anti-mouse IgG. Negative controls included replacing the primary antibody with an equal concentration of normal mouse IgG and incubation of tissue sections from animals that had not received a BrdU injection before sacrifice.
Quantitative Analysis of Cell Proliferation
One BrdU-stained histological section from each animal was prepared as described above. An average of six fields per animal was analyzed as previously described.24 The percentage of positive cells/section was then calculated and recorded in StatView 4.02 (Abacus Concepts) and analyzed by analysis of variance.
In Situ Hybridization
Probe Preparation
The in situ hybridization method used to detect
TGF-ß1 and TNF- mRNA has been described in detail
elsewhere.23
For TGF-ß1, control sense, or
antisense cRNA probe was transcribed from a pGEM vector containing an
~0.9-kb SmaI fragment of TGF-ß1 consisting
of nucleotides 421 through 1395 of the murine cDNA. This region
contains 764 bp of the amino-terminal glycopeptide (precursor) region
and 210 bp of the mature region and was a generous gift of Dr. Harold
L. Moses (Vanderbilt University School of Medicine, Nashville,
TN).25
For TNF-, a control sense or antisense cRNA probe
was transcribed from a pGEM3 vector containing an ~1.1-kb
PstI-EcoRI fragment of murine TNF- cDNA
(GenBank M13049), a generous gift of Dr. B. Beutler. Linearized
plasmids were used for in vitro transcription of
digoxigenin-11-UTP-labeled antisense and control sense riboprobes using
T7 and SP6 polymerase, respectively (Genius 4 RNA labeling kit,
Boehringer Mannheim Corp., Indianapolis, IN).
Hybridization
Hybridization in 4X SSC/50% formamide at 43°C, post-hybridization washes, and RNAse A digestion were carried out as previously described.23 Positive hybridization was detected using alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments followed by colorimetric development with NBT/BCIP as described by the manufacturer (Boehringer Mannheim).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice from two inbred strains (C57BL/6 and SJL) and their F1 hybrid
(B6SJLF1) all developed typical fibroproliferative lesions
consequent to asbestos exposure (not shown). The 129 strain failed to
develop lung lesions as assessed by qualitative histopathology. Here we
report that the 129 inbred strain is resistant to the development of
asbestos-induced lung lesions. We compared the response of 129 mice
with that of C57BL/6 mice, which develop lesions typical of those
observed in other animal models (Figure 1)
.23,24
One of the key
features of an early asbestos-induced fibroproliferative process is an
enlargement of bronchiolar-alveolar duct (BAD) regions due to selective
deposition of asbestos fibers at this anatomical region.13
Our initial observation was that the 129 mice consistently exhibited
reduced lesions compared with those observed in the C57BL/6 mice at BAD
junctions as early as 48 hours after exposure (Figure 1, C and D)
.
These differences persisted through 4 weeks, the last time point
considered in our experiments.
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, sections from the 48-hour and 4-week time points
after exposure were evaluated by video image analysis for bifurcation
area measurement. C57BL/6 mice from both of these time points exhibited
a mean bifurcation size significantly increased over controls. In
contrast, the mean bifurcation size of the exposed 129 animals was
elevated at the 48-hour time point but not at 4 weeks after exposure.
Additionally, mean bifurcation area in exposed 129 mice was
significantly less than that of exposed C57BL/6 mice at the time points
examined. Cell Proliferation
Because the mean lesion size in the 129 mice was significantly
less in comparison with the C57BL/6 animals, we investigated whether
cell proliferation at BADs and associated terminal bronchioles
correlated with these findings. Cells stained positively for BrdU
incorporation were counted at BADs and at terminal
bronchioles.24,26
These results are shown in Figure 2
. At BAD junctions (Figure 2A)
, the
percentage of BrdU-labeled cells at 48 hours in the C57BL/6 mice was
increased significantly over the percentage in unexposed mice and in
the lungs from 129 mice (P < 0.01 for both).
Although the percentage of BrdU-stained cells at BAD junctions in
sections from the 129 mice did achieve a significant increase
(P < 0.05) over controls, this increase was
significantly less (P < 0.05) than that
observed for the C57BL/6 over controls. At 1 week after exposure, the
percentage of BrdU-labeled cells in the exposed C57BL/6 lung sections
remained significantly elevated (P < 0.01) over
unexposed controls. By this time point, the percentage of labeled cells
at BAD junctions in 129 mice had returned to control levels.
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, we observed similar
increased proliferation at 48 hours after exposure in the C57BL/6
animals. This increase was again significantly greater than that
observed in the 129 mice (P < 0.01). The
increase in the percentage of labeled cells in C57BL/6 mice persisted
through 1 week after the exposure and remained significantly elevated
over controls (P < 0.05). As with the BAD
region, by 1 week after exposure, the percentage of BrdU-labeled cells
in the 129 mice had returned to control levels. Fiber Deposition in the Lung
Critical to the comparisons made above is a demonstration that the
numbers of fibers deposited in the lungs of both C57BL/6 and 129 mice
were not different. Therefore, the accessory lobe of the right lung of
each animal was reserved after sacrifice for fiber counts in two
separate experiments. Each lung sample was ashed and the fibers
transferred to a filter for counting by phase contrast light
microscopy. The numbers of fibers deposited in the lungs of animals of
each strain were the same (Figure 3)
.
Additionally, over time, both strains exhibited similar rates of fiber
clearance (Figure 3)
. We also have shown by scanning electron
microscopy that the pattern of initial fiber deposition in the lungs of
the 129 mice (Figure 4)
at BAD junctions
is essentially the same as has been previously reported in other rodent
model systems.13
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Morphometric analysis and cell counting data (Figures 1E and 2
, A
and B) clearly demonstrate a difference in mean lesion cross-sectional
area between the two inbred mouse strains. One known contribution to
this increased bifurcation area after a brief asbestos exposure is an
overall increase in the number of macrophages present at these
sites.10,13,27
Figures 1 and 4
clearly demonstrate that in
C57BL/6 and 129 mice, macrophages are attracted to sites of fiber
deposition. Thus, we asked whether there is a difference in the number
of macrophages attracted to first alveolar duct bifurcations 48 hours
after asbestos exposure in the two mouse strains. Alveolar macrophages
that could be clearly identified by light microscopy associated with
duct bifurcations were counted. Figure 5
shows that the mean numbers of macrophages attracted to first alveolar
duct bifurcations in the asbestos-exposed 129 animals were elevated
~2.5 times over controls, whereas the mean numbers of macrophages
attracted to bifurcations in the asbestos-exposed C57BL/6 animals were
~5 times that of controls. The increased response of the macrophages
in C57BL/6 mice is significantly elevated (P <
0.05) over that of the 129 animals.
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Gene and Protein Expression
Because TNF- appears to be a key mediator in pulmonary
fibrogenesis, we examined the expression pattern of this pleiotropic
cytokine at the sites of initial injury (Figure 6)
. In both the exposed C57BL/6 and 129
mice, expression of TNF- mRNA appears to be increased over unexposed
controls immediately after the exposure (Figure 6, C and D)
. This
increase is confined to the epithelium of the terminal bronchioles and
apparently precedes the up-regulation of TNF- protein. By 48 hours
after exposure in the C57BL/6 lung sections (Figure 6E)
, TNF- mRNA
expression is clearly abundant at BAD junctions as well as in adjacent
terminal bronchioles. In contrast, 129 mice (Figure 6F)
exhibited a
pattern of expression similar to that observed immediately after the
exposure and confined to the epithelium of the terminal bronchioles.
These sections frequently exhibited little positive signal outside of
the bronchiolar epithelial compartment, with the exception of an
occasional alveolar macrophage. By 1 week after exposure, the
expression pattern of TNF- mRNA returned to background levels (not
shown) comparable to unexposed animals (Figure 6A)
.
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mRNA precedes protein
expression. Although there is clear hybridization signal for TNF-
mRNA in the epithelium of the terminal bronchioles at the time point
immediately after the exposure, there is not yet any detectable protein
expression (not shown). By 48 hours after exposure, however, there is
prominent TNF- protein expressed at BAD junctions in the exposed
C57BL/6 mice (Figure 6G)
as identified by immunohistochemistry. There
is little apparent up-regulation of TNF- in the 129 mice (Figure 6H)
at these same sites, and expression generally is absent. As with gene
expression, TNF- protein expression returns to background levels by
1 week after exposure (not shown). The level of protein expression in
the 129 mice never exceeded the minimal levels achieved at 48 hours
after exposure (not shown). TGF-ß1 Gene and Protein Expression
Based on our previous findings in rats7
and mice
(J.-Y. Liu, unpublished data), after asbestos exposure we expected to
see TGF-ß1 gene expression up-regulated at sites of fiber
deposition in the C57BL/6 mice by in situ
hybridization.25
At 48 hours after exposure, there was a
robust signal for TGF-ß1 mRNA (Figure 7A)
at BADs and in adjacent terminal
bronchioles when compared with control animals. In contrast, the 129
mice (Figure 7B)
showed essentially no increase in TGF-ß1
expression at these sites at any time after the exposure. By 1 week
after exposure, TGF-ß1 mRNA levels in the C57BL/6 animals
returned to background levels, similar to unexposed animals (not
shown).
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shows TGF-ß1 protein clearly present at BADs and in
adjacent terminal bronchioles in the C57BL/6 animals 48 hours after the
initial exposure. In contrast, the 129 mice exhibit essentially no
TGF-ß1 staining in the bronchiolar regions and little at
the duct bifurcations (Figure 7D)
. These results closely follow the
mRNA expression pattern demonstrated by in situ
hybridization. As with the gene expression, TGF-ß1
protein expression returned to background levels in the C57BL/6 mice by
1 week after exposure, and expression in the 129 mice remained at
background levels at all time points after exposure. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TGF-ß1 expression, supporting the view that
these potent growth factors play a role in the development of
fibroproliferative lung lesions. In addition, 3) the 129 inbred
mouse strain represents a model that can be used to study the genes
that control interstitial fibrogenesis. The 129 mouse strain has been used extensively to develop knockout animals.28,29 To our knowledge, this strain has not been tested previously in regard to its pulmonary responses to inhaled fibrogenic particles. This is an important concept for those interested in the mechanisms through which inhaled toxic particles cause lung injury, and these animals could open opportunities to understand the genetic basis for the apparent protected phenotype observed. Here, we have shown that the 129 strain fails to develop a fibroproliferative response to a dose of asbestos fibers that we know causes fibrogenic lesions in several strains of mice24,30 and rats.7,27 Thus, we have begun to test our hypothesis that reduced expression of one or more peptide growth factors is fundamental to the apparent protection from an asbestos-induced fibroproliferative response in the 129 mouse strain.
A major problem confronting investigators interested in growth factor
biology is the multitude of highly active cytokines known to be
elaborated by lung cells.4
Consequent to injury, many of
these are up-regulated,6,7,23
suggesting an important role
in disease development. Previously, we carried out a series of studies
focused on three of the factors thought to participate in the
development of fibroproliferative lung disease. These are the isoforms
of PDGF,6
TGF-,23
and
TGF-ß1.7
These factors are of interest
because 1) PDGF is the most potent mesenchymal cell mitogen yet
described,31
2) TGF- is a powerful mitogen for
epithelial cells,32
and 3) TGF-ß1 is known as
a clear inducer of extracellular matrix proteins by mesenchymal
cells.33
TGF-ß1 generally blocks cell
proliferation, but under certain circumstances, TGF-ß1
has been demonstrated to be mitogenic for some cell types in
vitro.34,35
We showed in asbestos-exposed rats that
these growth factor genes and their concomitant proteins are
up-regulated significantly within hours after
exposure,6,7,23
and expression is limited to the anatomical
sites of asbestos deposition and lung injury,6,7,23
ie, the
bronchiolar-alveolar duct (BAD) junctions. BAD junctions are sites of
initial injury consequent to exposure to a number of inhaled
agents, including asbestos13
and silica,36
and
these anatomical regions are commonly used as focal points of study
after exposure to toxic materials.37
In addition to the factors discussed above, there is compelling
evidence that TNF- plays a critical role in fibroproliferative lung
disease.8,14,38
We have supported this finding by
demonstrating here that markedly lower TNF- expression in the lung
correlates with a reduced fibroproliferative response (Figure 6)
. With
this information and the knowledge that TNF- apparently can function
as an activator of expression of other growth factors, such as
TGF-ß,38,39
we postulate that TNF- acts as a central
mediator in controlling the development of interstitial lung fibrosis
(see further discussion of this point below). Also, inasmuch as the
numbers of macrophages responding to asbestos exposure are fewer in the
129 mice (Figure 5)
, a proven source of TNF- would be reduced.
Alveolar macrophages have been shown to produce TNF- in our mouse
model,40
and TGF-ß is a known product of lung
macrophages.33,34
The mechanisms controlling reduced
macrophage migration and how concomitant reduction in growth factor
release might affect lesion development in the 129 mice remain unknown
at this time.
If these selected growth factors discussed above have any role in
fibrogenic lung disease, it would follow that animals exhibiting a
reduced fibroproliferative reaction might have less expression of such
factors. However, a significant question in interpretation remains;
specifically, is a given factor reduced because it is intrinsically
genetically programmed as such, or is it reduced because there is less
of a lesion developing for some other reason, with fewer cells
available for growth factor expression? The comparison we present
between the C57BL/6 and 129 strains could shed some light on this
important issue. First, gene expression and proliferative rates in the
epithelial cells lining the most distal terminal bronchioles clearly
were different between the strains after asbestos exposure (Figures 2, 5, and 6)
, yet these well defined regions of lung exhibited little
apparent anatomical difference throughout the experiment, suggesting
the genetic explanation noted above. Furthermore, our studies on
knockout mice lacking both the 55- and 75-kd receptors for TNF- show
that these animals also are protected from the fibrogenic effects of
inhaled asbestos.40
Even though the levels of TNF-
expression are up-regulated after exposure in these knockout mice,
there is a clear lack of a typical fibroproliferative response to
asbestos, similar to that shown here for the 129 strain. In addition,
the protected knockout mice exhibit reduced expression of PDGF,
TGF-, and TGF-ß1,40
suggesting that
TNF- plays a central role in the activation of other growth factors
as discussed above. This is consistent with the findings of Piguet et
al8
who showed that TNF- is necessary for silica- and
bleomycin-induced fibrosis to develop. Perhaps the reduced expression
of TNF- exhibited by the 129 mice studied here is key to the
protected phenotype. Accordingly, recent experiments with primary mouse
lung fibroblasts cultured from the C57BL/6 and 129 inbred strains
suggest further that TNF- may indeed be important in the protected
phenotype observed in the 129 inbred strain. Our preliminary data show
that TNF- up-regulates production of pro-1 (I) collagen mRNA in
primary mouse lung fibroblasts from the C57BL/6 inbred
strain.41
TNF- is much less effective in up-regulating
this gene in the primary MLFs from the 129 inbred strain.
Additional data from these ongoing experiments indicate that primary
MLFs from the 129 strain exhibit reduced proliferative capacity
in response to both serum and PDGF.41
Such studies
culturing mesenchymal cells should prove highly valuable in
understanding the mechanisms controlling protection of the 129 mouse
strain.
Thus, we have shown that the 129 mouse strain fails to develop
fibroproliferative lesions consequent to asbestos exposure. These mice
exhibit a significantly reduced percentage of dividing cells and fewer
macrophages accumulating within the first 2 days after exposure, and
this results in essentially normal lungs through the 1-month period
studied. These animals have reduced expression of TNF- and
TGF-ß1 mRNA, and little TNF- or TGF-ß1
protein is detected at the sites of initial lung injury. The dose and
clearance rate of asbestos fibers in these animals were the same as
that in the C57BL/6 strain. We suggest that the reduced growth factor
expression plays some role in the lack of disease development, and our
ongoing experiments are designed to test this postulate.
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Acknowledgements |
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Footnotes |
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Supported by NIH grants RO1ES60766 and RO1HL60532, Tulane/Xavier Center for Bioenvironmental Research, and The Tulane Cancer Center.
Accepted for publication December 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-ß1 and myofibroblasts. Am J Pathol 1998, 153:825-8321. J Immunol 1992, 149:103-106[Abstract]
receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. Am J Pathol 1998, 153:1839-1847This article has been cited by other articles:
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 A. R. Brody, G. S. Warshamana, J.-Y. Liu, S.-Y. Tsai, D. A. Pociask, D. M. Brass, and D. Schwartz Identifying Fibrosis Susceptibility Genes in Two Strains of Inbred Mice Chest, March 1, 2002; 121(2007): 31S - 31S. [Full Text] [PDF]
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W. H. Chung, B. M. Bennett, W. J. Racz, J. F. Brien, and T. E. Massey Induction of c-jun and TGF-beta 1 in Fischer 344 rats during amiodarone-induced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1180 - L1188. [Abstract] [Full Text] [PDF]
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 P.J.A. Borm and R.P.F. Schins Genotype and phenotype in susceptibility to coal workers' pneumoconiosis. The use of cytokines in perspective Eur. Respir. J., July 1, 2001; 18(32_suppl): 127S - 133s. [Abstract] [Full Text] [PDF]
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 R. F. Robledo, S. A. Buder-Hoffmann, A. B. Cummins, E. S. Walsh, D. J. Taatjes, and B. T. Mossman Increased Phosphorylated Extracellular Signal-Regulated Kinase Immunoreactivity Associated with Proliferative and Morphologic Lung Alterations after Chrysotile Asbestos Inhalation in Mice Am. J. Pathol., April 1, 2000; 156(4): 1307 - 1316. [Abstract] [Full Text] [PDF]
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G. W. Hoyle, J. Li, J. B. Finkelstein, T. Eisenberg, J.-Y. Liu, J. A. Lasky, G. Athas, G. F. Morris, and A. R. Brody Emphysematous Lesions, Inflammation, and Fibrosis in the Lungs of Transgenic Mice Overexpressing Platelet-Derived Growth Factor Am. J. Pathol., June 1, 1999; 154(6): 1763 - 1775. [Abstract] [Full Text] [PDF]
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