RAPID COMMUNICATION
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The development of interstitial pulmonary fibrosis is associated with a variety of inflammatory mediators, including peptide growth factors and cytokines. In the work presented here, we have asked whether or not platelet-derived growth factor (PDGF)-A and -B genes and proteins are expressed in anatomic and temporal patterns consistent with this factor playing a role in the disease process. Using an established rat model of asbestos-induced fibroproliferative lung disease, we demonstrate elevated levels of PDGF-A and -B mRNAs in total lung RNA immediately after a single 5-h exposure to ~ 1,000 fibers/ml of chrysotile asbestos. In situ hybridization revealed the PDGF-A and -B in RNAs primarily in macrophages and bronchiolar-alveolar epithelial cells at sites of initial fiber deposition and lung injury. There was clear evidence of PDGF-A and -B mRNAs in interstitial cells as well. The pattern of in situ hybridization was entirely consistent with the appearance (established by immunohistochemistry) of PDGF-A and -B proteins by 24 h post-exposure in the same cell types. Both mRNAs and proteins remained detectable at the fiber deposition sites for almost 2 wk post-exposures. These findings are consistent with our previous studies showing increased mesenchymal cell proliferation and fibroproliferative lesions that progress at the sites where PDGF-A and -B are expressed. Although it is clear that multiple growth factors are produced simultaneously at sites of initial injury, we suggest that the PDGF isoforms could be playing a central role in the disease process based upon their potent mitogenic effects upon mesenchymal cells.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Platelet-derived growth factor (PDGF) could be a significant mediator of fibroproliferative disease (1, 2). This postulate follows a series of studies showing a primary role for PDGF in lung development (3), atherosclerotic disease (4), kidney sclerosis (5), and wound healing (6). PDGF is the most potent mitogenic factor yet found for a wide variety of mesenchymal cells (1, 2). The factor was originally described as the major growth promoting agent in serum (7), explaining in large part how sera support the long-term maintenance of mesenchymal cell cultures. PDGF is stored in the alpha granules of blood platelets and is released in vivo at sites of inflammation and injury (1, 6, 7).
PDGF consists of 3 isoforms that result from the disulfide bridging of peptides from 2 separate genes coding for
the A chain and B chain (8). This results in the AA, AB,
and BB isoforms, each of which is a potent mesenchymal
cell mitogen, but only in the dimeric forms. In addition, it
is clear that the 3 isoforms bind to specific membrane receptors, designated as alpha (
) and beta (
), which also
must dimerize on the cell surface to be biologically active
(9). The AA isoform binds only to the - receptor, AB
binds to the - and -, while the BB isoform binds to
all 3 receptors, stimulating cells through a tyrosine kinase
signaling pathway (10).
We have been studying the fibroproliferative disease process associated with asbestos-induced lung injury (11). This interstitial process results in increased numbers of mesenchymal cells and extracellular matrix as the disease develops its diffuse character after continued exposure (11). Our studies have focused on the earliest lesions associated with fiber inhalation (12, 13) in order to understand the initial cellular, biochemical and molecular events that could be mediating a fibroproliferative process in the lung. This is most likely to be the result of multiple interactive cell signaling pathways that activate oxygen radicals, peptide growth factors, and cytokines, which are released simultaneously and at varying times from several cell types during disease development. A major challenge facing investigators is to elucidate the precise roles for these potential mediators. Proving whether or not a given factor is expressed, in the appropriate cell types and in meaningful anatomic and temporal patterns, would allow investigators to establish those mediators that are most likely to be significant in disease development.
In the work presented here, we show for the first time in an animal model of fibroproliferative lung disease, the expression of PDGF-A and -B chain mRNAs and proteins, primarily in bronchiolar-alveolar epithelium as well as in macrophages and mesenchymal cells at sites of initial fiber deposition and cell injury. PDGF mRNA levels increase within the first 5 h of exposure, whereas protein is apparent 24 h later. Our data show that PDGF-A and -B chain expression remains elevated for at least 2 wk after a single 5-h exposure. The association of PDGF expression with the precise anatomic sites preceding development of fibroproliferative lesions supports the view that this potent peptide plays an integral role in the disease process.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals, Exposure and Tissue Preparation
Animals and exposures to chrysotile asbestos (10 mg/m3 respirable mass), iron (50 mg/m3) and air were described previously (14). From each exposure group of 35 animals, 5 animals were killed immediately post-exposure and at 1, 2, 4, 8, 14, and 30 days post-exposure. After cutting the renal artery, the right bronchus was tied off, and the right lung was removed and placed in liquid nitrogen. The remaining left lung was fixed by intratracheal perfusion with 4% paraformaldehyde as previously described (14). Tissue blocks were embedded in paraffin and sections stained with H & E for analysis or left unstained for in situ hybridization or immunohistochemistry as described below. The results reported here are combined from 2 separate exposures to the asbestos aerosol.
RNase Protection Assay for PDGF-A and -B mRNA Levels
Total lung RNA was prepared with the Ultraspec RNA Isolation System (Biotecx Laboratories, Houston, TX) as described by the supplier. Ribonuclease protection assays were performed as described previously (15). The 32P radiolabeled RNA probes were prepared by T3 transcription of a Nco I digested linear PDGF-A cDNA plasmid (described further below) and by T7 transcription of a Bam HI digested linear PDGF-B cDNA containing plasmid (described further below). For PDGF-A, the probe was 477 nucleotides and the protection product, 418 nucleotides. For PDGF-B the probe was 535 nucleotides and the protection product was 480 nucleotides.
In Situ Hybridization
Tissue and probe preparation. In situ hybridization to detect PDGF-A and PDGF-B mRNAs at the indicated times post-exposure was as described previously (14). Briefly, the antisense RNA probes of PDGF-A and -B were transcribed from the pBluescript SK+ vector containing an ~ 0.8 kb Sma I fragment and 0.5 kb fragment, respectively, derived from the cloned rat PDGF-A and PDGF-B cDNAs (16), kindly provided by Dr. Dai Katayose (NHLBI/NIH, Bethesda, MD). For PDGF-A, the Nco I digested plasmid was transcribed by T3 RNA polymerase to generate digoxigenin-labeled antisense, and the Bam HI digested plasmid was transcribed with T7 RNA polymerase to generate digoxigenin-labeled sense RNA probes. For PDGF-B, the Bam HI digested plasmid was transcribed with T7 RNA polymerase to prepare the antisense and the Hind III digested plasmid was transcribed with T3 RNA polymerase to generate the sense probe (Genius 4NA Labeling Kit; Boehringer Mannheim Corp.). Hybridization. Hybridization in 4× SSC + 50% formamide at 43°C and post-hybridization washes + RNase A digestion were carried out as described previously (14). The hybrids were detected with alkaline phosphatase conjugated anti-digoxigenin antibodies followed by visualization within NBT/NCIP chromogen as described by the manufacturer (Boehringer Mannheim). As routine controls in each experiment, no signal was detected if the probe was excluded or the sense probe was employed, or if the section was pretreated with ribonuclease A.Immunohistochemistry
Immunohistochemical staining for PDGF-AA, -BB and vimentin proteins was performed using the immunoperoxidase technique described previously (14). In summary, detection of AA and AB isoforms was with a rabbit anti-human PDGF-A chain IgG polyclonal antibody (5 µg/ml; ZymoGenetics Inc., Seattle, WA) pretreated with 0.1% pepsin at 37°C for 30 min. This antibody has < 1% cross-reactivity to the PDGF-BB chain and reacts with both human and rat tissues (17, 18). The PDGF-BB and -AB isoforms were detected with PGF 007, a mouse anti-human PDGF-BB monoclonal antibody (5 µg/ml; Mochida Pharmaceutical Co., Tokyo, Japan) that reacts with human and rat PDGF-BB (19, 20). PGF 007 was generated to a 25-amino acid peptide located near the COOH-terminus of B chain of PDGF (residues 73-97 of the mature B chain) and recognizes PDGF-BB and -AB, but not PDGF-AA (21). Detection of vimentin was with V9, a mouse anti-vimentin monoclonal antibody (4 µg/ml; Dako, Carpinteria, CA). Primary antibodies were incubated in 0.1% gelatin/1% BSA-PBS for 1 h at room temperature followed by a wash with 0.1% gelatin/1% BSA-PBS. After the incubation with the primary antibody, the slides were incubated with biotinylated goat anti-rabbit and -mouse IgG (Jackson Immunoresearch, West Grove, PA) diluted 1:4,000 in 0.1% gelatin/1% BSA-PBS for 1 h followed by 2 washes in the same buffer without antibody. Visualization of immune conjugates was achieved by oxidation of diaminobenzidine (DAB) by streptavidin-conjugated horseradish peroxidase as described previously (14). For negative controls, an equivalent dilution of normal rabbit or mouse IgG replaced the primary antibodies for all immunostaining.
Quantitative Analysis
Results were quantified as described previously (14). Briefly, two sections from each of the 5 animals at each time point of the 3 treatment groups were evaluated. Fifteen hundred epithelial cells and 1,000 interstitial cells were counted from 4 to 6 bronchiolar alveolar duct units, each including a terminal bronchiole, the alveolar walls between the terminal bronchiole and first alveolar duct bifurcation, as well as the first alveolar duct bifurcation itself. Stained cells were counted by light microscopy at ×400 magnification for PDGF-AA and PDGF-BB, -AB immunostaining to generate the percentages of positive cells. Epithelial and interstitial cells were identified by anatomic location relative to the basement membrane. Three-way analysis of variance (ANOVA) was carried out to determine differences among independent variables (i.e., trial, exposure time). One-way ANOVA was performed to establish differences between exposures at each time point.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Whole Lung mRNA by RNase Protection Assay
PDGF is a potent mitogen for mesenchymal cells with an essential role in lung development. Thus it could be a mediator of the fibrogenic effects caused by inhaled toxic agents. To determine whether or not there was increased PDGF mRNA that could be detected in whole lung homogenates of asbestos-exposed rats, we used the sensitive RNase protection assay (RPA) (15). RNA was extracted from the lungs of rats exposed for 5 h to an aerosol of chrysotile asbestos fibers at various times post-exposure to assess changes in the level of PDGF mRNA. The analysis included two simultaneously processed control groups that consisted of animals exposed to an aerosol of carbonyl iron spheres or sham-exposed to air. Equal amounts of lung RNA from an animal in each group were probed for PDGF-A and PDGF-B mRNAs by RNase protection (Figure 1). Unexposed animals expressed low levels of PDGF-A and PDGF-B mRNAs in the lung throughout the experimental time course (Figure 1). Inhaled asbestos produced a 3.5-4.0-fold increase in the level of both PDGF-A and PDGF-B mRNAs in the lung as determined by densitometry (data not shown). Higher levels were detected immediately after the 5-h exposure to asbestos (0 h) and remained high at the 2-wk time point post-exposure (Figure 1). In contrast, inhaled nonfibrogenic carbonyl iron particles, which deposit with a pattern similar to asbestos, did not induce a significant increase in PDGF-A or PDGF-B mRNA levels at any time (Figure 1). Control experiments with yeast tRNA verified the specificity (not shown) and demonstrated equal inputs of lung RNA in each assay (Figure 1). These data indicate that asbestos induces a rapid and prolonged increase in the levels of PDGF mRNA in the lung, but to make a specific association of PDGF expression with the sites of fiber deposition and lung injury requires in situ hybridization and immunostaining experiments described below.
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Vimentin Immunohistochemistry
In previous studies, we presented the histopathology and morphometry of the fibroproliferative lesions caused by brief exposures to chrysotile asbestos in rats and mice (11- 13). Here we add to the earlier findings the dramatic effect on the peribronchiolar-alveolar interstitium as demonstrated by immunohistochemistry of vimentin expression (Figure 2). This intermediate-filament protein becomes prominent as the interstitial mesenchymal cell populations expand and the macrophages (both alveolar and interstitial) are activated to migrate to the sites of fiber deposition and lung injury (13, 22). These vimentin-rich regions serve to highlight the anatomic locations upon which we have focused the studies reported below using immunohistochemistry and in situ hybridization.
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In Situ Hybridization
We predicted that gene expression for PDGF-A and -B would be most prominent at sites of initial fiber deposition and lung injury. As seen in Figures 3 and 4, this is the case. Several key controls are necessary to validate in situ hybridization as a specific marker of gene expression. First, in Figures 3A and 4A, we show that unexposed animals exhibit little labeling by antisense mRNA in any cell types. Second, the sense mRNA probes for PDGF-A and -B do not produce a signal within the tissues, even in obvious asbestos-induced lesions (Figures 3B and 4C). Iron-exposed rats also showed little labeling with antisense mRNA (not shown). PDGF-B chain message could be detected in a few cells immediately after the 5-h exposure (Figure 4B), and the signal persisted for at least 2 wk post-exposure in both A and B chain studies (Figures 3D and 4F).
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PDGF-A. (A) Low magnification view of the bronchiolar-alveolar duct region from a
sham (air)-exposed rat. These tissues were hybridized with
the antisense probe for PDGF-A, and essentially no staining
was observed. (B) In an animal exposed to chrysotile asbestos for 5 h, several fibroproliferative lesions are seen (arrows) 48 h post-exposure, but the tissue was treated with the
sense probe for PDGF-A, and no staining reaction is noted.
(C) 48 hours after asbestos exposure, multiple lesions of the
alveolar duct walls are observed (boxes). These tissues treated with the antisense probe demonstrate the distribution of PDGF-A mRNA in macrophages as well as in epithelial and interstitial cells of the lesions. Three of the lesions are boxed in this figure and are magnified in C1-3.
(C1-3) Here, epithelial cells (arrows), macrophages (M),
and interstitial cells (arrowheads) clearly have hybridized
with the antisense probe. (D) The signal has waned but is
present in the lesions and surrounding macrophages at 2 wk
post-exposure. Bars = 20 µm.
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In Figures 3C and 4C are shown typical asbestos-induced fibrogenic lesions hybridized with the antisense mRNA probes for PDGF-A and -B, respectively. There is clear labeling of increased numbers of bronchiolar-alveolar epithelial cells, macrophages, and interstitial cells, particularly at 48 h post-exposure (Figures 3C and 4D). We demonstrate in Figures 3D and 4F that the hybridization is reduced but still obvious at 14 days post-exposure. The labeled cells include Clara cells of the terminal bronchioles, cuboidal epithelial cells covering the alveolar duct bifurcations, alveolar and interstitial macrophages, as well as populations of interstitial cells, although the precise identity of each cell type in this compartment is not entirely clear. Scattered type II cells are labeled in the lung parenchyma adjacent to the bronchiolar-alveolar duct regions (Figures 3 and 4).
An estimate of the number of PDGF mRNA containing cells was carried out by reviewing slides from exposed animals, with the observer blinded as to time after exposure. Control animals were not included in this exercise because they are obviously negative. Table 1 shows time-related changes with the largest number of cells hybridized for both PDGF-A and -B mRNAs appearing in the tissues at 48 h post-exposure (see Figures 3C and 4D). The epithelial cells appeared to hybridize most intensely compared to macrophages and interstitial cells, but this feature does not lend itself well to quantitation.
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Immunohistochemistry
The first evidence of increased immunostaining for PDGF-A and -B chain proteins appeared at 24 h post- exposure. No staining could be ascertained immediately after the 5-h exposure (not shown) although the mRNAs were apparent at this time (see Figures 1, 3 and 4). The lungs of unexposed and iron-exposed rats exhibited little staining (Figures 5A and 6A). By 48 h post-exposure, the most intense reaction was apparent, with epithelial cells, macrophages, and interstitial fibroblasts clearly staining (Figures 5C and 6C). Non-immune serum did not produce staining of adjacent histologic sections (Figures 5B and 6B). Clara cells lining the bronchioles as well as type II alveolar cells and cuboidal epithelial cells covering the alveolar duct bifurcations exhibited brilliant immunohistochemical reactions (Figures 5 and 6). Spindle-shaped mesenchymal cells (i.e., fibroblasts or myofibroblasts) contained PDGF-A and -B in the lung interstitium (Figures 5D and 6D). The number of cells positive for the immunostain was maintained for 8 days and waned by 2 wk post-exposure (Figures 5I and 6H). Little staining was observed after 3 wk (not shown), consistent with the in situ hybridization data described above.
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Again, we quantified the number of stained cells with the observer blinded as to the time post-exposure (Figure 7). As can be seen in the light micrographs of immunostained tissues (Figures 5 and 6), it is not difficult to ascertain the numbers of stained cells in both alveolar and interstitial compartments, although it was not possible in every case to establish the precise cell types. Expressed as percentages of cells immunolabeled in either the epithelial or interstitial compartments, PDGF-A and -B peptides follow the same general time course (Figure 7). The major difference appears to be that the percentages of stained cells were fewer by 50% in the interstitium of the alveolar lesions compared to the stained cells in lesions of the interstitium of the bronchiolar-alveolar duct walls 48 h post- exposure (not shown). In addition, the percentages of stained epithelial cells, for both A and B proteins, were consistently higher at almost every time point after exposure than the values for the interstitial cells. There are several points to note concerning these quantitative data: (1) few stained cells could be identified immediately after exposure; (2) the unexposed and iron-exposed animals exhibited few labeled cells at any time during the observation period; and (3) by 1 mo post-exposure, the number of positive cells had returned to the normal low level.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have demonstrated that both PDGF-A and -B are increased in the epithelium, interstitium, and macrophages of the bronchiolar-alveolar duct regions of rat lungs consequent to a 5-h exposure to chrysotile asbestos fibers. The mRNAs coding for these proteins were upregulated during the 5-h exposure period (Figures 1, 3 and 4). This rapid mRNA increase and protein elaboration is consistent with the pattern of cell proliferation we have documented previously in this animal model (11, 12). For example, a single brief (1- or 3-h) exposure to asbestos induced a significant increase in the percentages of mesenchymal cells and vascular smooth muscle cells incorporating tritiated thymidine by 12 h post-exposure (12, 23), and the numbers and volume of these cells increased accordingly by 2 days after exposure (13, 23, 24). These cell types are major components of hyperplastic, hypertrophic interstitial lesions measured at 1 mo and 6 mo after a brief asbestos exposure (13, 25). Smooth muscle cells, fibroblasts, and myofibroblasts are the cell types most richly endowed with cell surface receptors for the PDGF isoforms (1, 2). The fact that these cell types are responding vigorously in vivo supports our working hypothesis that this potent growth factor is playing a role in the fibroproliferative response leading to fibrogenic lung disease. Further experiments using transgene and knockout technology are necessary to establish whether or not our postulate will hold true. Two recent papers, one using PDGF-A deficient mice (3), and the other using instillation of PDGF-BB into the trachea (26), strongly support a role for the PDGF isoforms in myofibroblast proliferation during lung development and in mediating interstitial fibrogenesis.
Clearly there are a number of growth factors and cytokines that induce cell proliferation (27). The major problem in establishing the mechanisms of fibrogenic disease is
due to this simultaneous elaboration of multiple factors
that could be playing a role. Thus, investigators must choose
the appropriate technologies to sort out those factors that
are most likely to be contributors to the process. Current
findings suggest that multiple factors are involved in the
development of fibrogenic lung disease. For example,
transforming growth factor beta (TGF-) is believed to be
central to the development of bleomycin-induced interstitial fibrosis (28). Also, transgenic mice overexpressing
TGF- develop severe interstitial pulmonary fibrosis (29),
and an antibody to tumor necrosis factor alpha (TNF-)
has been shown to reduce experimental silicosis in mice
(30). In addition, human lung tissues exhibiting various
stages of fibrosis have been stained for the presence of
multiple growth factors including TGF- (31), TGF-
(32), TNF- (33) and PDGF (34). Indeed, studies in animal models and human tissues support the concept of multiple factors contributing to the fibrogenic process. In the
experiments reported here, we opted to use in situ hybridization and immunohistochemistry to ask whether or not
PDGF-A and -B chains are present in the appropriate anatomic locations and temporal sequence associated with
developing fibrogenic lesions. While the data indicate a spatial and temporal pattern of expression consistent with
our postulate that PDGF is integral to fibroproliferative
lung disease, we are only slightly closer to knowing if the
growth factor is playing a mechanistic role. If the genes
and proteins were not expressed as predicted, it might be
possible to exclude PDGF as a significant mediator of the
early fibroproliferative response. Thus, we must continue
pursuit of the technologies that will allow definitive answers to the fundamental questions. In our view, the current methods to produce transgenic and knockout mice
hold the key.
Based upon what is known about gene and protein expression in animal models of lung injury, it is possible to
reconstruct a partial scenario of how several growth factors may be functioning. Using asbestos-induced lung fibrosis as a paradigm of lung injury, it is clear that multiple
events lead to the fibroproliferative process. For example,
elegant studies from Mossman's laboratory (35) show a
rapid upregulation of genes coding for antioxidant enzymes. This has been interpreted to mean that tissue-damaging oxygen radicals are being generated soon after asbestos exposure, and this could be the initial mechanism
through which inhaled fibers injure the bronchiolar-alveolar epithelium (36). Such injury consequently could activate the expression of epithelial cell mitogens like TGF-
(14), mesenchymal mitogens (e.g., PDGF), and inducers of
extracellular matrix elements like TGF-. This view of
events is supported by a recent study from our group
showing that a single 5-h exposure to asbestos upregulates
TGF- expression, primarily in macrophages and the bronchiolar-alveolar epithelium (14). As demonstrated here for
PDGF, TGF- was found only at sites of lung injury, and
not in unexposed or control iron-exposed animals. The major difference in the pattern of expression between PDGF
and TGF- is that PDGF is demonstrated in significantly
more interstitial cells than TGF-, and PDGF gene expression can be detected more rapidly, i.e., immediately after
the exposure versus 24 h post-exposure for TGF-. Whether
or not these findings tell us anything about the function of
these factors is not known at this time, particularly since it
is becoming apparent that cells responding to injury can
exhibit alterations in PDGF- receptor expression and respond accordingly (37). This concept of PDGF- receptor
upregulation by injury or membrane perturbation adds another variable to an already complex pathobiology, as we
have suggested previously (2). In addition, not only are
multiple "pro-growth" factors being elaborated as a result
of injury, there are "anti-growth" factors. The best characterized of these are prostaglandins and interferons that reduce fibroblast growth (38), interleukin 10 (39), and TGF-
(40). This potent factor not only blocks both epithelial and
mesenchymal cell proliferation, it also induces extra-cellular matrix production by fibroblasts (40), a distinct feature
of the fibroproliferative process. We have shown that
TGF- is expressed in these asbestos-exposed animals
(41) as part of the developing scenario of growth factor elaboration, but the distribution of interferons, arachidonic acid metabolites or interleukins remains undefined
in this model. Thus, as a result of deposition of fibers and
the consequent injury, macrophages are summoned by
complement proteins (22) and numerous fibers are phagocytized on the alveolar surfaces (2, 11, 13). Many of the inhaled fibers are transported to the lung interstitium by the
alveolar epithelium (42), activating growth factor genes in
macrophages, alveolar epithelium and interstitial mesenchymal cells, shown here for PDGF and referenced above
for TGF- and -. With the brief, intense exposures used
in our experiments, cell proliferation peaks rapidly and returns to normal within 1 or 2 wk, depending upon the duration of exposure (12, 14, 23, 25). This is consistent with
the growth factor expression shown here and in our earlier
studies (14). Even though cell proliferation and growth
factor presence wane, obvious fibroproliferative lesions
develop and persist for at least 6 mo post-exposure (25). Of course, we do not know if it is the epithelial, mesenchymal, or macrophage-derived factors, singly or in combination, that are the central mediators of lesion development.
We also propose that continuous or repeated exposure to
a toxic agent like asbestos will induce chronic expression
of the growth factors, thus inducing the diffuse nature of
the interstitial process (43).
New findings from our laboratory show that the p53 tumor suppressor protein is expressed with similar temporal
and anatomic features as the PDGF, TGF- (14), and
TGF- proteins described above (44). Our findings are
consistent with studies in a swine model of wound healing
after acute cutaneous injury where PDGF-B and PDGF-
receptor expression precede the appearance of p53 (45). PDGF appeared to be expressed during the cell proliferation stage of healing, and p53 became apparent at later
times. It was proposed that this inverse relationship of
PDGF and p53 expression reflected their reciprocal roles
in the healing process. However, the recent finding that
p53 can activate expression of the TGF- gene (46) suggests a potential role for the p53 protein at the earliest events critical to the initiation of fibroproliferative disease. We currently are using transgenic mouse models, asking
whether or not p53 is significant in cytokine gene expression in asbestos-exposed mice.
The rapid activation of PDGF expression shown here
in asbestos-exposed rats suggests that PDGF may initiate
or amplify other early events associated with asbestos-
induced lung injury. The rapid activation of transcription
factors such as AP-1 (47) or NF-
B (48) that is observed in
asbestos-exposed cells in culture could promote production of PDGF. Moreover, PDGF can stimulate the activity
of AP-1 (49) and NF-B (50). Thus, asbestos exposure may stimulate a positive feedback loop that promotes
mesenchymal cell proliferation via PDGF as we previously
proposed (2, 51). This postulate is reasonable because it
now is clear that asbestos exposure upregulates PDGF
gene expression and protein production in the interstitial
compartment of the lung at sites of developing fibroproliferative lesions (see Figures 3-7). Furthermore, new information from this model system shows an increase in
PDGF--receptor mRNA and protein in the lung interstitium following asbestos exposure (J. Lasky, unpublished
observations).
The main message we wish to convey is that the genes for multiple growth factors, anti-growth factors, antioxidants, and regulatory proteins are upregulated during the development of fibroproliferative lung disease. We show here that PDGF-A and -B chain expression fits neatly into the scenario as a potent mesenchymal cell mitogen. It obviously is not the only one, and we will not understand the precise role of each element in the process until the appropriate technologies are applied.
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Footnotes |
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Address correspondence to: Arnold R. Brody, Ph.D., Lung Biology Program and Department of Pathology, Tulane University Medical Center, 1430 Tulane Avenue; SL-79, New Orleans, LA 70112. E-mail: abrody{at}tmc.tulane.edu
(Received in original form March 10, 1997 and in revised form April 11, 1997).
The authors want to thank MOCHIDA Pharmaceutical Co., Ltd. for the gift of PGF 007 and the staff of Tulane Medical Center Anatomic Histopathology Laboratory for technical assistance.Acknowledgments: This work was supported by NIH grants RO1 ES06766 (A.R.B.), R29 ESO7856 (G.F.M.), and K08HL03374 (J.A.L.). The authors also acknowledge the continued support of the Tulane Center for Bioenvironmental Research and The Tulane Cancer Center.
Abbreviations ANOVA, analysis of variance; IHC, immunohistochemistry; ISH, in situ hybridization; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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