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A R Brody

Publications and source records attributed to A R Brody.

At least 19 recordsLinked to original sources
PubMed1998

Dexamethasone activates expression of the PDGF-alpha receptor and induces lung fibroblast proliferation.

Corticosteroids (CSs) are commonly used for anti-inflammatory therapy in asthma and in interstitial lung diseases. In attempting to understand the mechanisms through which CSs control cell proliferation, we have carried out experiments to test the effects of dexamethasone (Dex) on the growth of lung fibroblasts. Using mouse 3T3 fibroblasts as well as early-passage rat lung fibroblasts (RLFs), we show that the quiescent cells in 1% serum or in serum-free media proliferate significantly in response to the addition of 10(-7) to 10(-9) M Dex. Increases as high as fourfold in cell numbers were recorded for the RLFs after 48 h in culture. A polyclonal antibody to the AB isoform of human platelet-derived growth factor (PDGF) blocked the proliferative response. As expected, the fibroblasts produced primarily PDGF-A chain, and the RLFs exhibited few PDGF-alpha receptors (PDGF-R alpha), the receptor type necessary for binding the AA isoform. Accordingly, we determined that Dex upregulated PDGF-R alpha mRNA and protein. Therefore, we can postulate that Dex-induced fibroblast proliferation is mediated, at least in part, by PDGF-AA, which binds to the PDGF-R alpha.

3T3 Cells↗
PubMed1998

Upregulation of the PDGF-alpha receptor precedes asbestos-induced lung fibrosis in rats.

Platelet-derived growth factor-AA (PDGF-AA) and its matching alpha receptor (PDGF-R alpha) are upregulated in rat lung fibroblasts (RLFs) after exposure to chrysotile asbestos fibers in vitro, which results in asbestos-induced RLF proliferation. We now report our in vivo observations, which show an increase in the expression of PDGF-R alpha mRNA, but not PDGF-beta receptor mRNA, in asbestos-exposed rat lungs when compared with RNA from air-exposed (sham) and iron-exposed lungs. Western analysis of membrane preparations confirmed the observations on mRNA expression by demonstrating an increase in PDGF-R alpha peptide expression in the asbestos-exposed rat lungs, compared with that in the air-exposed lungs. Immunohistochemistry for the PDGF-R alpha was performed on air- and asbestos-exposed rat lungs and revealed a clear increase in staining within interstitial and subepithelial compartments in the exposed animals. These observations, along with our previous report demonstrating an increase in the PDGF-AA isoform expression immediately after asbestos-exposure, suggest a scenario in which a potent lung mesenchymal cell mitogen, PDGF-AA, and its alpha-receptor are upregulated prior to the development of a fibroproliferative lung lesion, and thus may play a central role in the pathogenesis of asbestos-induced lung fibrosis.

Animals↗
PubMed1997

Occupational lung disease and the role of peptide growth factors.

Occupational lung disease can take many forms, including obstructive airway disease, asthma, and restrictive parenchymal fibrosis. It would not be useful to attempt an all-encompassing review of such a broad topic, even if one were restricted to the most recent literature. Thus, I have chosen a small corner of one disease process that relates to work ongoing in my laboratory, ie, the potential role of growth factors in the pathogenesis of fibroproliferative lung disease. This process is the cornerstone of interstitial pulmonary fibrosis, which is caused by inhaling such commonly used materials as asbestos and silica. Diffuse interstitial pulmonary fibrosis results from chronic exposures in the workplace, and the initial lesions of the fibroproliferative process that culminate in interstitial pulmonary fibrosis are readily produced in the laboratory by exposing rats and mice. The disease could be mediated by a combination of peptide growth factors and cytokines that are expressed at sites of lung injury.

Animals↗
PubMed1997

Inhaled asbestos fibers induce p53 expression in the rat lung.

Humans and rodents exposed to an aerosol of asbestos fibers develop lung injury that can lead to a fibroproliferative response culminating in excessive scarring and impaired lung function. To define the early events that precede asbestos-induced fibrotic lung disease, rats were exposed to an aerosol of chrysotile asbestos fibers for 5 h. At various times after exposure, the lungs of the asbestos-exposed animals were evaluated immunohistochemically for expression of the p53 tumor suppressor protein, a growth regulatory protein. p53 became detectable by immunostaining at the predicted sites of fiber deposition (the bronchiolar-alveolar duct bifurcations) by 24 h after exposure. The number of cells positive for p53 immunostaining increased to a maximal level at 8 days after exposure, decreased by 14 days and returned to a low basal level at the 30-day time point. Control groups of rats that were unexposed or exposed to an aerosol of iron beads were negative for p53 immunostaining throughout the 30-day assessment period. Simultaneous detection of the proliferating cell nuclear antigen (PCNA) at the sites of fiber deposition in the asbestos-exposed animals agrees with our previous finding that p53 binds and regulates the PCNA promoter.

Administration, Inhalation↗
PubMed1997

Rapid activation of PDGF-A and -B expression at sites of lung injury in asbestos-exposed rats.

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 approximately 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.

Animals↗
PubMed1997

Analyzing the genes and peptide growth factors expressed in lung cells in vivo consequent to asbestos exposure and in vitro.

Inhalation of fibrogenic particles causes injury to the bronchiolar-alveolar epithelium. Consequently, there is a rapid proliferative response as the epithelium recovers and interstitial mesenchymal cells divide and produce connective tissue. In our model of brief (5-hr) exposure to chrysotile asbestos (approximately 1000 fibers/cc) in rats and mice, these events result in focal scarring at the bronchiolar-alveolar duct junctions in a histopathologic pattern identical to that seen in asbestos-exposed individuals. After 3 consecutive days of exposure, these lesions persist for at least 6 months postexposure. We postulate that cell proliferation and production of extracellular matrix is mediated in large part by three peptide growth factors, transforming growth factors alpha and beta (TGF-alpha and -beta), and platelet-derived growth factor (PDGF) A- and B-chains. To test this hypothesis in part, we have asked whether the genes that code for these growth factor proteins are activated at sites of asbestos-induced lung injury. If these genes were not activated, it would be reasonable to suspect that other potent growth factors and cytokines released during lung injury could be the primary mediators of fibroproliferative lung disease. In the studies reported here, we show, by in situ hybridization (ISH) and immunohistochemistry, that the four genes and their concomitant proteins are expressed within 24 hr in the bronchiolar-alveolar epithelium and underlying mesenchymal cells. RNase protection assay and ISH showed that the PDGF gene was upregulated during the first 5 hr of exposure and all the gene products remained above control levels for at least 2 weeks postexposure. TGF-alpha is a potent mitogen for epithelial cells, whereas the PDGF isoforms are potent growth factors for mesenchymal cells. TGF-beta retards fibroblast growth but stimulates extracellular matrix synthesis. Further studies using gene knockouts, appropriate antibodies, or antisense technology will be necessary to prove whether any of the growth factors are playing a significant role in fibrogenic lung disease. In addition, we have carried out a series of studies using type II alveolar epithelial cells purified from adult mouse lungs and maintained for up to 8 weeks in serum-free culture. These cells exhibit high transepithelial resistance values and they release TGF-beta 1 and -beta 2. This cell type also has been cultured from TGF-alpha knockout mice, resulting in monolayers with increased transepithelial resistance. This combination of studies in vivo and in vitro will allow us to pursue the mechanisms through which growth factors mediate lung fibrosis.

Animals↗
PubMed1996

Interferon-gamma modulates lung macrophage production of PDGF-BB and fibroblast growth.

Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. We have studied here the cytokine interferon-gamma (IFN-gamma) which is known to prime macrophages for increased PDGF production. Thus, we postulated that IFN-gamma would act as a positive regulator of PDGF-BB secretion by rat alveolar macrophages, and in addition we asked whether or not the IFN-gamma (a known anti-mitogenic cytokine) would block the growth response of primary lung fibroblasts to the PDGF-BB. Macrophages incubated with IFN-gamma or iron spheres alone for 24 h secreted 2.5-fold more PDGF-BB than control macrophages incubated in serum-free medium. Preincubation of macrophages with IFN-gamma prior to the addition of iron spheres synergistically increased PDGF-BB production 2-10-fold after 24 h. In contrast, when IFN-gamma was added to quiescent rat lung fibroblasts (RLFs) in the presence of PDGF-BB, the cytokine induced a concentration-dependent decrease in cell growth, while IFN-gamma alone did not affect proliferation. [125I]PDGF-BB receptor assays showed that neither preincubation nor coincubation of RLF with IFN-gamma affected PDGF-BB binding to its receptors.

Animals↗
PubMed1996

Pulmonary fibrogenesis after three consecutive inhalation exposures to chrysotile asbestos.

Previously, this laboratory developed a model of asbestos-induced pulmonary fibrogenesis in rats and mice after a brief (1 to 3-h) inhalation exposure. However, typical human environmental exposures would be repeated, although at lower concentrations than those used in our animal model. Here we have extended this model to encompass repeated exposures and consequent long-term effects. Groups of rats were exposed to chrysotile aerosol (10 mg/m3) for 3- to 5-h periods over 3 consecutive days. Lung fiber burden and pathologic features were studied for as long as 6 mo after exposure. We found that many of the longest (> or = 8 microm) fibers were retained in the lung for at least 6 mo, whereas shorter fibers were cleared more rapidly. The three exposures to chrysotile caused a large increase in DNA synthesis in the epithelium of terminal bronchioles and more proximal airways. When compared with a single exposure, the triple exposure caused an enhanced inflammatory response as well as a prolonged period of increased DNA synthesis in the proximal alveolar region. Hyperplastic, fibrotic lesions subsequently developed in the same region and persisted for at least 6 mo after exposure. These findings will be valuable in directing future studies of the mechanisms of pulmonary fibrosis in this model.

Administration, Inhalation↗
PubMed1996

Isolation and primary culture of murine alveolar type II cells.

Previous attempts to culture mouse alveolar type II (ATII) cells have been hampered by limited purity and cell recovery. We have now obtained culturable ATII cells from female C57BL/6 mice at a purity of 92% +/- 3 (mean +/- SD; n = 20), with viabilities of 96% +/- 2 and total yields of 5.1 +/- 0.7 X 10(6) cells per mouse. Crude lung cell suspensions were prepared by intratracheal instillation of Dispase and agarose followed by mechanical disaggregation of the lungs. Crude cell suspensions were purified by negative selection using a biotinylated-antibody, streptavidin-coated biomagnetic particle system. Cell purities were determined by Pap staining and confirmed ultrastructurally. Purified ATII cells were cultured on fibronectin-coated chamber slides and maintained for up to 5 days in DMEM with 10% fetal bovine serum. Cultures exhibited minimal contamination by Clara cells, mesenchymal cells, or endothelial cells, and the epithelial nature of the cultures was confirmed by positive cytokeratin staining in at least 97% of the cells through day 5. Day 3 cultures demonstrated osmium tetroxide/tannic acid-stained granules consistent with lamellar bodies in 76% +/- 3.6 of the cells. The cultures displayed features distinct from those previously described for adult rat ATII cells, including irregularly-shaped cells and the formation of numerous cytoplasmic projections in direct contact with other cells. These studies indicate that excellent yields of highly purified, culturable ATII cells can be obtained from genetically defined mice. These techniques may provide powerful new models for the study of parenchymal lung disease in vitro.

Animals↗
PubMed1996

Up-regulated expression of transforming growth factor-alpha in the bronchiolar-alveolar duct regions of asbestos-exposed rats.

It has become apparent that the numerous growth factors and cytokines are produced during the development of fibroproliferative lung disease. Investigators must sort out which combinations of these factors are playing mechanistic roles in the disease process. Here we demonstrate that transforming growth factor (TGF)-alpha, a potent epithelial and mesenchymal cell mitogen, is upregulated specifically at the sites of asbestos fiber deposition in the lungs of rats exposed for 5 hours. Unexposed animals and those exposed to high concentrations of iron spheres exhibited no increase in TGF-alpha expression at any time during the experiment. Inhaled asbestos fibers deposit initially at the bronchiolar-alveolar duct regions and alveolar macrophages accumulate at these sites within hours. Non-isotopic in situ hybridization and immunohistochemistry were used to show that the mRNA that codes for TGF-alpha along with the peptide were clearly up-regulated at the bronchiolar-alveolar duct regions by 24 hours after the single asbestos exposure. The numbers of labeled cells demonstrated that expression of the mRNA and protein remained significantly above background for at least 2 weeks after exposure along with increased cell proliferation assessed by staining for proliferating cell nuclear antigen. This, to our knowledge, is the first demonstration of TGF-alpha expression at sites of lung injury in developing fibroproliferative disease. This finding supports the hypothesis that the growth factor is involved in the dramatic epithelial and mesenchymal proliferation we documented previously, although additional experiments will be essential to establish the precise role of TGF-alpha.

Administration, Inhalation↗
PubMed1995

Bromo-deoxyuridine (BRDU) uptake in the lungs of rats inhaling amosite asbestos or vitreous fibres at equal airborne fibre concentrations.

Rats were exposed, by inhalation, to target airborne fibre concentrations of 1000 f/ml (PCOM fibres by WHO criteria) of a long amosite asbestos sample and a vitreous fibre sample; the target was closely attained for both fibre samples. The size distributions of the two fibre samples was closely similar. Rats were placed in the chambers for 7 hours and then, following a further 16 hours in room air, were injected with bromo-deoxyuridine (BRDU). The presence of BRDU-positive cells in terminal bronchioles/alveolar ducts was assessed in blocks taken from various parts of the left lung, from apex to base. There were significant differences in the proliferative responses between animals but there were also significant differences between the treatments. Lungs from rats exposed to vitreous fibres showed no greater response than the controls, but there was a markedly greater proliferative response in the lungs of rats inhaling long amosite. There was a decreasing gradient of proliferative response from the apex of the lung to the base with all treatments. This could be explained by different degrees of deposition in different areas of the lung. Similar amounts of fibre accumulated in the lungs of rats exposed to the two fibre types and it is unlikely that dissolution could be important over the timescale used here. We conclude that, when amosite asbestos deposits in the lungs of rats it stimulates a proliferative response and that deposition of an equal number of similar-sized vitreous fibres has no effect.

Air Pollutants↗
PubMed1995

Cytokine-binding proteins in the lung.

Numerous cytokines and growth factors signal the normal processes of tissue maintenance and remodeling in the lung, yet the aberrant expression of these peptide mediators is involved in a variety of pulmonary diseases. Furthermore, several different binding proteins function in controlling the extracellular levels of many of these cytokines in the lung. For example, a variety of cytokines and growth factors bind to and are regulated by the ubiquitous proteinase inhibitor, alpha 2-macroglobulin. The insulin-like growth factors are controlled by a specific class of six different insulin-like growth factor binding proteins. The transforming growth factor-beta family and fibroblast growth factors interact with extracellular matrix proteins. Several growth factor receptors are shed into the extracellular milieu where they retain a functional binding domain and thereby act as specific binding proteins. Cytokine-binding proteins appear to have a diversity of functions and may serve as extracellular cytokine reservoirs, protective shields against proteolytic degradation of cytokines, modifiers of cytokine-induced biological activity, or as clearance avenues for cytokines. The wide spectrum of cytokine-regulating molecules is important in cell-cell communications under normal conditions, whereas cytokine-binding protein dysfunction could contribute to a number of pulmonary diseases.

Animals↗