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Bleomycin Stimulates Production of Transforming Growth Factor-β by Rat Pulmonary Artery Endothelial Cells
increased cellularity were disrupted and showed type II pneumocyte hyperplasia with intense intracellular TGF-P staining. The extracellular form of TGF-13 was localized in the subepithelial matrix of bronchiolar epithelium type II pneumocytes, suggesting that TGF-P may be both produced and secreted by bronchiolar epithelial cells and type II pneumocytes. Lung sections with no evidence of inflammation or IPF did not stain with either form of anti-TGF-13 antibody. All positive staining sections were controlled by either replacing TGF-P antibody by normal rabbit serum lgG or by blocking TGF-p antibody with TGF-13 peptide l 30 prior to staining. The presence of TGF-13 both in the animal model of pulmonary fibrosis and in IPF suggests that TGF-13 may be pathogenic to the pulmonary fibrotic response.
Transforming Growth Factor-p Production by Dog Mastocytoma Cells* Storage and Release from Mast Cell Granules D. ftmnlngton, M.D.; P. Tlwmo.s, M.D.; A. Lopez, M.D.; and WGold, M.D.
T
ransforming growth factor-13 (TGF-13) is a multifunctional peptide implicated in animal models of wound healing and pulmonary fibrosis. It is a potent mediator of extracellular matrix production by lung fibroblasts, and has diverse effects on a variety of lung cells. Histologic studies in pulmonary fibrosis show markedly increased numbers of mast cells as well as evidence of mast cell secretion. However, no potent fibrogenic mediators are known to be produced by mast cells. We have demonstrated that four dog mastocytoma cell lines produce TGF-p. Each cell line expressed TGF-13, mRNA. Constitutive release of TCF-P was determined by bioassay (inhibition of mitogenesis of mink lung epithelial cells), blockade of its biologic effect by neutralizing antibody, and characteristic migration on Western blot. Quantitation of TGF-P by comparing concentration-response curves for mastocytoma cell-conditioned media and for purified porcine platelet TCF-P revealed baseline secretion of approximately 1 ng/ml per 10' cells, an amount similar to that produ£--ed by LPS-activated human monocytes. Like monocytes, dog mastocytoma cells produced TGF-P in a latent form, inactive until treated with acid. In addition to spontaneous secretion, TCF-13 was rapidly released by the degranulating agents calcium ionophore and phorbol ester, which suggests the possibility of intracellular storage. Pure mastocytoma granules were isolated by nitrogen cavitation and sucrose gradient centrifugation. Isolated granules contained TCF-P bioactivity and produced an appropriate band on Western blot. In addition, mastocytoma cell growth is strongly inhibited by TCF-13. or by mastocytoma cell-conditioned medium after transient acidification, which suggests that, in addition *Fro~ the Cardiovascular Research Institute, Cancer Research
Institute, and Department of Medicine, University of California, San Francisco.
to regulation of proliferation of other lung cells, TCF-13 may play an autocrine role in the regulation of mast cell proliferation. These studies suggest that mast cells may play an important part in inftammatory and fibrotic reactions in the lung via regulated secretion ofTCF-13.
Bleomycin Stimulates Production
of Transforming Growth Factor-p
by Rat Pulmonary Artery Endothelial Cells*
S. H. Phan, M.D.; M. Gharee-Kerrooni, D.V.M.; F. \\blber; and U. S. Ryan, Ph.D.
Bleom.ycin-induced pulmonary fibrosis is characterized by mcreased lung collagen synthesis. The mechanism ~f upregulation oflung collagen synthesis is unclear, although
mcreased secretion of cytokines with fibrogenic properties is thought to be important in regulating this response. However, the cellular source of these cytokines remains to be identified. We examined rat pulmonary artery endothelial cells as a possible source of mediator(s) that could stimulate fibroblast collagen production. Confluent endothelial cells were treated with 0-500 nw ml bleomycin and the 16-h-conditioned media collected and analyzed for collagen synthesis stimulatory activity, with rat lung fibroblasts used as target cells. The results show that bleomycin caused the secretion of such an activity by rat pulmonary artery endothelial cells in a dose-dependent manner, reaching maximal levels at 100 nwml of bleomycin. The activity was specific for collagen synthesis, which was stimulated up to 4 times control values, while noncollagenous proteins were stimulated only 2-fold. The activity was acid stable and by gel filtration analysis was found to be heterogeneous, with major peaks having estimated molecular weights of6-25 kD. It was significantly reduced on treatment with anti-transforming growth factor-13 (TGF-13) IgG, but not with nonspecific lgG. The presence of TGF-13 in bleomycin-treated endothelial cell-conditioned media was confirmed by a standard bioassay that used mink lung epithelial cells, and the responsiveness of rat lung fibroblasts to TGF-13 was confirmed by using pure porcine TGF-13. Acidification failed to increase the observed TCF-13 activity, which suggested that most of the secreted cytokine was in the active form with minimal amounts in latent form. Northern blot analysis of endothelial cell RNA revealed upregulation ofTGF-13 mRNA in bleomycin-treated cells. These results indicate that the collagen synthesis stimulatory activity in conditioned media of bleomycin-treated rat pulmonary artery endothelial cells is mostly due to TGF13. Such a direct mechanism of stimulation by bleomycin may be important in the pathogenesis of pulmonary fibrosis induced by this drug, since these concentrations of bleomycin are likely to be encountered in vivo. *From the University of Michigan Medical School, Ann Arbor, and the Monsanto ComllW\y. St Louis, MO (U .S.R.). Work was supporte
Transforming Growth Factor-p Production by Dog Mastocytoma Cells* Storage and Release from Mast Cell Granules D. ftmnlngton, M.D.; P. Tlwmo.s, M.D.; A. Lopez, M.D.; and WGold, M.D.
T
ransforming growth factor-13 (TGF-13) is a multifunctional peptide implicated in animal models of wound healing and pulmonary fibrosis. It is a potent mediator of extracellular matrix production by lung fibroblasts, and has diverse effects on a variety of lung cells. Histologic studies in pulmonary fibrosis show markedly increased numbers of mast cells as well as evidence of mast cell secretion. However, no potent fibrogenic mediators are known to be produced by mast cells. We have demonstrated that four dog mastocytoma cell lines produce TGF-p. Each cell line expressed TGF-13, mRNA. Constitutive release of TCF-P was determined by bioassay (inhibition of mitogenesis of mink lung epithelial cells), blockade of its biologic effect by neutralizing antibody, and characteristic migration on Western blot. Quantitation of TGF-P by comparing concentration-response curves for mastocytoma cell-conditioned media and for purified porcine platelet TCF-P revealed baseline secretion of approximately 1 ng/ml per 10' cells, an amount similar to that produ£--ed by LPS-activated human monocytes. Like monocytes, dog mastocytoma cells produced TGF-P in a latent form, inactive until treated with acid. In addition to spontaneous secretion, TCF-13 was rapidly released by the degranulating agents calcium ionophore and phorbol ester, which suggests the possibility of intracellular storage. Pure mastocytoma granules were isolated by nitrogen cavitation and sucrose gradient centrifugation. Isolated granules contained TCF-P bioactivity and produced an appropriate band on Western blot. In addition, mastocytoma cell growth is strongly inhibited by TCF-13. or by mastocytoma cell-conditioned medium after transient acidification, which suggests that, in addition *Fro~ the Cardiovascular Research Institute, Cancer Research
Institute, and Department of Medicine, University of California, San Francisco.
to regulation of proliferation of other lung cells, TCF-13 may play an autocrine role in the regulation of mast cell proliferation. These studies suggest that mast cells may play an important part in inftammatory and fibrotic reactions in the lung via regulated secretion ofTCF-13.
Bleomycin Stimulates Production
of Transforming Growth Factor-p
by Rat Pulmonary Artery Endothelial Cells*
S. H. Phan, M.D.; M. Gharee-Kerrooni, D.V.M.; F. \\blber; and U. S. Ryan, Ph.D.
Bleom.ycin-induced pulmonary fibrosis is characterized by mcreased lung collagen synthesis. The mechanism ~f upregulation oflung collagen synthesis is unclear, although
mcreased secretion of cytokines with fibrogenic properties is thought to be important in regulating this response. However, the cellular source of these cytokines remains to be identified. We examined rat pulmonary artery endothelial cells as a possible source of mediator(s) that could stimulate fibroblast collagen production. Confluent endothelial cells were treated with 0-500 nw ml bleomycin and the 16-h-conditioned media collected and analyzed for collagen synthesis stimulatory activity, with rat lung fibroblasts used as target cells. The results show that bleomycin caused the secretion of such an activity by rat pulmonary artery endothelial cells in a dose-dependent manner, reaching maximal levels at 100 nwml of bleomycin. The activity was specific for collagen synthesis, which was stimulated up to 4 times control values, while noncollagenous proteins were stimulated only 2-fold. The activity was acid stable and by gel filtration analysis was found to be heterogeneous, with major peaks having estimated molecular weights of6-25 kD. It was significantly reduced on treatment with anti-transforming growth factor-13 (TGF-13) IgG, but not with nonspecific lgG. The presence of TGF-13 in bleomycin-treated endothelial cell-conditioned media was confirmed by a standard bioassay that used mink lung epithelial cells, and the responsiveness of rat lung fibroblasts to TGF-13 was confirmed by using pure porcine TGF-13. Acidification failed to increase the observed TCF-13 activity, which suggested that most of the secreted cytokine was in the active form with minimal amounts in latent form. Northern blot analysis of endothelial cell RNA revealed upregulation ofTGF-13 mRNA in bleomycin-treated cells. These results indicate that the collagen synthesis stimulatory activity in conditioned media of bleomycin-treated rat pulmonary artery endothelial cells is mostly due to TGF13. Such a direct mechanism of stimulation by bleomycin may be important in the pathogenesis of pulmonary fibrosis induced by this drug, since these concentrations of bleomycin are likely to be encountered in vivo. *From the University of Michigan Medical School, Ann Arbor, and the Monsanto ComllW\y. St Louis, MO (U .S.R.). Work was supporte