Cyclooxygenase-2 Deficiency Results in a Loss of the Anti-Proliferative Response to Transforming Growth Factor-β in Human Fibrotic Lung Fibroblasts and Promotes Bleomycin-Induced Pulmonary Fibrosis in Mice
2001; Elsevier BV; Volume: 158; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64092-8
ISSN1525-2191
AutoresCarmel Beulin Keerthisingam, Gisli Jenkins, Nicholas K. Harrison, Norma A. Hernández-Rodríguez, Helen Booth, Geoffrey J. Laurent, Stephen L. Hart, Martyn Foster, Robin J. McAnulty,
Tópico(s)Pulmonary Hypertension Research and Treatments
ResumoProstaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-β1. However, in patients with pulmonary fibrosis, PGE2 levels are decreased. In this study we examined the effect of TGF-β1 on PGE2synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-β1-induced PGE2 synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-β1. This failure to induce PGE2 synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE2synthesis in the presence of increasing levels of profibrotic mediators such as TGF-β1 may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis. Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-β1. However, in patients with pulmonary fibrosis, PGE2 levels are decreased. In this study we examined the effect of TGF-β1 on PGE2synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-β1-induced PGE2 synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-β1. This failure to induce PGE2 synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE2synthesis in the presence of increasing levels of profibrotic mediators such as TGF-β1 may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis. Pulmonary fibrosis is characterized by inflammatory cell infiltration, fibroblast proliferation, and excess deposition of extracellular matrix proteins including collagen in the lung parenchyma. This results in the loss of normal alveolar structure and impaired lung function. The disease can occur as an isolated pulmonary disorder in response to known or unknown causes1Turner-Warwick M In search of a cause of cryptogenic fibrosing alveolitis (CFA): one initiating factor or many?.Thorax. 1998; 53: S3-S9Crossref PubMed Google Scholar and in association with connective tissue disorders such as systemic sclerosis2Harrison NK Myers AR Corrin B Soosay G Dewar A Black CM Du Bois RM Turner-Warwick M Structural features of interstitial lung disease in systemic sclerosis.Am Rev Respir Dis. 1991; 144: 706-713Crossref PubMed Google Scholar and rheumatoid arthritis.3Gabbay E Tarala R Will R Carroll G Adler B Cameron D Lake FR Interstitial lung disease in recent onset rheumatoid arthritis.Am J Respir Crit Care Med. 1997; 156: 528-535Crossref PubMed Scopus (325) Google Scholar The pathogenesis of pulmonary fibrosis is incompletely understood. However, fibroblast proliferation and collagen synthesis are known to be regulated, at least in part, by a complex interaction between stimulatory and inhibitory mediators.4McAnulty RJ Laurent GJ Collagen and its regulation in pulmonary fibrosis.in: Phan SH Thrall RS Pulmonary Fibrosis. Marcel Dekker, New York1995: 135-171Google Scholar Several stimulatory mediators including transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), insulin-like growth factor-1, and thrombin as well as inhibitory mediators such as interferon, glucocorticoids, epidermal growth factor, and prostaglandin E (PGE)2 have been suggested to play a role in the pathogenesis of pulmonary fibrosis.4McAnulty RJ Laurent GJ Collagen and its regulation in pulmonary fibrosis.in: Phan SH Thrall RS Pulmonary Fibrosis. Marcel Dekker, New York1995: 135-171Google Scholar PGE2 is a potent inhibitor of fibroblast proliferation5Oliver MH Harrison NK Bishop JE Cole PJ Laurent GJ A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.J Cell Sci. 1989; 92: 513-518Crossref PubMed Google Scholar, 6McAnulty RJ Hernandez-Rodriguez NA Mutsaers SE Coker RK Laurent GJ Indomethacin suppresses the anti-proliferative effects of transforming growth factor-β isoforms on fibroblast cell cultures.Biochem J. 1997; 321: 639-643Crossref PubMed Scopus (113) Google Scholar and collagen synthesis.7Goldstein RH Polgar P The effect and interaction of bradykinin and prostaglandins on protein and collagen production by lung fibroblasts.J Biol Chem. 1982; 257: 8630-8633Abstract Full Text PDF PubMed Google Scholar, 8Saltzman LE Moss J Berg RA Hom B Crystal RG Modulation of collagen production by fibroblasts: effects of chronic exposure to agonists that increase intracellular cyclic AMP.Biochem J. 1982; 204: 25-30Crossref PubMed Scopus (52) Google Scholar It is normally present in the lung at much higher concentrations than in plasma9Ozaki T Rennard SI Crystal RG Cyclooxygenase metabolites are compartmentalized in the human lower respiratory tract.J Appl Physiol. 1987; 62: 219-222PubMed Google Scholar and is the major eicosanoid product of fibroblasts.10Korn JH Fibroblast prostaglandin E2 synthesis. 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In addition, a number of pro-inflammatory mediators such as TGF-β1, interleukin (IL)-1β, tumor necrosis factor-α, and PDGF induce fibroblasts to synthesize PGE2.6McAnulty RJ Hernandez-Rodriguez NA Mutsaers SE Coker RK Laurent GJ Indomethacin suppresses the anti-proliferative effects of transforming growth factor-β isoforms on fibroblast cell cultures.Biochem J. 1997; 321: 639-643Crossref PubMed Scopus (113) Google Scholar, 12Diaz A Varga J Jimenez SA Transforming growth factor-β stimulation of lung fibroblast prostaglandin E2 production.J Biol Chem. 1989; 264: 11554-11557Abstract Full Text PDF PubMed Google Scholar, 13McAnulty RJ Chambers RC Laurent GJ Regulation of fibroblast procollagen production. 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Potential implications for the evolution of the inflammatory process.Am J Respir Cell Mol Biol. 2000; 22: 628-634Crossref PubMed Scopus (95) Google Scholar but the functional effects of this have not been investigated. Furthermore, mice deficient in COX-2 exhibit fibroproliferative disorders of the heart and kidneys22Morham SG Lagenbach R Loftin CD Tiano HF Vouloumanos N Jennette JC Mahler JF Kluckman KD Ledford A Lee CA Smithies O Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 23Dinchuk JE Car BD Focht RJ Johnston JJ Jaffee BD Covington MB Contel NR Eng MB Collins RJ Czerniak PM Gorry SA Trzaskos JM Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.Nature. 1995; 378: 406-409Crossref PubMed Scopus (903) Google Scholar but there is no data on the fibroproliferative response in the lungs of these animals. Evidence suggests that TGF-β1 plays a key role in the pathogenesis of pulmonary fibrosis. It is a potent stimulator of collagen synthesis and regulator of fibroblast proliferation.6McAnulty RJ Hernandez-Rodriguez NA Mutsaers SE Coker RK Laurent GJ Indomethacin suppresses the anti-proliferative effects of transforming growth factor-β isoforms on fibroblast cell cultures.Biochem J. 1997; 321: 639-643Crossref PubMed Scopus (113) Google Scholar, 13McAnulty RJ Chambers RC Laurent GJ Regulation of fibroblast procollagen production. 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Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein.Biochem J. 1995; 307: 63-68PubMed Google Scholar In this study we investigated the effects of TGF-β1 on PGE2 synthesis, proliferation, and collagen production by lung fibroblasts isolated from human fibrotic and nonfibrotic lung. In addition, we have assessed the role of COX-1 and COX-2 in mediating the effects of TGF-β1 on fibroblast PGE2synthesis by Northern analysis and using selective COX inhibitors. We have also performed in vivo experiments examining the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We provide evidence, for the first time, to demonstrate that fibroblasts from patients with pulmonary fibrosis have a limited capacity to up-regulate PGE2 synthesis in response to TGF-β1 and that in control fibroblasts this response is mediated via COX-2. The lack of stimulation of PGE2 synthesis by the fibroblasts derived from fibrotic lung correlates with a loss of the anti-proliferative response to TGF-β1. We also demonstrate that mice deficient in COX-2 are more susceptible to bleomycin-induced lung injury. Thirty-five lung fibroblast cell lines were studied. The fibrosis group consisted of 17 cell lines derived from patients with pulmonary fibrosis (idiopathic pulmonary fibrosis, n = 9; systemic sclerosis, n = 7). All biopsies from which cell lines were derived showed histological evidence of pulmonary fibrosis. In addition one cell line (CCL-134) established from a patient with idiopathic pulmonary fibrosis was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The control group consisted of 18 cell lines that were established from lung tissue derived from various sources. Thirteen were biopsy samples from patients undergoing lung resection for localized tumor. Tissue was taken from areas of macroscopically normal lung parenchyma, distal to any tumor mass. Two cell lines were established from patients who died of non-lung related causes. In addition to these, another three human lung fibroblast cell lines were obtained from the ATCC (CCI-201, CCI-204, and CCL-200). Fibroblast cell lines were established from explant cultures.34Jordana M Schulman J McSharry C Irving LB Newhouse MT Jordana G Gauldie J Heterogeneous proliferative characteristics of human adult lung fibroblasts lines and clonally derived fibroblasts from control and fibrotic tissue.Am Rev Respir Dis. 1988; 137: 579-584Crossref PubMed Scopus (205) Google Scholar Briefly, lung tissue biopsies were cut into 1-mm3 fragments and placed ∼10 mm apart on the surface of culture dishes with Dulbecco’s modified Eagle’s medium (Life Technologies, Paisley, UK) supplemented with 10% (v/v) newborn calf serum (Imperial Laboratories, Andover, UK), penicillin (100 U/ml), streptomycin (100 μg/ml), and 2.5 μg/ml amphotericin B (all from Life Technologies, Paisley, UK). Fibroblasts were observed growing out of the tissue fragments after 6 to 8 days, developing into a near confluent monolayer of cells after 3 to 4 weeks. Experiments were conducted on cells between passages 3 and 17. Fibroblast cell lines were characterized immunohistochemically to confirm their purity. Staining with antibodies to cytokeratin, von Willebrand factor, and desmin was negative, indicating that the cultures did not contain significant numbers of epithelial, mesothelial, endothelial, or smooth muscle cells. Greater than 95% of the cells stained positively for vimentin, and between 20 and 30% of the cells were also positive for α-smooth muscle actin, confirming the fibroblast/myofibroblast phenotype of the cell lines. Cell proliferation was assessed using either a spectrophotometric assay based on the uptake and subsequent elution of methylene blue as described previously5Oliver MH Harrison NK Bishop JE Cole PJ Laurent GJ A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.J Cell Sci. 1989; 92: 513-518Crossref PubMed Google Scholar or by measuring the incorporation of3H-thymidine into DNA. Briefly, 96-well microtiter plates were seeded with 6 × 103 cells/well in Dulbecco’s modified Eagle’s medium containing 0.4% newborn calf serum. After a 24-hour preincubation, serum-free media was added containing TGF-β1 (R&D Systems Europe Ltd., Oxon, UK) at concentrations between 0 and 640 pg/ml. The final concentration of newborn calf serum in the media was 0.2% (v/v). Changes in cell number were assessed 72 hours later. Results were expressed as percentage change in mean absorbance compared with cells exposed to medium alone. To measure thymidine incorporation, [3H]-thymidine (Amersham, Buckinghamshire, UK) was added to each well to give a final concentration of 37 KBq/well. Changes in DNA synthesis were assessed at various times up to 72 hours. Thymidine incorporated into DNA was harvested onto glass fiber filters (ICN Flow, Oxfordshire, UK), radioactivity measured, and values expressed as percentage change in mean disintegrations per min (dpm) as compared to cells exposed to medium alone. In experiments to block PGE2 synthesis, indomethacin at a final concentration of 1 μg/ml (Sigma, Poole, England) was added to the cells 30 minutes before the addition of TGF-β1. Hydroxyproline was measured as an index of fibroblast procollagen production using previously described methods.13McAnulty RJ Chambers RC Laurent GJ Regulation of fibroblast procollagen production. Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein.Biochem J. 1995; 307: 63-68PubMed Google Scholar Cells were grown to confluence in 2.4-cm diameter wells in Dulbecco’s modified Eagle’s medium supplemented with 5% newborn calf serum. Once confluent, cells were incubated for a further 24 hours. The media was removed and replaced with 1 ml of preincubation medium containing 4 mmol/L glutamine, 50 μg/ml ascorbic acid (Sigma), 0.2 mmol/L proline (Sigma), and 0.4% newborn calf serum (v/v) and incubated for 24 hours. The media was then replaced with either 0.5 ml of fresh preincubation medium alone or preincubation media containing indomethacin (1 μg/ml) and incubated for 30 minutes. Finally 0.5 ml of media or media containing TGF-β1 (1 ng/ml final concentration) was added and incubated for 24 hours before harvesting. Parallel sets of plates were seeded and treated in the same way to determine cell number. To assess procollagen production, the cell layer and medium were combined and proteins precipitated in 67% (v/v) ethanol at 4°C overnight. The precipitated proteins were recovered by vacuum filtration onto polyvinylidene difluoride filters (pore size, 0.45 μm; Millipore Ltd., Watford, UK) and hydrolyzed in 2 ml of 6 mol/L HCl at 110°C overnight. Hydroxyproline was isolated and quantified by reverse-phase high-pressure liquid chromatography of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-CI)-derivatized hydrolysates as described previously.13McAnulty RJ Chambers RC Laurent GJ Regulation of fibroblast procollagen production. Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein.Biochem J. 1995; 307: 63-68PubMed Google Scholar Values were corrected for the amount of hydroxyproline present in the cell layer and culture medium at the start of the incubation period as well as cell number and expressed either as pmol of hydroxyproline/105 cells/hour or as a percentage increase greater than basal procollagen production. PGE2 was measured in the medium from cells cultured in the same way as described for the determination of procollagen production. In addition to indomethacin, cells were also preincubated with the COX-2 selective inhibitor; NS-398 (Biomol Research Labs; Plymouth, UK) at a final concentration of 5 μg/ml35Wong E Bayly C Waterman HL Riendeau D Mancini JA Conversion of prostaglandin G/H synthase-1 into an enzyme sensitive to PGHS-2-selective inhibitors by a double His513? Arg and lle523? Val mutation.J Biol Chem. 1997; 272: 9280-9286Crossref PubMed Scopus (133) Google Scholar or the COX-1 preferential inhibitor, piroxicam (Sigma) at a final concentration of 2.5 ng/ml36Frolich JC A classification of NSAIDs according to the relative inhibition of cyclooxygenase isoenzymes.Trends Pharmacol Sci. 1997; 18: 30-34Abstract Full Text PDF PubMed Scopus (262) Google Scholar Cells were exposed to the selective and nonselective inhibitors for 30 minutes before the addition of TGF-β1 (1 ng/ml) or preincubation medium. After a further 24-hour incubation period, PGE2 was measured using a specific enzyme immunoassay (Amersham, Bucks, UK). Results were expressed as pg of PGE2 per 105 cells or per ml of media. Cells were seeded into 10-cm Petri dishes at a concentration of 5 × 105 cells/dish and treated in an identical manner to those cultures used for the procollagen assay. Cells were exposed to TGF-β1 (1 ng/ml) for various times up to 24 hours. Total RNA was extracted from the cells using Trizol reagent (Life Technologies, Paisley, UK) in accordance with the manufacturer’s instructions. Five to 10 μg of RNA was fractionated by electrophoresis through a 1% (w/v) agarose/formaldehyde gel, transferred to a nylon membrane (Hybond N, Amersham, Bucks, UK) by Northern transfer, and fixed by UV crosslinking. To assess COX-1 and COX-2 mRNA levels, membranes were hybridized with a [32P]dCTP-labeled human cDNA probe for COX-1 (Biogenesis, Poole, UK), or a [32P]dCTP-labeled human cDNA probe for COX-2 (kindly donated by T. Hla, Dept. of Molecular Biology, Holland Lab, American Red Cross, Rockville, MD). mRNA levels were quantitated by densitometric laser scanning. COX-2+/+ (wild-type, strain SV129/C57BL/6 F2) and COX-2-deficient mice (COX-2−/−, obtained from the Jackson Laboratory Bar Harbor, ME (stock numbers 101045 and 002476), aged 6 weeks received a single intratracheal instillation of saline (0.9%) or saline containing bleomycin sulfate (1 mg/kg body weight) in a volume of 50 μl and were killed after 14 days by pentobarbitone overdose. Lungs were harvested from between four and six mice of each genotype for histological analysis. The vasculature was perfused with heparinized phosphate-buffered saline (PBS) and the lungs fixed by intratracheal instillation of freshly prepared 4% paraformaldehyde in PBS at a pressure of 25 cm H2O. The trachea was ligated just caudal to the larynx and the thoracic contents were removed and immersed in fixative overnight, transferred to 15% sucrose in PBS before dehydrating and embedding in paraffin wax. Sections (5 μm) were cut and stained with Masson’s trichrome. The extent of fibrosis was scored in a blinded manner by three independent observers based on a previously described method.37Aschroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1178) Google Scholar Each lung lobe was scored on a scale of 0 to 4 and a mean derived from the five lobe scores for each individual mouse. For comparisons between patient groups, data were expressed as the median (range) and statistical differences were determined using the Mann-Whitney U test. To compare the effects of TGF-β1, indomethacin, and the COX selective inhibitors on individual cell lines, data were expressed as the mean ± SEM and statistical differences were evaluated using the Student’s two-tailed unpaired t-test for single group comparisons and Newman-Keuls one-way analysis of variance (analysis of variance) for multiple group comparisons. Fibrosis scores were evaluated by calculating the mean ± SEM, followed by single comparisons between individual treatment groups using the Student’s two-tailed unpaired t-test. Data were considered to be statistically significant when P < 0.05. Analysis of the nonfibrotic lung fibroblast cell lines suggested that these fell into two distinct phenotypic groups in terms of their PGE2 production, basally and in response to TGF-β1, as well as their functional responses to TGF-β1 (see below). The groups consisted of those cell lines that produced PGE2 basally that was further stimulated by incubation with TGF-β1, and those that produced little or no PGE2 basally and that were not stimulated further by TGF-β1. We therefore divided this group on the basis of basal and TGF-β1-induced PGE2 production. Group II (n = 4) consisted of cell lines isolated from nonfibrotic lung where basal PGE2 production was lower than the highest level produced by cell lines derived from fibrotic lung and was not stimulated by incubation with TGF-β1. Group I (n = 6) contained all other cell lines derived from nonfibrotic lung and group III (n = 6) contained the cell lines derived from fibrotic lung. Basal and TGF-β1-induced PGE2 levels for cell lines in the three groups are shown in Figure 1. Under basal conditions, levels of PGE2 synthesis in group I ranged from 30 to 2,176 pg/105 cells (median, 321 pg/105 cells) and TGF-β1(1 ng/ml) enhanced this further by up to 16-fold (median, 798 pg/105 cells; range, 446 to 3,077 pg/105 cells). Basal levels of PGE2 synthesis by g
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