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. 2001 Apr;158(4):1411-22.
doi: 10.1016/s0002-9440(10)64092-8.

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

C B Keerthisingam  1 , R G Jenkins, N K Harrison, N A Hernandez-Rodriguez, H Booth, G J Laurent, S L Hart, M L Foster, R J McAnulty
Affiliations

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

C B Keerthisingam et al. Am J Pathol. 2001 Apr.

Abstract

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, 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-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) 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 PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.

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Figures

Figure 1.
Figure 1.
Basal and TGF-β1-induced lung fibroblast PGE2 production. Fibroblasts were grown to confluence and then incubated for 24 hours in media alone (a) or in TGF-β1 at a concentration of 1 ng/ml (b). Cell lines from groups I and II were established from nonfibrotic lung and were categorized according to their ability to synthesize PGE2. Group III cell lines were derived from lung tissue from patients with pulmonary fibrosis. Results are expressed in pg of PGE2 per 10 cells. Each point represents an individual cell line and is derived from the mean for six replicate cultures. The median is indicated by the horizontal bar.
Figure 2.
Figure 2.
Effect of TGF-β1 on lung fibroblast proliferation. Proliferation was assessed 72 hours after the addition of TGF-β1 at the indicated concentrations using a spectrophotometric assay in representative fibroblast lines from group I (a), group II (b), and group III (c). Results are expressed as percentage change compared to cells exposed to media alone. Mean absorbance at 650 nm for cells grown in media alone were 0.149 ± 0.004, 0.180 ± 0.012, and 0.349 ± 0.011 in groups I, II, and III, respectively. Each point represents the mean ± SEM for six replicate cultures. *, P < 0.05; **, P < 0.005; and ***, P < 0.000,5 respectively.
Figure 3.
Figure 3.
Effect of a low and high concentration of TGF-β1 on lung fibroblast proliferation. Fibroblast proliferation was assessed after the addition of TGF-β1 at 5 pg/ml or 160 pg/ml. Results are expressed as percentage change compared to cells exposed to media alone. Each point represents the mean for six replicate cultures.
Figure 4.
Figure 4.
Effect of TGF-β1 on lung fibroblast procollagen production. Fibroblasts were grown to confluence and procollagen production was assessed 24 hours after the addition of TGF-β1 (1 ng/ml). Values were corrected for cell number and then expressed as percentage change compared with cells exposed to media alone. Each point represents an individual cell line and is derived from the mean of six replicate cultures. The median is indicated by the horizontal bar.
Figure 5.
Figure 5.
Effect of indomethacin on TGF-β1-induced lung fibroblast proliferation. Fibroblasts were pretreated for 30 minutes with indomethacin (1 μg/ml) before the addition of TGF-β1 (160 pg/ml) and proliferation was assessed 48 hours later in representative cell lines from groups I, II, and III. Results are expressed as percentage change compared with cells exposed to media alone. The mean absorbance at 650 nm for cells grown in media alone was 0.149 ± 0.004, 0.349 ± 0.011, and 0.154 ± 0.004 in groups I, II, and III, respectively. Each bar represents the mean ± SEM for six replicate cultures.
Figure 6.
Figure 6.
Effect of indomethacin on basal and TGF-β1-induced lung fibroblast procollagen production. Representative fibroblast cell lines from groups I, II, and III were grown to confluence and pretreated with indomethacin (1 μg/ml) for 30 minutes before the addition of TGF-β1 (1 ng/ml). Procollagen synthesis was assessed 24 hours later. Values for basal and TGF-β1-induced procollagen production (pmol hyp/10 cells/hour) from which percentage changes were calculated are as follows: group I: basal 86 ± 4, TGF-β1 146 ± 9; group II: basal 83 ± 4, TGF-β1 207 ± 16; group III: basal 284 ± 10, TGF-β1 922 ± 74. Each value represents the mean ± SEM for six replicate cultures.
Figure 7.
Figure 7.
Effect of COX selective inhibitors on TGF-β1-induced lung fibroblast PGE2 production. Fibroblasts were grown to confluence and then pretreated for 30 minutes with either NS-398 (a) or piroxicam (b) before the addition of TGF-β1 (1 ng/ml) for 24 hours in representative cell lines from groups I, II, and III. Results are expressed in pg of PGE2 per ml of media. Each bar represents the mean ± SEM for six replicate cultures. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005, respectively.
Figure 8.
Figure 8.
Effect of TGF-β1 on COX-1 and COX-2 mRNA levels in lung fibroblasts. Representative fibroblast cell lines from groups I, II, and III were grown to confluence and incubated with media alone or media containing TGF-β1 (1 ng/ml) for 6 hours before extraction of total RNA. Hybridized probe was detected by autoradiography and quantitated by densitometric laser scanning. mRNA levels for COX-2 (a) and COX-1 (b) are expressed in arbitrary densitometry units after correcting for levels of 28S RNA.
Figure 9.
Figure 9.
Extracellular matrix protein deposition after bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. COX-2+/+ (a, c, and e) and COX-2−/− (b, d, and f) mice received saline (a and b) or bleomycin sulfate (c–f) via intratracheal instillation. Lungs were harvested after 14 days and sections stained with Masson’s trichrome. In saline-treated animals (a and b) normal lung architecture was preserved. In bleomycin-treated COX-2+/+ mice, mild inflammation and moderate alveolar wall thickening was observed (c and e). In contrast, widespread inflammation and destruction of alveolar structures occurred in COX-2−/− mice instilled with bleomycin (d and f). Original magnifications: ×400 (a, b, e, and f); ×100 (c and d).
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