Prostaglandin E₂ protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction

Abstract

Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 μg/h), or the prostacyclin analog iloprost (0.33 μg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.

Fig. 1.

Surgical implantation of subcutaneous prostaglandin E₂ (PGE₂) osmotic minipumps results in elevated serum PGE₂ and cAMP levels. A: mice were given vehicle osmotic minipumps for 7 days or minipumps containing PGE₂ for the designated time points. Serum PGE₂ levels were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). B: serum was collected 1 wk after implantation of osmotic minipumps containing either PGE₂ or iloprost. ELISA showed a similar induction in cAMP levels in both PGE₂- and iloprost-treated mice. *P < 0.05 vs. saline-treated group; n = 3–5 per group for serum PGE₂ analysis and 9–11 per group for serum cAMP analysis.

Fig. 2.

Bronchoalveolar lavage fluid (BALF) PGE₂, but not 6-keto-PGF_1α, is elevated in response to bleomycin. Mice were treated once with either saline or 1 mg/kg bleomycin, and BALF PGE₂ and 6-keto-PGF_1α levels were measured 7 days later by LC/MS/MS. *P < 0.05 vs. saline-treated group; n = 6–7 per group.

Fig. 3.

PGE₂ and iloprost attenuate bleomycin-induced weight loss and decline in lung function. Mice were given vehicle, PGE₂, or iloprost via minipumps and treated with bleomycin 1 wk later. A: body weights were recorded after 1 wk of bleomycin (Bleo) administration. B: static compliance (Cst) was measured via Flexivent 21 days after bleomycin administration. Bleomycin-induced decrease in Cst observed in vehicle-treated animals was significantly attenuated in PGE₂-treated mice. *P < 0.05 vs. vehicle + bleomycin group, †P < 0.05 vs. vehicle + saline group; for body weight analysis, n = 4–8 mice for saline groups, n = 9 for bleomycin groups. For lung function analysis, n ≥ 6 for saline groups, n ≥ 11 for bleomycin groups.

Fig. 4.

Cellular infiltration in PGE₂- and iloprost-treated mice. A: although no differences were observed in total cell numbers among any of the bleomycin-treated groups, PGE₂ and iloprost both inhibited lymphocyte influx into BALF 7 days after bleomycin challenge. B: no differences in total cell numbers were observed among any of the bleomycin-treated groups 21 days after challenge. In contrast, PGE₂, but not iloprost, inhibited lymphocyte infiltration into BALF at this later time point. *P < 0.05 vs. vehicle + bleomycin group, †P < 0.05 vs. vehicle + saline group; n ≥ 4 for saline groups, n ≥ 9 for bleomycin groups.

Fig. 5.

Cell adhesion molecule and inflammatory cytokine levels in bleomycin-treated mice. A: bleomycin challenge resulted in elevated lung TNF-α, intercellular adhesion molecule (ICAM), and chemokine C-C motif ligand 2 (CCL2) mRNA levels after 7 days (determined by real-time RT-PCR). TNF-α and ICAM mRNA levels were not affected by PGE₂ or iloprost treatment. In contrast, PGE₂ significantly reduced CCL2 mRNA levels. B: BALF CCL2 protein levels were significantly reduced in PGE₂- and iloprost-treated mice that received bleomycin (determined by Bio-Plex). *P < 0.05 vs. vehicle + bleomycin group, †P < 0.05 vs. vehicle + saline group; n ≥ 4 for saline groups, n ≥ 8 for bleomycin groups.

Fig. 6.

Effect of PGE₂ and iloprost on bleomycin-induced collagen production and development of lung fibrosis. A: lung collagen content 21 days after bleomycin challenge was lower in mice treated with PGE₂ or iloprost. B: representative histology of Masson's trichrome-stained slides. C: calculated fibrosis scores based on histopathological assessment of Masson's trichrome-stained sections revealed a protective effect of PGE₂ in bleomycin-treated lungs. *P < 0.05 vs. vehicle + bleomycin group, †P < 0.05 vs. vehicle + saline group; n ≥ 6 for saline groups, n ≥ 15 for bleomycin groups.

Fig. 7.

Therapeutic effect of PGE₂ and iloprost on lung dysfunction and cellular infiltration following bleomycin. A: mice were challenged with bleomycin and then given vehicle, PGE₂, or iloprost via minipumps 14 days later. Static compliance was measured via FlexiVent 21 days after bleomycin administration. Bleomycin-induced decrease in Cst observed in vehicle-treated animals was not affected by PGE₂ or iloprost. B: PGE₂ and iloprost both inhibited total cell and lymphocyte influx into BALF when administered 14 days after bleomycin challenge. *P < 0.05 vs. vehicle + bleomycin group, †P < 0.05 vs. vehicle + saline group; n ≥ 8 for saline groups, n ≥ 21 for bleomycin groups.

Fig. 8.

Therapeutic effect of PGE₂ and iloprost on bleomycin-induced collagen production and development of lung fibrosis. Lung collagen content (A) and calculated fibrosis scores (B) were not altered in mice receiving PGE₂ or iloprost 14 days after bleomycin challenge. †P < 0.05 vs. vehicle + saline group; n ≥ 8 for saline groups, n ≥ 21 for bleomycin groups.