Lung myofibroblasts are characterized by down-regulated cyclooxygenase-2 and its main metabolite, prostaglandin E2

Abstract

Background: Prostaglandin E2 (PGE2), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing α-smooth muscle actin (α-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE2 in human lung myofibroblasts and establish whether fibroblast-myofibroblast transition (FMT) and EMT are associated with COX-2 and PGE2 down-regulation.

Methods: Fibroblasts obtained from IPF patients (n = 6) and patients undergoing spontaneous pneumothorax (control, n = 6) and alveolar epithelial cell line A549 were incubated with TGF-β1 and FMT and EMT markers were evaluated. COX-2 and α-SMA expression, PGE2 secretion and cell proliferation were measured after IL-1β and PGE2 incubation.

Results: Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1β showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1β. TGF-β1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-β1 induced slight COX-2 expression at 4 h (without increase in myofibroblasts) and 24 h, but not at 72 h. Both IPF and control cultures incubated with TGF-β1 for 72 h showed diminished COX-2 induction, PGE2 secretion and α-SMA expression after IL-1β addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-β1 for 72 h showed down-regulated COX-2 expression and low basal PGE2 secretion in response to IL-1β. Immuno-histochemical analysis of IPF lung tissue showed no COX-2 immuno-reactivity in myofibroblast foci.

Conclusions: Myofibroblasts are associated with COX-2 down-regulation and reduced PGE2 production, which could be crucial in IPF development and progression.

Figure 1. Co-immunofluorescence of COX-2 (red) and α-SMA (green) of a representative control fibroblast culture.

In basal conditions (left), it shows no COX-2 expression and isolated α-SMA-positive cells, and after IL-1β treatment for 24 h (right), it shows high COX-2 expression and a lack of COX-2 immunoreactivity in α-SMA-positive cells. Nuclei were stained with DAPI (blue). (Original magnification X200).

Figure 2. Induction of fibroblast-myofibroblast transition (FMT) in control and IPF fibroblasts by TGF-β1 incubation.

(A) Quantification of α-SMA positive cells (myofibroblasts) by immunofluorescence. Results are presented as percentage of total cells. (B) Representative immunofluorescence showing the effect of 5 ng/ml TGF-β1 for 72 h in control fibroblasts. The expression of α-SMA is shown in green and the nuclei were stained with DAPI (blue) (Original magnification X200). (C) Representative Western blot of COX-2 and β-actin in two fibroblast cultures obtained from both control and IPF patients in response to TGF-β1 treatment at 4, 24 and 72 h. Densitometric analysis of COX-2 expressed as a ratio versus β-actin is also shown. N = 6 Control cells. N = 4–6 IPF cells. (D) Collagen Iα1 mRNA measured by real-time PCR with or without TGF-β1 treatment (5 ng/ml) for 72 h in control and IPF fibroblasts. *P<0.05 compared to respective untreated cells, ^#P<0.05 and ^##P<0.01 compared to control group.

Figure 3. Effect of IL-1β stimulation on a myofibroblast-enriched population.

(A) Protein levels in control and IPF fibroblasts of COX-2, COX-1, α-SMA and β-actin at 72 h with or without TGF-β1 treatment (5 ng/ml), and at subsequent 24 h in the presence or absence of IL-1β (10 ng/ml), measured by Western blot. (B) Densitometric analysis of COX-2 expressed as a ratio versus β-actin. (C) Quantification of COX-2 positive cells by immunofluorescence. Results are presented as percentage of total cells. (D) COX-1/β-actin ratio at 72 h in the presence or absence of TGF-β1 (5 ng/ml). *P<0.05 compared to untreated cells, ^#P<0.05 compared to IL-1β treated cells, ^†P<0.05 and ^‡P<0.01 compared to control group in the same conditions. (E) Representative co-immunofluorescence image of COX-2 (red) and α-SMA (green) of a myofibroblast-enriched culture obtained from control fibroblasts stimulated with IL-1β for 24 h. Nuclei were stained with DAPI (blue). Note that myofibroblasts do not express COX-2 in response to IL-1β and the different shape of the COX-2 positive cells (Original magnification X400).

Figure 4. Induction of epithelial-mesenchymal transition (EMT) in A549 cells by TGF-β1 incubation and effect of IL-1β in transformed cells.

Effect of 5 ng/ml TGF-β1 for 72 h in A549 cell culture. (A) Representative Western blot of four replicates of E-cadherin and ERK 1/2. (B) Representative immunofluorescence showing the expression of F-actin (stained with phalloidin, red) and bright field (Original magnification X400). (C) Collagen Iα1 mRNA measured by real-time PCR. Effect of IL-1β for 24 h (1 ng/ml) in A549 cells after incubation with 5 ng/ml TGF-β1 (72 h). (D) Representative Western blot and densitometric analysis of COX-2 expression versus ERK 1/2. *P<0.05 compared to untreated cells, ^#P<0.01 compared to IL-1β-treated cells. (E) Immunofluorescence of COX-2 (green) and F-actin (stained with phalloidin, red) in cells cultured in absence (upper) or presence (lower) of TGF-β1. In both images, cells were stimulated with IL-1β (1 ng/ml). Nuclei were stained with DAPI (blue). Note the almost complete absence of COX-2 staining in TGF-β1-treated cells (original magnification X400).

Figure 5. Measurement of PGE₂ secretion after FMT or EMT induction.

Control and IPF fibroblasts (A) and A549 cells (B) were incubated for 72 h in the presence or absence of TGF-β1 (5 ng/ml), followed by treatment in the presence or absence of IL-1β (10 ng/ml or 1 ng/ml, respectively) for 24 h. Supernatant was then collected and total PGE₂ was measured using Prostaglandin E₂ EIA Kit (Cayman). Results are expressed as PGE₂ secreted/µg total protein content. *P<0.05 compared to untreated cells, ^†P<0.05 and ^#P<0.01 compared to IL-1β-treated cells.

Figure 6. Cell proliferation measured by the analysis of DNA replication.

For this purpose, control and IPF fibroblasts (A) and A549 cells (B) were incubated for 72 h in the presence or absence of TGF-β1 (5 ng/ml), followed by treatment in the presence or absence of IL-1β (10 ng/ml or 1 ng/ml, respectively), PGE₂ (5 ng/ml) and the selective COX-2 inhibitor Celecoxib (10 µM). Cell proliferation was then analyzed by measuring the incorporation of the modified nucleoside EdU into the DNA using the Click-iT® commercial kit. Cells were incubated with 10 µM EdU for 2 h before being harvested for flow cytometry measurements. Results are expressed as the percentage of EdU positive cells. *P<0.05 and **P<0.01 compared to respective untreated cells, ^#P<0.05 compared to control group in the same conditions, ^†P<0.05 compared to non-treated TGF-β1 cells in the same conditions.

Figure 7. Immuno-histochemical detection of COX-2 and α-SMA in healthy lung tissue (A–B) and in idiopathic pulmonary fibrosis tissue (C–D).

Immuno-localizations of COX-2 (A–C) and α-SMA (B–D) are shown. The image shows slight basal expression of COX-2 and isolated α-SMA staining related to α-SMA-positive cells in control tissue. In contrast, increased α-SMA staining (black arrows) and absence of COX-2 expression (white arrows) are observed in fibroblastic foci. Metaplastic epithelium presents widespread expression of COX-2 (Original magnification X200).