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).
Table 1.
Quantification of immunofluorescence of COX-2, α-SMA and COX-2+ α-SMA.
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).
Table 2.
Quantification of immunofluorescence of COX-2, α-SMA and COX-2+ α-SMA.
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).