Tumor necrosis factor-alpha (TNF-α) modulates the synthesis of prostaglandin E
) in alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs). (a) Immunofluorescent staining of cyclooxygenase-2 (COX2) (first panel, green, fluorescein isothiocyanate filter) and α-SMA (second panel, red, Texas Red filter) in MF cultures with or without the COX2 inhibitor diclofenac (Diclo). The third panel illustrates merged images of Höchst 33248-stained nuclei (blue, DAPI filter) as well as immunofluorescence for COX2 and α-SMA. α-SMA-positive MFs derived from hip joint capsules express the enzyme COX2. The fourth panel shows merged images of the negative controls. Scale bars are shown in the lower right corner of each panel. (b) MFs of three different donors were exposed to 10 μg/mL diclofenac. The expression of α-SMA (45 kDa), COX2 (72 kDa), and β-actin (42 kDa) as a loading control was evaluated by using Western blots. A characteristic double band for the COX2 protein corresponding to the expected molecular weight represents different glycosylated forms of the enzyme. (c) Gas chromatographic/mass spectrometric analysis revealed a time-dependent increase of PGE2 concentration in MF cultures upon TNF-α stimulation with a peak after 24 and 48 hours. This effect was completely blocked by diclofenac. DAPI, 4'-6-diamidino-2-phenylindole.