FIGURE 1.

Up-regulation of cytokine-dependent PG production and COX-2 does not require sPLA₂-IIA enzyme activity. A, FLS cells, grown to 80–90% confluence, were stimulated with TNF (10 ng/ml), alone or in combination with the activity-impaired mutant of sPLA₂-IIA (H48Q) (4 μg/ml), or with sPLA₂-IIA (WT) (4 μg/ml) in the presence or absence of the COX-2-selective inhibitor NS-398 (1 μm) for 16 h in DMEM/Ham's F-12 containing 0.1% BSA. PGE₂ concentration was measured in cell culture supernatants and total cellular protein was determined as described under “Experimental Procedures.” Data are combined mean ± S.E. of triplicate determinations from cell cultures derived from each of 4 patients. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's unpaired t test) relative to unstimulated cells unless indicated. B, cells were treated for 16 h as indicated, lysed, and protein extracts were subjected to electrophoresis and Western blot analysis as described under “Experimental Procedures.” A representative Western blot from one cell culture (RA79) is shown. Bands were quantified by densitometry as described. COX-2 density for each sample was normalized relative to β-actin density and the COX-2/β-actin ratio for each treatment was then normalized relative to control for each cell culture. Data are mean ± S.E. of three independent cell cultures.