Figure 2.

Inhibitory effects of pyrrophenone on lipid mediator biosynthesis in activated human PMN. (a) PMN suspensions (37°C, 5 × 10⁶ cells ml⁻¹) were treated with increasing concentrations of pyrrophenone and stimulated with either 300 nM fMLP, 300 nM PAF or 100 nM thapsigargin as described in Methods. Incubations were stopped by the addition of 0.5 volume of a cold (4°C) stop solution (MeOH/MeCN, 1/1) containing 12.5 ng of both 19-OH-PGB₂ and PGB₂ (as internal standards) and 5-LO products were analyzed by RP-HPLC as described in Methods. (b) PMN suspensions were treated with pyrrophenone and stimulated with 100 nM thapsigargin as described above. Incubations were stopped by the addition of 1 volume of EtOH containing 5 ng of ²H₄-PAF. PAF and lyso-PAF were extracted and analyzed by LC/MS/MS as described in Methods. (c) PMN suspensions (37°C, 10⁷ cells ml⁻¹) were treated 4 h with 700 pM GM-CSF, 1.5 nM TNF-α and 10 μM cytochalasin B for optimal COX-2 expression. Cell suspensions were incubated with increasing concentrations of pyrrophenone, then stimulated with 100 nM A23187 for 5 min. Incubations were stopped and PGE₂ was analyzed by ELISA in PMN supernatants as described in Methods. In all experimental settings, ADA (0.3 U ml⁻¹) and pyrrophenone were added 10 min before the addition of the agonists. Data represent the mean (±s.e.m.) of three separate experiments, each performed in duplicate.