Pathway for ATP-induced rapid MMP-9 release. (A) PBMCs (1 × 107/mL) were pretreated with or without ATP, or with KN62 plus ATP or PMA as indicated in “Materials and methods.” Fresh cells (surface staining) or fixed cells (intracellular staining) were stained with anti–MMP-9 mAb and FITC-conjugated secondary Ab. The lymphocyte or monocyte populations were identified and gated by forward and side scatter. Flow cytometry histograms show representative data from 1 of 5 subjects with wild-type P2X7 receptors. (B) PBMCs (1 × 107/mL) with wild-type P2X7 receptors were pretreated with or without brefeldin A (10 μg/mL), monensin (5 μg/mL), or cycloheximide (50 μg/mL) for 60 minutes in NaCl medium with 0.1 mM CaCl2 and washed once. Cells then were resuspended at 2 × 107/mL in the same medium with the same concentration of inhibitors, followed by incubation with or without 1 mM ATP (gray bar) or 100 nM PMA (dark gray bar) at 37°C for 30 minutes. MMP-9 concentrations in supernatant were measured by ELISA. Results from individuals were normalized and shown as mean ± SEM (n = 4).
