Figure 7.

ATP modulation of ROS production during macrophage polarization. (A–C) ROS production was monitored over 20 min with the fluorescent probe H₂HFF-BSA in M1 (A, C) or M2 (B) macrophages from P2X₇R^+/+ (A, B) or P2X₇R^−/− (C) mice stimulated with ATP (5 mM), MTX (0.2 nM) or a combination of ATP and MTX; traces are the average of three to four independent cultures and are representative of three independent experiments. (D) Concentration–inhibition curves of ROS production induced by MTX (0.2 nM, 20 min) in the presence or absence of different concentrations of ATP (squares), PPi (circles) or clodronate (triangles) in P2X₇R^−/− M1 macrophages; n=3 for each point. (E) Average of three different experiments for ROS production at 20 min by M1 or M2 polarized macrophages from P2X₇R^+/+ (open bars) or P2X₇R^−/− (grey bars) mice stimulated as in (A–C), in the presence or absence of PPi (5 mM), clodronate (1 mM) or N-acetyl cystein (NAC, 20 mM); ^*P=0.0005; ^**P=0.0003; ^***P=0.0002. (F) P2X₇R^−/− M1 macrophages were treated with cytochalasin B (CB; 2.5 μg/ml, 5 min before MTX) and ROS production was induced by 20 min incubation at 37°C with 0.2 nM of MTX in the absence or presence of 5 mM of ATP or 3 mM of PPi; n=4 independent cultures and representative of two independent experiments.