Role of PDE4 in superoxide anion generation through p44/42MAPK regulation: a cAMP and a PKA-independent mechanism.

We investigated the generation of reactive oxygen species (ROS) from bronchoalveolar lavage (BAL) cells of either control or LPS-exposed rats and the effects of PDE4 inhibitors on ROS production. The PDE4 inhibitors, rolipram and Ariflo (cilomilast, SB 207499) dose-dependently (0.1-10 microm) inhibited fMLP-induced superoxide anion (O(2)(*-)) production (IC(50)s: 0.03 and 0.55 microm, respectively) in BAL cells of Wistar rats collected 3 h after an LPS-aerosol (200 micrograms ml(-1), 1 h). These BAL contained 85-95% neutrophils (BAL cells enriched in neutrophils). In contrast, BAL cells collected at the end of the challenge contained only macrophages and in these conditions, rolipram and Ariflo (0.1-10 microm) could only inhibit 25 and 45% of fMLP-induced O(2)(*-) release, respectively. We also observed that the inhibition of p44/42(MAPK) by PD98059 (1-10 microm) increased O(2)(*-) release by BAL cells enriched in neutrophils, but not by macrophages, and prevented the inhibition of O(2)(*-) production induced by PDE4 inhibitors. Western blot analysis showed that PDE4 inhibitors strongly activated p44/42(MAPK) in BAL cells enriched in neutrophils but not in macrophages. And in these cells, PDE4 and p44/42(MAPK) were co-immunoprecipitated by a polyclonal anti-PDE4 antibody. The following cell permeable-cAMP analogues, dbcAMP (10 microm-1 mm), 8-CPT-cAMP (1 mm) and 8-pMeOPT-2'-O-Me-cAMP (0.5 mm), could not reduce fMLP-induced O(2)(*-) production and both PKA inhibitors, PKA inhibitor 14-22 amide myristoylated (50 nm-1 microm) and H-89 (100 nm-1 microm), did not affect the decrease of O(2)(*-) release induced by PDE4 inhibitors in BAL cells enriched in neutrophils. These data suggest that PDE4 inhibitors decreased fMLP-induced O(2)(*-) release in BAL cells enriched in neutrophils but not in macrophages, through p44/42(MAPK) activation by a cAMP- and a PKA-independent mechanism.