Figure 8. PM-induced ROS generation and priming of adenylyl cyclase are required for β₂ agonist–mediated worsening of IL-6 release.

We treated (A) MH-S cells and (B) primary alveolar macrophages (AM) with PM (10 μg/cm²) or control (medium) and measured IL-6 levels in the absence or presence of a mitochondrially targeted antioxidant (Mito-Q or control [TPP]) or (C) a nontargeted antioxidant (EUK-134) (20 μM). (D) We treated MH-S cells with PM or control and with forskolin (50 μM) or control and measured cAMP levels 1 minute after forskolin treatment. (E) We treated MH-S cells with antimycin A (AA) (1 μM) or vehicle and with forskolin (50 μM) and measured IL-6 in the medium 24 hours later in the absence or presence of stigmatellin (1 μM). (F) We treated MH-S cells with PM or control and with forskolin (50 μM) or control and measured IL-6 levels 24 hours later in the absence or presence of stigmatellin. (G) We treated MH-S cells with PM or control and with EUK-134 or control and performed immunoblotting against cAMP CREB in the nuclear and cytoplasmic fractions 4 hours later. (H) We measured IL-6 levels in control and CREB shRNA–transfected MH-S cells after treatment with PM or PBS. (I) We treated control and p65-shRNA–transfected cells with PM or control and with albuterol or control and measured IL-6 levels. *P < 0.05, PM vs. PBS; **P < 0.05, albuterol or forskolin vs. control; ^‡P < 0.05, Adrb1^+/+Adrb2^–/– vs. Adrb1^+/+Adrb2^+/+, AC inhibitor, mito-Q, EUK-134, or stigmatellin vs. control, CREB or p65 vs. control shRNA.