Figure 3.

cAMP modulates LPS- and PMA/ionomycin-stimulated macrophage cytokine profile. Human monocyte-derived macrophages were plated out at 1 × 10⁵ cells per well in a flat-bottomed 96-well plate and pretreated with dibutyryl cAMP for 1 hr prior to stimulation with 50 ng/ml PMA/0·5 µg/ml ionomycin (a, b) or 1 ng/ml LPS (c, d) and incubated for 24 hr at 37°/5% CO₂, after which time supernatants were harvested and assayed for IL-10 (a, c) and TNF-α (b, d) by ELISA. Data are mean cytokine levels in pg/ml of triplicate culture supernatants ± SD, showing a representative of four replicate experiments. Western blot analysis demonstrates (e) cAMP modulation of activated phospho-PKCζ: Lane 1, macrophage control; 2, macrophage + PMA/ionomycin; 3, macrophage + cAMP; and (f) costimulation required for CREB activation: Lane 1, macrophage control; 2, macrophage + PMA/ionomycin; 3, macrophage + PMA/ionomycin + cAMP; 4, macrophage + cAMP. Loading controls are presented as total PKCζ and CREB blots below the corresponding phospho-Westerns. Data are representative of three replicate experiments. *P≤0·05, **P≤0·01, ***P≤0·001.