Figure 4

Elevation of intracellular cAMP augments PMA/ionomycin-stimulated macrophage IL-10 production and suppresses TNF-α. 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 hour prior to stimulation with 50 ng/ml PMA and 0.5 μg/ml ionomycin and incubated for 24 hours at 37°C in a 5% CO₂ humidified atmosphere, after which time supernatants were harvested and assayed for IL-10 (a) and TNF-α (b) by ELISA. Data are mean cytokine levels in pg/ml of triplicate culture supernatants ± SD, showing a representative of n = 7 replicate experiments. Western blot analysis of activated phospho-CREB (c) shows cAMP modulation of CREB upon macrophage stimulation by PMA/ionomycin. Lane 1, macrophage control; 2, macrophage + PMA/ionomycin; 3, macrophage + PMA/ ionomycin + cAMP. Data are representative of n = 3 replicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. CREB, cAMP response element binding protein; P-ATF, activating transcription factor-1; P-CREB, phospho-CREB; PMA, phorbol 12-myristate 13-acetate; TNF-α, tumour necrosis factor α.