Figure 2
VIP suppresses LPS induction of macrophage TNF-α with little effect on IL-10 production. Human-monocyte-derived macrophages were plated out at 1 × 105 cells per well in a flat-bottomed 96-well plate and pretreated with VIP (a,b), rolipram (c,d) or dibutyryl cAMP (e,f) for 1 hour prior to stimulation with 1 ng/ml LPS and were incubated for 24 hours at 37°C in a 5% CO2 humidified atmosphere, after which time supernatants were harvested and assayed for TNF-α and IL-10 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 (g) shows VIP modulation in the presence or absence of PDE inhibition. Lane 1, LPS-stimulated macrophage control; 2, LPS-stimulated macrophage + 10-6 M VIP; 3, LPS-stimulated macrophage + 10-6 M VIP + 10 μM rolipram. Data are representative of n = 3 replicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. CREB = cAMP response element binding protein; LPS, lipopolysaccharide; P-ATF, activating transcription factor-1; P-CREB, phospho-CREB; rol., rolipram; TNF-α, tumour necrosis factor α; VIP, vasoactive intestinal peptide.