Figure 5

PGI₂ analogs modulate IL-10 and TNF-α expression via the IP-EP2/EP4-cAMP pathway, and iloprost also modulated IL-10 and TNF-α expression via the EP1-Ca²⁺ pathway in human mDCs. Isolated human mDCs were treated with iloprost or treprostinil for 24 h. Iloprost (10⁻⁸ to 10⁻⁷ mol/L) (A) and treprostinil (10⁻⁷ mol/L) (B) increased intracellular cAMP levels in human mDCs. Forskolin, an adenyl cyclase activator, enhanced IL-10 expression (C) and suppressed poly I:C-induced TNF-α expression (D). To verify the involvement of EP1-Ca²⁺ pathway in the modulatory effect of iloprost, cells were treated with iloprost (10⁻⁷ mol/L) for 2 h with or without pretreatment of EP1 receptor antagonist SC 19220 (10⁻⁵ mol/L) and were detached and labeled with Fluo-3-AM. The change of intracellular Ca²⁺ was analyzed using flow cytometry. One experiment representative of three is shown as a histogram plot graph (E). (F) Iloprost increased cytosol Ca²⁺ level by the measurement of the mean fluorescence intensity (MFI), and the EP1 receptor antagonist SC 19220 partly reversed the effect. BAPTA-AM, an intracellular free Ca²⁺ chelator, partly reversed the modulatory effects of iloprost on IL-10 (G) and poly I:C-induced TNF-α expression (H). Results represent the mean ± SD of three independent experiments using mDCs from three subjects for the cAMP assay, the flow cytometry analysis for intracellular Ca²⁺ and the BAPTA-AM experiment, and of six independent experiments using mDCs from six subjects for the forskolin experiment. *P < 0.05 compared with vehicle-treated cells (A, B, C, F, G) or poly I:C-treated cells (D, H). ^#P < 0.05 compared with iloprost-treated cells.