Figure 4.

Role of EP receptors and cyclic AMP (cAMP) in prostaglandin E2 (PGE₂)-mediated suppression of toll-like receptor (TLR)-induced interferon-α (IFN-α) secretion. (a) EP receptor messenger RNA (mRNA) expression. Real-time reverse transcription–polymerase chain reaction (RT-PCR) was performed from mRNA obtained from freshly isolated whole peripheral blood mononuclear cells (PBMC) and plasmacytoid dendritic cells (PDC). The diagram shows the mean from six independent donors ± standard error of the mean (SEM). Mean values are shown as numbers. (b and c) PDC were pre-incubated with PGE₂, forskolin or EP receptor agonists for 4 hr before stimulation with cytosine–phosphate–guanosine oligodesoxynucleotide (CpG ODN) 2006. After 24 hr, IFN-α production was determined in the supernatants. The diagram summarizes the mean values ± SEM of n = 4 individual donors. Only values for CpG ODN-stimulated samples are shown. Individual values were normalized to the untreated CpG ODN control. (b) Forskolin. **P (CpG/CpG+Forskolin) = 0·004. (c) EP-receptor agonists: sulprostone (EP1 and EP3 agonist) and 11-deoxy-PGE₁ (EP2 and EP4 agonist). *P (CpG/CpG+PGE2) = 0·01; *P (CpG/CpG+100nm 11-deoxy-PGE1) = 0.04; *P (CpG/CpG+100 nm Sulprostone) = 0·01. (d) Inhibition of smad signaling. PDC were pre-incubated with SB-431542 (SB-4315.) for 20 min before the addition of transforming growth factor-β (TGF-β) for a 4-hr pre-incubation period and the addition of CpG ODN 2006 for another 24 hr. IFN-α production in the supernatants was measured using enzyme-linked immunosorbent assays (ELISAs). Only CpG ODN-stimulated conditions are shown. The mean values ± SEM of four individual donors are shown. P** [medium/TGF-β] = 0·0001.