Human monocyte-derived DCs secrete AREG. (A) Quantitative RT-PCR data for the EGFR ligands regulated by ATPγS. Ratios were obtained at 2, 6, 12, and 24 hours for 2 independent preparation of human MoDCs (means ± SEM) using SYBR Green technology. mRNA expression in ATPγS-treated cells and untreated cells has been normalized for each gene (AREG = amphiregulin, EREG = epiregulin, HB-EGF = heparin-binding EGF-like factor), and each time point using 2 housekeeping genes (B2M and SDHA). (B) AREG release by human MoDCs. DCs were stimulated by ATP (300μM), ATPγS (100μM), UTP (100μM), PGE₂ (500nM), or Ado (10μM) in the absence or the presence of LPS (100 ng/mL) for 24 hours. Supernatants of treated DCs were collected for ELISA measurements of human AREG. Results represent the mean ± SEM of 6 independent experiments at least. The Student t test was performed using Prism 5.0 software (GraphPad; *P < .05; **P < .01; ***P < .001). (C) Effect of NECA and suramin on AREG release by human MoDCs. DCs were stimulated by ATP (300μM), ATPγS (100μM), NECA (1μM), or ATPγS (100μM) plus suramin (10μM) in the absence or the presence of LPS (100 ng/mL) for 24 hours. Supernatants of treated DCs were collected for ELISA measurements of human AREG. Results represent the mean ± SEM of 3 independent experiments. The Student t test was performed using Prism 5.0 software (*P < .05; **P < .01; ***P < .001). (D) HA-VSMC growth is increased by AREG secreted by MoDCs. DCs were stimulated by ATPγS (100μM) in presence of LPS (100 ng/mL) for 24 hours. Supernatants were treated or not with an anti–human AREG blocking antibody for 3 hours and incubated with HA-VSMCs for 48 hours. HA-VSMCs were then counted with hemocytometer. Data (mean ± SEM) are representative of 5 independent experiments. The Student t test was performed using Prism 5.0 software (***P < .001).