Figure 6: Upregulation of CD14 expression in Itgam^−/− BM-DCs matured with CpG DNA rectifies TLR4 endocytosis but not TRIF-dependent signalling events.

BM-DCs were treated with 0.5 μM CpG DNA (CpG-matured) or left untreated (immature) for 18 h. (a) Surface expression of CD14 was assessed. Cells were then stimulated with 1 μg ml⁻¹ of LPS and the surface levels of TLR4 were measured using flow cytometry with the Sa15-21 mAb at the times indicated. Displayed are the MFI of specific receptor staining at each time point (MFI at time 0 as 100%). (b) Immunoblot analysis of phosphorylated (p-)IRF3 in cell extracts from CpG-matured and -immatured DCs after stimulation with 1 μg ml⁻¹ of LPS for 45 and 90 min. Quantification of band intensity showing the fold increase in p-IRF3 levels relative to untreated cells and normalized to total IRF3 levels. Data are mean±s.e.m. from three independent experiments. (c) The levels of RANTES secreted were measured using ELISA 24 h after LPS stimulation (1 μg ml⁻¹). Data are representative of three independent experiments (mean±s.e.m., n=3). n.s=nonsignificant, *P<0.05, **P<0.01 (t-test).