Figure 4: LPS induces CD11b endocytosis in DCs.

(a) W/T or Itgam^−/− BM-ΜΦs and BM-DCs were treated with LPS (1 μg ml⁻¹) for the times indicated. Receptor endocytosis was monitored with flow cytometry using the Sa15-21 mAb. Displayed are the MFIs of specific receptor staining (TLR4 or FcγR1) at each time point after LPS stimulation (MFI at time 0 as 100%). Data are representative of three independent experiments (mean±s.e.m., n=3). (b) W/T BM-ΜΦs and BM-DCs were treated with LPS (1 μg ml⁻¹), and surface levels of TLR4 and CD11b were assessed using flow cytometry at the times indicated. Shown are the MFIs of specific receptor staining at each time point (MFI at time 0 as 100%). Data are representative of three independent experiments (mean±s.e.m., n=3). (c) W/T BM-DCs were left untreated (upper panel) or were treated with 1 μg ml⁻¹ of LPS (lower panel) for 30 min. Cells were fixed, permeabilized and labelled with EEA1-alexa488 (green), CD11b-V450 (cyan) and CD14-alexa555 (red) antibodies, and analysed using confocal microscopy. Images are representative of at least two independent experiments. Scale bars=10 μm. Left of the merged image plotted the florescence spectra of each channel in the region of interest (ROI; yellow line). (d) W/T BM-DCs left untreated (circle) or treated with Dynasore (square) or dimethylsulphoxide (cross) were stimulated with LPS (1 μg ml⁻¹). Surface levels of TLR4 and CD11b were measured at the times indicated. Displayed are the MFIs of specific receptor staining at each time point (MFI at time 0 as 100%). Data are representative of three independent experiments (mean±s.e.m., n=3). *P<0.05, **P<0.01, ***P<0.005 (t-test).