Immature MoDCs internalize Ag by macropinocytosis. (A) Flow cytometric analysis of internalization of fluorescein-coupled 500 kDa dextran by immature versus LPS-matured MoDCs. (B-E) Immature MoDCs were pretreated with cytochalasin D, wortmannin, or amiloride for 30 minutes before addition of dextran (B), F(ab)′2 (C), HRP (D), or Lucifer Yellow (E) for 30 minutes. Cells were analyzed by flow cytometry. Results are expressed as a percentage of control nontreated (NT) cells. Data are mean ± SEM of 3 independent experiments. (F) Immature MoDCs were transfected or not with plasmids encoding the wild-type (DynWT) or the dominant-negative form (DynK44A) of dynamin2 coupled to GFP and then incubated with 500 kDa dextran, followed by flow cytometry. Mean fluorescence intensities (MFI) are plotted. (G) Immature MoDCs were treated or not (NT) with dynasore and incubated with fluorescent dextran, transferrin, or F(ab)′2 for 30 minutes at 37°C. Cells were analyzed by flow cytometry. Results are expressed as a percentage of control nontreated (NT) cells. Data are mean ± SEM of 3 independent experiments. (H) Immature MoDCs were incubated with dextran, F(ab)′2, or HRP for 30 minutes and then fixed and analyzed by wide field microscopy and deconvolution. A medial optical section is shown. Bar represents 5 μm. (I) Immature MoDCs were pulsed for 1 hour with 50 μg/mL F(ab)′2 and then fixed and prepared for electron microscopy after immunogold labeling on thawed cryosections (10 nm-gold particles). The white arrowheads indicate label in late endosomal vacuoles; and black arrowhead, label in a tubular structure. The black arrow points to a label in a tubular extension of an early endosomal vacuole. P indicates plasma membrane; E, early endosomal vacuole; G, Golgi; M, mitochondrium; and L, late endosomal/lysosomal vacuole. Bar represents 200 nm.
