Figure 6.

Chemokines superinduce IFN-I production by enhancing clathrin-mediated DNA uptake in pDCs. (A–F) Purified pDCs were cultured with fluorescent CpG-AF488 (0.25 μM) with or without CXCL4 (10 μg/ml), and fluorescence was quantified by flow cytometry. Representative histogram (A) and mean fluorescence intensity (MFI; B) of CpG-AF488 uptake at 15 and 60 min are shown (n = 6). Representative histogram (C) and MFI (D) of CpG-AF488 uptake at 4°C and 37°C are shown (n = 5). (E–H) pDCs from HDs were cultured with medium only or either CpG-AF488 or nonfluorescent CpG, alone or with MβCD, CPZ, or Dyn. (E and F) Fluorescence was quantified by flow cytometry; representative histogram (E) and MFI (normalized to CpG-AF488 + CXCL4; F) are shown (n = 4–7). (G and H) Normalized gene expression level of IFN-α (G) and IL-6 (H) quantified by Q-PCR (n = 5). Unstim, unstimulated. (I and J) pDCs from HDs (n = 4) were incubated with medium only or CpG, alone or with CPZ. Normalized gene expression level of IFN-α (I) and IL-6 (J) quantified by Q-PCR. All results are represented as means ± SEM. Statistical significance was evaluated using Mann–Whitney U test, and only comparisons that are significant are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.