FIGURE 1.

NETs are engulfed by monocyte-derived phagocytes. (A) Freshly isolated primary human neutrophils from healthy adult donors were exposed to 25 nM PMA for 2 h. NETs were detected by immunofluorescence staining of the following components: MPO (red), LL-37 (green), and DNA (blue). Scale bars are 20 μm for the upper and 10 μm for the lower panels. (B) HMDMs and MDDCs were incubated with Hoechst 33342 for 15 min to visualize nuclear DNA, whereas purified NETs were prestained with the cell-impermeable DNA dye SYTOX Green for 15 min. HMDMs or MDDCs were then washed to remove the Hoechst staining and incubated with SYTOX Green–labeled NETs for 1 h. Thirty minutes prior to the end of incubation, LysoTracker Red was added to visualize lysosomes. This dual DNA staining allows for the differentiation between internalized NETs (arrows) and the cell nuclei of the phagocytic cells. Confocal images are representative of three independent experiments. Scale bars in (B) are 5 μm for all panels. Refer to Supplemental Fig. 1 for results on costaining with EEA1 and LAMP1. (C and D) Cell viability of HMDMs (C) and MDDCs (D) exposed to NETs or control media for 1 h, then cultured in the relevant culture medium for 24 h prior to the assessment of viability. Data shown are mean values ± SD of two independent experiments.