Figure 6.

NADPH oxidase is required to make NETs. (a–d) Immunostaining for NET components (green, neutrophil elastase; red, histone–DNA complex) of neutrophils isolated from a CGD patient. (a) Unstimulated neutrophils showed a typical lobulated nucleus and a distinct pattern of cytoplasmic granules. After S. aureus (b, blue; MOI 20) and 10 nM PMA (c) stimulation, we observed no substantial morphological changes. (d) Stimulation with 100 mU/ml GO elicited NET formation in neutrophils from CGD donors. (e–h) Transmission electron micrographs. Neutrophils isolated from CGD patients 3 h after PMA activation (e and f) showed no morphological signs of NET formation. The nuclei are still lobulated and granules are clearly visible. (g and h) GO-stimulated neutrophils isolated from CGD patients at the moment of NET formation. The plasma membrane ruptured, allowing the release of NETs. The nuclear membrane disintegrated, allowing the mixing of nuclear and granular components. (i) ROS production of PMA-activated neutrophils obtained from healthy donors in the absence or presence of 10 μM DPI and of PMA-activated neutrophils from CGD donors. ROS production was completely blocked by DPI to basal levels, as with neutrophils from CGD donors. (j) Quantification of NET-DNA of normal neutrophils and neutrophils from CGD patients. CGD neutrophils did not make NETs after S. aureus or PMA activation, but stimulation with GO elicited NETs to normal levels. The experiment was repeated at least five times with neutrophils from independent donors, with similar results. The data shown is a representative triplicate experiment and presented as a mean value ± the SD. Bars: (a–e and g) 10 μm; (f and h) 1 μm.