Figure 4.

Neither apoptosis nor necrosis induce NETs. Neutrophils were stimulated with 20 ng/ml anti-Fas antibodies for 18 h to induce apoptosis, incubated with 25 μg/ml of pore-forming secreted toxins from S. aureus for 15 min to induce necrosis, or activated with 10 nM PMA for 4 h to induce NETs. (a–c) Transmission electron micrographs. (a) Neutrophil treated with anti-Fas antibodies show characteristic apoptotic morphology, including nuclear condensation and fragmentation as well as cytoplasmic vacuolization. (b) Features of necrosis in neutrophils treated with toxins are the loss of segregation into eu- and heterochromatin and of the nuclear lobules. The nuclear envelope, as well as the granules, remains intact. (c) Neutrophils undergoing NET-forming active cell death exhibit a morphology clearly different from both apoptosis and necrosis. The nuclear membranes are entirely fragmented while most of the granules are dissolved, allowing direct contact and mixing of nuclear, cytoplasmic, and granular components. Bars, 2 μm. (d) TUNEL analysis of different forms of cell death reveals that 60% of the anti-Fas–treated neutrophils have fragmented DNA, whereas toxin- and PMA-treated neutrophils do not. n = 200 cells/condition. (e) Neither apoptosis nor necrosis lead to NET formation, as revealed by quantifying extracellular DNA. The experiment was repeated at least three 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.