FIG 5.
Increased NETosis in WKO neutrophils. A, Neutrophil nuclear shapes were scored as lobulated (resting), delobulated (pre-NETotic), or spread (NETotic), as shown in the representative images. Bars represent means ± SEMs of 100 to 200 cells per genotype. Scale bar = 5 μm. B, Representative images show MPO associated to DNA as an index of NETosis. Plots show Manders coefficient (mean ± SEM) for MPO (red) in DNA (blue) calculated on 150 to 200 cells per genotype. C, Spontaneous NETosis in isolated neutrophils. Data are expressed as percentage of fluorescence on the maximum fluorescence signal obtained by cell lysis. Bars are means ± SEMs of 6 independent experiments. D, NETosis induced by WKO sera. BM neutrophils (1 × 105) from WT mice were incubated in medium (NS) or with a 1:100 dilution of sera from WT or WKO mice (2–4 months old). NETosis in Fig 5, C and D, was quantified by using a SYTOX Green plate assay. E, Schematic representation of experimental design. F, WT splenic pDCs were incubated with cell-free supernatants from resting WT or WKO splenic neutrophils. IFN-α transcripts (left) and protein (right) were evaluated by using RT-PCR and ELISA after 4 or 24 hours, respectively. Bars represent means ± SEMs of 6 and 4 independent experiments, respectively. *P < .05, **P < .01, and ***P < .001, Mann-Whitney test.