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. 2017 Oct 24;114(45):E9618–E9625. doi: 10.1073/pnas.1708247114

Fig. 2.

Fig. 2.

ANCA induces NET generation in human and murine neutrophils by RIPK1- and MLKL-dependent necroptosis signaling. (A) Isolated TNFα-primed human neutrophils were stimulated with a monoclonal antibody to MPO (αMPO mab) or an isotype control (ctrl). NETs were detected by Sytox green staining. NET generation was reduced by both necrostatin-1s and necrosulfonamide (NAS) preincubation. (B) Corresponding statistical analysis of the percentage of NET-producing neutrophils from three independent experiments with different neutrophil donors. (C) Isolated TNFα-primed murine neutrophils from WT or Ripk3−/− mice were stimulated with murine polyclonal anti-MPO IgG, and NET generation was quantified by Sytox green staining. Neutrophils from Ripk3−/− mice displayed reduced NET generation. (D) Corresponding statistical analysis of the percentage of NET-producing neutrophils from three independent experiments. (E–H) p-MLKL immunohistochemistry on isolated human neutrophils. αMPO mAb-stimulated TNFα-primed neutrophils displayed a strong p-MLKL signal that was absent in isotype control-stimulated cells; E and F show 63× magnification. G and H depict higher digital magnification of the marked areas in E and F. Error bars indicate means ± SEM. Comparisons were made using t test or ANOVA; *P < 0.05, **P < 0.01.