Figure 4.

NE partially degrades core histones during NET formation. (A) Histone cleavage during NET formation is inhibited by NEi. Western immunoblotting against histones in lysates of naive (N) and PMA-activated neutrophils (PMA) in the presence (+NEi) or in the absence of NEi (untreated). (B and C) Quantitation of chromatin decondensation for the samples shown in A. (B) Untreated neutrophils. (C) Neutrophils treated with NEi. Shown are naive neutrophils at 0 h (gray) or 4 h (black), activated with PMA for 1 (yellow), 2 (orange), 3 (red), or 4 h (blue). (D) NE is not significantly externalized before NET formation. The time course of the release of NE into the medium by neutrophils activated with PMA measured by ELISA is shown. MNase was added to solubilize NE bound to DNA. Samples were normalized to NE levels from plated naive neutrophils lysed with 0.1% Triton X-100 (Total).