Figure 3.

Neutrophil elastase (NE) and myeloperoxidase (MPO) are indispensable for light-induced NETosis. (A) Representative images of neutrophil nuclei stained by Hoechst after exposure to UVA (70 J/cm² of 375 nm) or blue light (214 J/cm² of 470 nm) in the presence of specific inhibitors. NETosis is clearly reduced upon inhibition of MPO (4-ABAH, 100 μM), NE (GW-311616A, iNE, 5 μM) and, to a lower extent, of PAD enzymes (Cl-amidine, 200 μM). However, neutrophils are still able to undergo NETosis in the presence of ROCK 1/2 inhibition by Y-27632 (20 μM). (B) Quantification of NETotic cells after irradiation with LED-light of 375 or 470 nm, respectively, in the presence of Y-27632, Cl-amidine, 4-ABAH, or iNE. The inhibition of MPO and NE significantly reduces light-induced NET formation. Inhibition of PAD-enzymes decreases the number of NETotic cells induced by LED-light. However, NETosis appears independent of ROCK 1/2 activity. Statistics: two-tailed paired t-test. ^*p < 0.05, ^**p < 0.01. N = 4–5 independent experiments. Error bars = SEM. Cells kept in RPMIcomp. + 10 mM HEPES. (C) Light-induced NETosis (70 J/cm² of 375 nm) occurs independently of z-VAD-FMK (20 μM, pan-caspase inhibitor) and is reduced by Nec-1 (50 μM, RIP1 kinase inhibition). Statistics: two-tailed paired t-test. ^*p < 0.05. N = 4 independent experiments. Error bars = SEM. Cells were kept in RPMIcomp. + 10 mM HEPES.