Figure 5.

Light-induced NETosis depends on extracellular reactive oxygen species (ROS) generation. (A) Light-induced NETosis is significantly inhibited by the ROS scavenger Trolox (50 μM) and by exclusively scavenging external ROS by a combination of catalase/SOD (2,000 and 50 U/ml). NETosis appears to be independent of intracellular ROS production by NADPH oxidase, as shown by inhibition with DPI (1 μM) or mitochondrial ROS, inhibited by MitoTEMPO (5 μM). Statistics: two-tailed paired t-test. ^*p < 0.05. N = 4 independent experiments. Error bars = SEM. Cells were kept in RPMIcomp. + 10 mM HEPES. (B) 70 J/cm² of 375 nm light clearly induces NET formation in HBSScomp. + riboflavin (2 mg/l) + tryptophan (1 mM). The obtained NET rates are comparable with NETosis induced in RPMIcomp. (containing 0.2 mg/l riboflavin) + 10 mM HEPES. In contrast, irradiation in HBSScomp. alone or supplemented only with riboflavin (0.2 or 2 mg/l) does not lead to NET formation. N = 3–5 independent experiments. Error bars = SEM. (C) Induction of extracellular H₂O₂ levels by light measured by AmplexRed. Irradiation of HBSScomp. (red) + 2 mg/l riboflavin + 1 mM tryptophan or RPMIcomp. (0.2 mg/l riboflavin, 0.024 mM tryptophan) + 10 mM HEPES (blue) induces stable extracellular H₂O₂ levels between 60 and 100 μM. Similar H₂O₂ levels are measurable after irradiation of neutrophils in these two media. In the presence of neutrophils, the H₂O₂ levels are continuously reduced over 30 min to around 30–40 μM. In HBSScomp. without riboflavin and tryptophan, neither direct irradiation nor irradiation in the presence of neutrophils causes any increase in H₂O₂ levels. “+” = addition of 2 mg/l riboflavin and 1 mM tryptophan. “(+)” = 0.2 mg/l riboflavin and 0.024 mM tryptophan within RPMI. N = 3–4 independent experiments. Error bars = SEM.