Induction of ROS by HL5B. HL5B or IgG were added to MonoMac1 cells in the concentrations indicated. (A) Induction of intracellular ROS production as determined by L-012 chemiluminescence. (B) Induction of extracellular ROS production as determined by luminol/HRP chemiluminescence. (C) Effects of apocynin (100μM), PEG-SOD (100 U/mL), and allopurinol (100μM) on intracellular ROS production. (D) Quantification by HPLC of 2HE and ethidium generated in response to incubation with HL5B and IgG. (E) Flow cytometric analysis of ROS production induced by HL5B and the effect of inhibitors. Cells were loaded for 2 minutes with H₂HFF-BSA at 37°C followed by incubation with HL5B (100 ng/mL) alone or with NFA (0.1mM), PEG-SOD (500 U/mL) or NAC (10mM) as indicated. Tert-butyl-hydroperoxide (25μM) serves as a positive control. Means ± SD of MFI values of 5 independent experiments are presented. *P < .005, **P < .001 for HL5B versus HL5B plus inhibitor. (F) Relative expression of TLR8 normalized to β-actin. Values represent mean ± SD of at least 5 independent experiments. *P < .005, **P < .001 for HL5B versus HL5B plus inhibitor. (G) ROS generation analyzed by confocal microscopy. MonoMac1 cells were loaded for 2 minutes with H₂HFF-BSA and stimulated for 15 minutes with HL5B or IgG (500 ng/mL each) and imaged by confocal fluorescence microscopy with identical instrument settings. The acidic organelles were visualized by the use of Lyso-Tracker (50nM). Fluorescent H₂HFF-BSA indicative of ROS production was only observed after incubation with HL5B. The majority colocalized with LysoTracker (arrows). ROS production could be almost completely blocked by incubation with PEG-SOD (500 U/mL).