Oxidant production in human and murine neutrophils with defects in p40phox. Neutrophils from the patient with NCF4 mutations showed substantial decreases in both total and intracellular oxidant responses when challenged with heat-killed A fumigatus hyphal particles (A), Zymosan (B), or SOZ (C) compared to healthy controls. Contrary to human neutrophil data, p40phoxR58A/R58A mouse neutrophils show a robust total and intracellular oxidant burst to these agonists (heat-killed A fumigatus hyphae (G), Zymosan (H) and SOZ (I)). Intracellular ROS response kinetics for human neutrophils are presented in panels D through F and murine intracellular response kinetics presented in panels J through L. Oxidant production by human or murine neutrophils was determined in 96-well plates using 125μM Luminol (total extracellular and intracellular) and Luminol with superoxide dismutase (SOD; 225 U/mL; intracellular). After addition of fungal agonists, chemiluminescence was recorded over 50 minutes in a SpectrMax L Luminometer. All experiments were performed in at least duplicates (±SD). Data shown are the total integrated relative light units (RLU) expressed as % of control (N = 2-3 independent experiments) or the RLU over time from a representative experiment.
