Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: Role of NADPH oxidase and mitochondria

Earlier studies have demonstrated that mast cells produce reactive oxygen species (ROS), which play a role in regulating Ca2+ influx, while in other cell types ROS are produced in a Ca2+-dependent manner. We sought to determine whether ROS are produced downstream of the extracellular Ca2+ entry in mast cells. Thapsigargin (TG), a receptor-independent agonist, could evoke a robust burst of intracellular ROS. However, this response was distinct from the antigen-induced burst of ROS with respect to time course and dependence on Ca2+ and phosphatidylinositol-3-kinase (PI3K). The antigen-induced ROS generation occurred immediately, while the TG-induced ROS generation occurred with a significant lag time (∼ 2 min). Antigen but not TG caused extracellular release of superoxide (O2−)/hydrogen peroxide (H2O2), which was blocked by diphenyleneiodonium, apocynin, and wortmannin. A capacitative Ca2+ entry resulted in the generation of O2− in the mitochondria in a PI3K-independent manner. Blockade of ROS generation inhibited TG-induced mediator release. Finally, when used together, antigen and TG evoked the release of leukotriene C4, tumor necrosis factor-α, and interleukin-13 as well as ROS generation synergistically. These results suggest that ROS produced upstream of Ca2+ influx by NADPH oxidase and downstream of Ca2+ influx by the mitochondria regulate the proinflammatory response of mast cells.