Peroxynitrite modulates release of inflammatory mediators from guinea pig lung mast cells activated by antigen-antibody reaction

Background: Peroxynitrite (ONOO-), the product of the reaction between the superoxide anion (center dot O-2(-)) and nitric oxide (NO), is produced during inflammatory disease and may be a major cytotoxic agent. No reports are available as to whether ONOO- generates or modulates inflammatory mediator release from activated guinea pig lung mast cells. In this study, we explored the modulatory role of intracellular ONOO- on inflammatory mediator release ( histamine and leukotrienes) from activated mast cells. Methods: Guinea pig lung mast cells were purified by the enzyme digestion, and by using the rough and discontinuous Percoll density gradients. Mast cells were sensitized with IgG1 (anti-ovalbumin) antibody and challenged with ovalbumin ( OVA). The intracellular ROS formation was determined by following the oxidative production of 2', 7'-dichlorofluorescein diacetate (DCFHDA), dihydrorhodamine 123 (DHR), and anti-nitrotyrosine antibody immunofluorescence. Histamine was assayed using a fluorometric analyzer, leukotrienes by radioimmunoassay, intracellular Ca2+ levels by confocal scanning microscopy, and PLA(2) activity using prelabeling of [H-3] arachidonic acid. Results: ROS detected by DCFH-DA weakly increased in mast cells activated with OVA (1.0 g/ml), and the ROS so generated was inhibited by ebselen ( 50 mu M). However, the ROS detected by DHR increased 3-fold under the same conditions. Peroxynitrite scavengers sL-MT, DMTU, and inhibitor FeTPPS inhibited ROS formation but the NADPH oxidase inhibitor diphenyleneiodonium (DPI) only partially inhibited this formation. Dimethyl thiourea (DMTU) and seleno-L -methionine (sL-MT) inhibited the tyrosine nitration of cytosolic proteins, the release of histamine and leukotrienes, Ca2+ influx, and the PLA(2) activity evoked by mast cell activation. Conclusion: The data obtained suggests that the ROS generated by the antigen/antibody reaction activated mast cells is ONOO-, and that this modulates the release of inflammatory mediators via Ca2+ -dependent PLA(2) activity. Copyright (C) 2005 S. Karger AG, Basel.