FcεRI Signaling of Mast Cells Activates Intracellular Production of Hydrogen Peroxide: Role in the Regulation of Calcium Signals

Abstract

Earlier studies, including our own, revealed that activation of mast cells is accompanied by production of reactive oxygen species (ROS) that help to mediate the release of the inflammatory mediators, including histamine and eicosanoids. However, little is known about the mechanisms of ROS production, including the species of oxidants produced. In this study we show that in both the RBL-2H3 mast cell line and bone marrow-derived mast cells, FcεRI cross-linking stimulates intracellular oxidative burst, including hydrogen peroxide (H2O2) production, as defined with the oxidant-sensitive dyes dichlorofluorescein and scopoletin and the selective scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). The oxidative burst was observed immediately after stimulation and was most likely due to an NAD(P)H oxidase. Experiments using selective pharmacological inhibitors demonstrated that activation of tyrosine kinases and phosphatidylinositol-3-kinase is required for induction of the oxidative burst. Blockade of the oxidative burst by diphenyleneiodonium impaired the release of preformed granular mediators, such as histamine and β-hexosaminidase, and the secretion of newly synthesized leukotriene C4, whereas selective scavenging H2O2 by ebselen impaired leukotriene C4 secretion, but not degranulation. Sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when ROS production was blocked. In contrast, selective depletion of H2O2 caused a considerable decrease and delay of the calcium response. Finally, tyrosine phosphorylation of phospholipase Cγ and the linker for activation of T cells, an event required for calcium influx, was suppressed by diphenyleneiodonium and ebselen. These studies demonstrate that activation of the intracellular oxidative burst is an important regulatory mechanism of mast cell responses.

BLT2 Is Upregulated in Allergen-Stimulated Mast Cells and Mediates the Synthesis of Th2 Cytokines

“…These data suggest that Nox1 acts downstream of ROS generation in Ag-stimulated mast cells, thus mediating the synthesis of Th2 cytokines. Consistent with our observation, DPI-sensitive flavoenzymes such as Nox were previously suggested to be involved in Ag-mediated ROS production in mast cells (44,45). We found that transient transfection with a BLT2 expression plasmid alone caused a modest increase in Nox1 transcript levels, ROS generation, and Th2 cytokine synthesis and that the addition of BLT2 ligands further increased these effects (Figs.…”

Amaranthus spinosusLinn. inhibits mast cell-mediated anaphylactic reactions

“…Compound 48/80 has been shown to penetrate cellular membranes and directly activate G proteins. This activation involves insertion of an aromatic ring of Compound 48/80 into the membrane and interaction of a positively-charged domain of the molecule with the COOH-terminal portion of the α-subunit of G protein (Fukuishi et al, 1997;Brooks et al, 1999;Suzuki et al, 2003). In the in vitro studies performed here, EAFAS attenuated the rate of mast cell degranulation in a dose-related manner as observed microscopically by toluidine blue staining.…”

Estradiol activates mast cells via a non-genomic estrogen receptor-α and calcium influx

“…Imaging of intracellular Ca 2+ fluxes was performed as previously described (Suzuki et al, 2003). Briefly, RBL-2H3 cells (5 × 10 4 ) were plated in the wells of chamber slides (Nalge Nunc Int, Naperville, IL) and cultured for 2 days.…”