Diphenyleneiodonium Prevents Reactive Oxygen Species Generation, Tyrosine Phosphorylation, and Histamine Release in RBL-2H3 Mast Cells

Mast cells have the ability to react to multiple stimuli, implicating these cells in many immune responses. Specific signals from the microenvironment in which mast cells reside can activate different molecular events that govern distinct mast cells responses. We previously demonstrated that hydrogen peroxide (H2O2) promotes IL-4 and IL-6 mRNA production and potentates FcεRI-induced cytokine release in rat basophilic leukemia RBL-2H3 cells. To further evaluate the effect of an oxidative microenvironment (which is physiologically present in an inflammatory site) on mast cell function and the molecular events responsible for mast cell cytokine production in this environment, we analyzed the effect of H2O2 treatment on IL-4 production in bone marrow-derived, cultured mast cells. Our findings show that nanomolar concentrations of H2O2 induce cytokine secretion and enhance IL-4 production upon FcεRI triggering. Oxidative stimulation activates a distinct signal transduction pathway that induces Fyn/PI3K/Akt activation and the selective phosphorylation of p38 MAP kinase. Moreover, H2O2 induces AP-1 and NFAT complexes that recognize the IL-4 promoter. The absence of Fyn and PI3K or the inhibition of p38 MAPK activity demonstrated that they are essential for H2O2-driven IL-4 production. These findings show that mast cells can respond to an oxidative microenvironment by initiating specific signals capable of eliciting a selective response. The findings also demonstrate the dominance of the Fyn/p38 MAPK pathway in driving IL-4 production.

Section: Discussionmentioning

confidence: 99%

Selective Activation of Fyn/PI3K and p38 MAPK Regulates IL-4 Production in BMMC under Nontoxic Stress Condition

Frossi

Rivera

Hirsch

et al. 2007

“…More recently, the mast cell line rat basophilic leukemia 2H3 (RBL-2H3) has been shown to release low-level ROS when stimulated with Ag or calcium ionophore (A23187) using the detection techniques of luminol and MCLA (38,39). Although this release was inhibitable by both DPI and SOD, no steady baseline readings were shown before stimulation, although appropriate controls were shown on stimulation.…”

Section: Discussionmentioning

confidence: 99%

A Comparison of Reactive Oxygen Species Generation by Rat Peritoneal Macrophages and Mast Cells Using the Highly Sensitive Real-Time Chemiluminescent Probe Pholasin: Inhibition of Antigen-Induced Mast Cell Degranulation by Macrophage-Derived Hydrogen Peroxide

Swindle

Hunt

Coleman

2002

Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another’s function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 μM) or ionomycin (1 μM), but not OVA (1 μg/ml), released high-level ROS, levels of which peaked after 3–7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degranulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell activation and promote resolution of IgE-mediated inflammation.

“…The RBL-2H3 cells obtained from the National Institute of Health Sciences (Japan Collection of Research Biosources; cell number JCRB0023) were grown in DMEM (Sigma-Aldrich) supplemented with 10% FCS (Life Technologies, Tokyo, Japan) in a 5% CO 2 -containing atmosphere as described previously (25,26). The RBL-2H3 cells were harvested by incubating them in HBSS-supplemented bicarbonate (pH7.4) containing 1 mM EDTA/0.25% trypsin for 5 min at 37°C.…”

Section: Cell Culturementioning

confidence: 99%

“…We previously reported that the RBL-2H3 mast cell line produces ROS upon Fc⑀RI cross-linking and releases them into the extracellular spaces (25,26). The production of ROS is sensitive to diphenyleneiodonium (DPI), a broad-spectrum inhibitor of flavoprotein-containing oxidoreductases (25).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Suzuki

Yoshimaru

Matsui

et al. 2003

Self Cite

134

109

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.