FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent

Abstract

We investigated the enzymes responsible for FcεRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcεRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcεRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91phox- or p47phox-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcεRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcεRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcεRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcεRI-dependent degranulation and cytokine production.

Prostaglandin E2 Activates and Utilizes mTORC2 as a Central Signaling Locus for the Regulation of Mast Cell Chemotaxis and Mediator Release

“…These include NADPH oxidases, mitochondria electron transportation, nitric-oxide synthases, and arachidonic acid metabolism. Of these, COX-1-driven arachidonic acid metabolism appears to be a major source of ROS in antigen-stimulated mast cells (32). Our data were obtained with two independent approaches: the use of a selective COX-1 inhibitor and COX-1 knockdown by targeted shRNA; the results from these approaches strongly support the conclusion that COX-1 is the major enzyme responsible for ROS production in PGE 2 -stimulated BMMCs (Fig.…”

“…Thus, a similar role for mTORC2-dependent AKT activation in PGE 2 -mediated CCL2 may be predicted. However, the role for the mTORC2-mediated ROS production in this response is unexpected in light of the observation that antigen-mediated production of the cytokines IL-6 and TNF-␣ in BMMCs was unaffected by inhibition of COX-1 (32). These data indicate that alternative pathways may regulate chemokine and cytokine production in mast cells in response to different stimuli.…”

“…PGD 2 Measurements-The release of PGD 2 from PGE 2 -stimulated cells was measured as described (32). Briefly, cytokine-deprived BMMCs were stimulated with PGE 2 (100 nM) for 20 min, and then cell-free supernatants were analyzed for PGD 2 by competitive enzyme immunoassay (Cayman Chemicals), according to the manufacturer's instructions.…”

“…Intracellular ROS Detection-Intracellular ROS were measured as described (32,33). Briefly, cytokine-deprived BMMCs were preincubated with or without the indicated inhibitors for 10 -20 min.…”

“…We and others have demonstrated that arachidonic acid metabolism through the actions of COX-1 is one major source for generation of ROS in immune cells including mast cells (32,33,46). The production of PGD 2 , a product of COX-1 activity, in PGE 2 -stimulated cells would be expected to increase ROS generation in mast cells.…”