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The Phosphoinositide 3-Kinase-Dependent Activation of Btk Is Required for Optimal Eicosanoid Production and Generation of Reactive Oxygen Species in Antigen-Stimulated Mast Cells¹

Hye Sun Kuehn^*, Emily J. Swindle^*, Mi-Sun Kim^*, Michael A. Beaven, Dean D. Metcalfe^* and Alasdair M. Gilfillan^2,*

^* Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, and Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Activated mast cells are a major source of the eicosanoids PGD₂ and leukotriene C₄ (LTC₄), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A₂, thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC₄ and PGD₂, respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcRI-dependent PGD₂, LTC₄, and ROS production in bone marrow-derived mast cells of Btk^–/– mice. The FcRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A₂ phosphorylation but rather appears to regulate the generation of ROS, LTC₄, and PGD₂ by contributing to the necessary Ca²⁺ signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the National Institute of Allergy and Infectious Diseases and National Heart, Lung, and Blood Institute Divisions of Intramural Research within the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Alasdair M. Gilfillan, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11C206, 10 Center Drive, Mail Stop Code 1881, Bethesda, MD 20892-1881. E-mail address: agilfillan{at}niaid.nih.gov

³ Abbreviations used in this paper: LTC₄, leukotriene C₄; cPLA₂, cytosolic phospholipase A₂; COX, cyclooxygenase; ROS, reactive oxygen species; BMMC, bone marrow-derived mast cell; 5-LO, 5-lipoxygenase; WT, wild type; HuMC, human MC; SA, streptavidin; DCF, dichlorofluorescein.