Supplementary Material for: NOX2-Derived ROS-Mediated Surface Translocation of BLT1 Is Essential for Exocytosis in Human Eosinophils Induced by LTB4

<b><i>Background:</i></b> Leukotriene B₄ (LTB₄) is a proinflammatory lipid mediator that elicits eosinophil exocytosis, leading to allergic inflammation. However, the detailed intracellular signaling mechanisms of eosinophil exocytosis induced by LTB₄ are poorly understood. Herein, we report that NADPH oxidase (NOX)2-derived reactive oxygen species (ROS)-mediated BLT1 migration to the cell surface is required for exocytosis in human eosinophils induced by LTB₄. <b><i>Methods:</i></b> Peripheral blood eosinophils were purified and stimulated for up to 60 min with LTB₄. The signaling role of NOX2-derived ROS in BLT1-dependent exocytosis in LTB₄-stimulated eosinophils was investigated. <b><i>Results:</i></b> Stimulating eosinophils with LTB₄ induced intracellular ROS production and surface upregulation of the exocytosis marker protein CD63 via BLT1-mediated signaling. LTB₄ induced p47^phox phosphorylation and 91^phox expression required for NOX2 activation in a BLT1-dependent manner. Pretreatment with NOX2 inhibitors, but not mitochondria inhibitor, prevented LTB₄-induced ROS generation and exocytosis. At 30 min after stimulation with LTB₄, BLT1 expression at the cell surface was upregulated. LTB₄-triggered surface upregulation of BLT1 was also blocked by inhibition of ROS generation with NOX2 inhibitors. Moreover, stimulation for 30 min with LTB₄ resulted in the interaction of BLT1 with NOX2 by immunoprecipitation. LTB₄-induced ROS generation, surface upregulation of BLT1 and exocytosis was also inhibited by pretreatment with a lipid raft disruptor, protein kinase C inhibitor, or Src kinase inhibitor. <b><i>Conclusion:</i></b> These results suggest that NOX2-derived ROS-mediated BLT1 trafficking to the cell surface plays a key role in the exocytosis of human eosinophils induced by LTB₄.