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

<p><b><i>Background:</i></b> Leukotriene B<sub>4</sub> (LTB<sub>4</sub>) is a proinflammatory lipid mediator that elicits eosinophil exocytosis, leading to allergic inflammation. However, the detailed intracellular signaling mechanisms of eosinophil exocytosis induced by LTB<sub>4</sub> 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 LTB<sub>4</sub>. <b><i>Methods:</i></b> Peripheral blood eosinophils were purified and stimulated for up to 60 min with LTB<sub>4</sub>. The signaling role of NOX2-derived ROS in BLT1-dependent exocytosis in LTB<sub>4</sub>-stimulated eosinophils was investigated. <b><i>Results:</i></b> Stimulating eosinophils with LTB<sub>4</sub> induced intracellular ROS production and surface upregulation of the exocytosis marker protein CD63 via BLT1-mediated signaling. LTB<sub>4</sub> induced p47<sup>phox</sup> phosphorylation and 91<sup>phox</sup> expression required for NOX2 activation in a BLT1-dependent manner. Pretreatment with NOX2 inhibitors, but not mitochondria inhibitor, prevented LTB<sub>4</sub>-induced ROS generation and exocytosis. At 30 min after stimulation with LTB<sub>4</sub>, BLT1 expression at the cell surface was upregulated. LTB<sub>4</sub>-triggered surface upregulation of BLT1 was also blocked by inhibition of ROS generation with NOX2 inhibitors. Moreover, stimulation for 30 min with LTB<sub>4</sub> resulted in the interaction of BLT1 with NOX2 by immunoprecipitation. LTB<sub>4</sub>-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 LTB<sub>4</sub>.</p>