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

Background: 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. Methods: 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. Results: Stimulating eosinophils with LTB4
induced intracellular ROS production and surface upregulation of the
exocytosis marker protein CD63 via BLT1-mediated signaling. LTB4 induced p47phox phosphorylation and 91phox
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. Conclusion: 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.