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

Abstract

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 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. 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.

SNAP23-Dependent Surface Translocation of Leukotriene B 4 (LTB 4 ) Receptor 1 Is Essential for NOX2-Mediated Exocytotic Degranulation in Human Mast Cells Induced by Trichomonas vaginalis-Secreted LTB 4

“…We demonstrated the relationship between the high-affinity LTB 4 receptor, BLT1, and NOX2 in LTB 4 -stimulated eosinophils (34). Although NOX2 generates ROS and NOX2-derived ROS are generally coupled to exocytotic degranulation in human eosinophils stimulated with LTB 4 (64,65), information regarding the functional role of NOX2 in regulating BLT1-mediated exocytotic degranulation in TvSP-stimulated mast cells remains elusive.…”

“…SNAP23 localizes to the lipid rafts of the plasma membrane (34). Using coimmunoprecipitation (co-IP), we examined whether SNAP23 could interact with BLT1 in TvSP-stimulated HMC-1 cells.…”

“…Similarly, in neutrophils, NF-B and CREB were involved in IL-8 production induced by TvSP in BLT-dependent signaling (36,37). More recently, we showed that LTB 4 -stimulated eosinophils showed upregulation of CD63, a marker of exocytosis, via surface translocation of BLT1 in a nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-derived ROS-dependent manner (34). In addition, LTB 4 can make a signaling interaction between BLT1 and NOX2 in HMC-1 cells, leading to exocytosis (34).…”

“…In previous studies, we found that TvSP or T. vaginalis-derived LTB 4 can induce exocytosis and interleukin-8 (IL-8) production in human granulocytes (34)(35)(36)(37). TvSP induced upregulation of CD63 expression and reactive oxygen species (ROS) generation in HMC-1 cells via activation of mitogen-activated protein kinases (MAPKs) (35).…”

“…More recently, we showed that LTB 4 -stimulated eosinophils showed upregulation of CD63, a marker of exocytosis, via surface translocation of BLT1 in a nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-derived ROS-dependent manner (34). In addition, LTB 4 can make a signaling interaction between BLT1 and NOX2 in HMC-1 cells, leading to exocytosis (34). These results suggest that T. vaginalis-derived LTB 4 can induce exocytosis in mast cells via surface trafficking of BLT1.…”