title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Environmental Medical Biology, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Arim
authors.initials: A
authors.lastname: Min
authors.forename: Young Ah
authors.initials: YA
authors.lastname: Lee
authors.forename: Kyeong Ah
authors.initials: KA
authors.lastname: Kim
authors.forename: Jamel
authors.initials: J
authors.lastname: El-Benna
authors.forename: Myeong Heon
authors.initials: MH
authors.lastname: Shin
mesh_terms: D004804:Eosinophils / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D007975:Leukotriene B4 / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018102:Receptors, Leukotriene B4 / Q000378:metabolism*; D060149:Tetraspanin 30 / Q000276:immunology
chemical_list: C554586:CD63 protein, human; C513240:LTB4R protein, human; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D018102:Receptors, Leukotriene B4; D060149:Tetraspanin 30; D007975:Leukotriene B4; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases