title: Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.
abstract: BACKGROUND
  The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA.
  
  
  METHODS
  Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2β, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system.
  
  
  RESULTS
  The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2β or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis.
  
  
  CONCLUSIONS
  iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.
authors.affiliation: From the Department of Acute Critical Care and Disaster Medicine (S.M., M.K., Y.O.), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University; Trauma and Acute Critical Care Medical Center (J.A., K.M.), Medical Hospital of Tokyo Medical and Dental University; and Department of Biology (T.K.), Faculty of Science, Ochanomizu University, Tokyo, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Mikami
authors.forename: Junichi
authors.initials: J
authors.lastname: Aiboshi
authors.forename: Tetsuyuki
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Kojima
authors.forename: Koji
authors.initials: K
authors.lastname: Morishita
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Otomo
mesh_terms: D002478:Cells, Cultured; D002633:Chemotaxis / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D054522:Group VI Phospholipases A2 / Q000037:antagonists & inhibitors / Q000276:immunology*; D006801:Humans; D042541:Intracellular Space / Q000276:immunology; D009504:Neutrophils / Q000276:immunology*; D010196:Pancreatic Elastase / Q000378:metabolism; D054467:Phospholipases A2 / Q000276:immunology*; D054510:Phospholipases A2, Cytosolic / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D017382:Reactive Oxygen Species; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; D054467:Phospholipases A2; D054510:Phospholipases A2, Cytosolic; D010196:Pancreatic Elastase