title: 5-Lipoxygenase activating protein (FLAP) dependent leukotriene biosynthesis inhibition (MK591) attenuates Lipid A endotoxin-induced inflammation.
abstract: The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan; Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi, Taiwan.
authors.forename: Wen-Feng
authors.initials: WF
authors.lastname: Fang
authors.affiliation: Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado, Denver and Denver Health Medical Center, Denver, Colorado, United States of America.
authors.forename: Ivor S
authors.initials: IS
authors.lastname: Douglas
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan; Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi, Taiwan.
authors.forename: Chin-Chou
authors.initials: CC
authors.lastname: Wang
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Hsu-Ching
authors.initials: HC
authors.lastname: Kao
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Ya-Ting
authors.initials: YT
authors.lastname: Chang
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Chia-Cheng
authors.initials: CC
authors.lastname: Tseng
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Kuo-Tung
authors.initials: KT
authors.lastname: Huang
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Huang-Chih
authors.initials: HC
authors.lastname: Chang
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan; Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi, Taiwan.
authors.forename: Meng-Chih
authors.initials: MC
authors.lastname: Lin
mesh_terms: D058946:5-Lipoxygenase-Activating Protein Inhibitors / Q000494:pharmacology*; D058927:5-Lipoxygenase-Activating Proteins / Q000502:physiology*; D000818:Animals; D017209:Apoptosis; D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D054426:Chemokine CXCL2 / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology*; D015289:Leukotrienes / Q000096:biosynthesis*; D008050:Lipid A / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D011804:Quinolines / Q000494:pharmacology*; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D058946:5-Lipoxygenase-Activating Protein Inhibitors; D058927:5-Lipoxygenase-Activating Proteins; C548593:Alox5ap protein, mouse; D054426:Chemokine CXCL2; C404915:Cxcl2 protein, mouse; D007211:Indoles; D015289:Leukotrienes; D008050:Lipid A; D011804:Quinolines; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; C078266:MK 0591; D001094:Arachidonate 5-Lipoxygenase