title: Extra domain A of fibronectin primes leukotriene biosynthesis and stimulates neutrophil migration through activation of Toll-like receptor 4.
abstract: OBJECTIVE
  There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling.
  
  
  METHODS
  The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo.
  
  
  RESULTS
  Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 α inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4).
  
  
  CONCLUSIONS
  These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.
authors.affiliation: Trudeau Institute, Québec, Québec, Canada.
authors.forename: Julie S
authors.initials: JS
authors.lastname: Lefebvre
authors.forename: Tania
authors.initials: T
authors.lastname: Lévesque
authors.forename: Serge
authors.initials: S
authors.lastname: Picard
authors.forename: Guillaume
authors.initials: G
authors.lastname: Paré
authors.forename: Annie
authors.initials: A
authors.lastname: Gravel
authors.forename: Louis
authors.initials: L
authors.lastname: Flamand
authors.forename: Pierre
authors.initials: P
authors.lastname: Borgeat
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002264:Carboxylic Acids / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D005260:Female; D005353:Fibronectins / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007975:Leukotriene B4 / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D015289:Leukotrienes / Q000096:biosynthesis*; D055666:Lipopeptides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011759:Pyrrolidines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D057705:Transendothelial and Transepithelial Migration / Q000187:drug effects
chemical_list: D000893:Anti-Inflammatory Agents; D001578:Benzopyrans; D002264:Carboxylic Acids; D016207:Cytokines; D005353:Fibronectins; D007093:Imidazoles; D015289:Leukotrienes; D055666:Lipopeptides; C560312:Pam2CSK4 lipopeptide; D020033:Protein Isoforms; D011759:Pyrrolidines; D011994:Recombinant Proteins; D013449:Sulfonamides; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C507035:ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate; C455981:pyrrophenone; D007975:Leukotriene B4; C402365:resiquimod; C089698:CP 105696