title: Endotoxin-induced activation of equine digital vein endothelial cells: role of p38 MAPK.
abstract: The endothelium plays a major role in the pathogenesis of endotoxemia. Binding of endotoxin (lipopolysaccharide; LPS) to endothelium initiates a range of signalling events, including activation of p38 mitogen activated protein kinases (MAPKs) that are involved in the initiation of inflammatory responses. In the present study we have examined whether clinically relevant concentrations of LPS can activate p38 MAPK in equine endothelial cells and have investigated the role of the kinase in neutrophil adhesion and mediator release. Cultured equine digital vein endothelial cells (EDVEC) were exposed to LPS and phosphorylation of p38 MAPK was assessed by Western blotting using phospho-specific antibodies. Neutrophil adhesion was quantified by assaying myeloperoxidase and the release of prostacyclin (PGI(2)) was measured by radioimmunoassay of its stable breakdown product 6-keto-PGF(1alpha). The effects of the p38 MAPK inhibitors, SB203580 and PD169316, on neutrophil adhesion and 6-keto-PGF(1alpha) release were examined, as was the effect of an anti-E-selectin antibody on neutrophil adhesion to LPS-activated EDVEC. LPS treatment significantly increased p38 MAPK phosphorylation, which was maximal after a 1h LPS incubation using a concentration of 10(-5)g/ml (EC(50) = 2 x 10(-7)g/ml). Neutrophil adhesion to LPS-stimulated endothelial cells (maximal at 10(-6)g/ml; EC(50) = 6 x 10(-10)g/ml) was significantly inhibited in the presence of p38 MAPK inhibitors (reduced from a maximum of 33+/-6% to 13+/-4% adhesion at 10(-6)M SB203580 and to 21+/-4% adhesion at 10(-6) M PD169316), or an anti-E-selectin antibody (reduced from 17+/-1% adhesion to 6+/-1% adhesion). 6-keto-PGF(1alpha) release was increased in a concentration-dependent manner following LPS exposure (maximal at 10(-6)g/ml; EC(50) = 1 x 10(-9)g/ml), and was significantly inhibited by p38 MAPK blockade (reduced from 1.6+/-0.3 x 10(-9)g/ml to 4+/-1 x 10(-10)g/ml and 4+/-2 x 10(-10)g/ml with 10(-6) M SB203580 or 10(-6) M PD169316, respectively). This study has demonstrated that clinically relevant concentrations of LPS activate p38 MAPK in equine endothelial cells and that both neutrophil adhesion to LPS-activated EDVEC and PGI(2) release are dependent upon p38 MAPK phosphorylation. These results reveal a key role for p38 MAPK in the response of the endothelium to LPS and suggest that inhibition of this kinase may reduce inflammatory events in the endotoxemic horse.
authors.affiliation: Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK. abrooks@rvc.ac.uk
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Brooks
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Menzies-Gow
authors.forename: Caroline
authors.initials: C
authors.lastname: Wheeler-Jones
authors.forename: Simon R
authors.initials: SR
authors.lastname: Bailey
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Cunningham
authors.forename: Jonathan
authors.initials: J
authors.lastname: Elliott
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011464:Epoprostenol / Q000378:metabolism; D006724:Hoof and Claw / Q000098:blood supply*; D006736:Horses / Q000502:physiology*; D007093:Imidazoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009504:Neutrophils / Q000502:physiology; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D014680:Veins / Q000187:drug effects / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D008070:Lipopolysaccharides; D011725:Pyridines; D011464:Epoprostenol; D048051:p38 Mitogen-Activated Protein Kinases; C408604:2-(4-nitrophenyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole; C093642:SB 203580