Stimulation of P2X(7) enhances Haemophilus somnus lipooligosaccharide-mediated apoptosis of bovine endothelial cells

Haemophilus somnus causes a variety of diseases in the bovine, including pneumonia, thrombotic meningoencephalitis and myocarditis. Despite the common finding of vasculitis in H. somnus infected tissues, little is known about how H. somnus damages endothelial cells. In this project, I demonstrated that H. somnus organisms, and its lipooligosaccharide (LOS), induced endothelial cell apoptosis as detected using multiple techniques.; The role of caspase activation in LOS-mediated endothelial cell apoptosis was also examined. H. somnus LOS induced caspases-3, 8 and 9 activation. Caspase-8 activity rose more rapidly than caspase-9. Likewise, a caspase-8 inhibitor significantly reduced LOS-mediated apoptosis, while blocking caspase-9 had no effect.; It is not clear which endothelial receptor(s) are involved in transducing the apoptotic signal from LOS. I focused on P2X₇, a member of the P2 purinergic receptor family that modulates lipopolysaccharide signaling in murine and human macrophages. Selective inhibitors of P2X ₇, oATP and PPADS, significantly reduce LOS-mediated endothelial cell apoptosis. oATP significantly blocked LOS-mediated caspase-8 activation, but not caspase-9. Treatment of endothelial cells with the P2X₇ agonists, BzATP or ATP, significantly enhanced LOS-mediated caspase-3 activation, whereas ADP, a P2y agonist, failed to significantly alter caspase-3 activation. These data suggest that P2X₇ participates in the cellular pathways (i.e. caspase-8 and 3 activation) responsible for transducing the apoptotic signal from H. somnus LOS.; P2X₇ has been reported to be involved in the processing and secretion of IL-1β by macrophages. The role of IL-1β in LOS-mediated apoptosis in bovine endothelial cells was examined. LOS induced caspase-1 activation in endothelial cells, which cleaves IL-1β to its active form. Conversely, blocking caspase-1 enhanced LOS-mediated apoptosis. Treatment of endothelial cells with IL-1β diminished LOS-mediated apoptosis, while IL-1ra enhanced LOS-mediated apoptosis. These data suggest that release of endogenous IL-1β appears to diminish apoptosis of endothelial cells in response to LOS.; Other mechanisms of endothelial cell damaged were examined. H. somnus LOS induced the generation of reactive oxygen species (ROS) and nitric oxide (NO) from bovine endothelial cells. Membrane permeable ROS scavengers and nitric oxide synthase inhibitors diminished LOS-mediated caspase-3 activation. These data suggest that ROS and NO play a role in LOS-mediated caspase-3 activation.