Pasteurella (Mannheimia) haemolytica leukotoxin-induced cytolysis of bovine leukocytes: role of arachidonic acid and its regulation

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor t hat contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis o nly in ruminant leukocytes and platelets. Cytolysis of many cell types is m ediated by arachidonic acid (AA) and its generation by phospholipases is re gulated by G-protein-coupled receptors. However, the contribution of Lkt-in duced AA generation to cytolysis and the signalling cascade underlying AA g eneration in bovine leukocytes have not been determined. We have determined whether PLA mediates Lkt-induced cytolysis and delineated the signalling m echanisms underlying AA generation in bovine leukocytes. Bovine lymphoma ce lls were used as an experimental system to investigate the Lkt-induced [H-3 ] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5 ,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis t oxin, inhibitors of cytosolic phospholipase A(2) (cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Fur thermore, Western blot analysis revealed the presence of G(i), G(s) and G(q ) type G-proteins. These results demonstrate that AA metabolites from cPLA( 2) activation contribute to Lkt-induced cytolysis and Gi type G-proteins, C a2+ and PKC, regulate the cPLA(2) activity. (C) 2001 Academic Press.