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 that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether PLA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells 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 toxin, inhibitors of cytosolic phospholipase A(2) (cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, 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, Ca2+ and PKC, regulate the cPLA(2) activity. (C) 2001 Academic Press.