Pasteurella haemolytica leukotoxin-induced increase in phospholipase A2 activity in bovine neutrophils

Exposure of bovine neutrophils to Pasteurella haemolytica leukotoxin (LKT) stimulates the production of leukotriene B4 (LTB4), which is believed to be an important chemotactic agent in the development of acute fibrinopurulent pneumonic infection in cattle. The involvement of phospholipase A2 (PLA2) in LKT-induced synthesis of LTB4 was studied by using bovine neutrophils labeled with 3H-arachidonate ([3H]AA). Incubation of isolated neutrophils with [3H]AA resulted in incorporation of radioactivity in the PLA2 substrates phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Exposure of radiolabeled neutrophils to LKT caused concentration- and time-dependent release of radioactivity and redistribution of radioactivity in neutrophil membranes consistent with utilization of phosphoglyceride substrate and release of free fatty acid and eicosanoid products. These LKT-induced effects could be inhibited by pretreatment with arachidonyl trifluoromethyl ketone, an inhibitor of type IV cytoplasmic PLA2, and were dependent on extracellular calcium. These results support the conclusion that LKT-induced synthesis of LTB4 involves a calcium-mediated increase in PLA2 activity.