Pasteurella haemolytica leukotoxin induced apoptosis of bovine lymphocytesinvolves DNA fragmentation

It has been reported that Pasteurella haemolytica leukotoxin (LKT) induces morphologic changes in bovine leukocytes consistent with apoptosis in vitro , but DNA fragmentation was not observed. We investigated whether bovine ly mphocytes undergo DNA fragmentation during LKT-induced apoptosis. Bovine pe ripheral blood lymphocytes were isolated by density gradient centrifugation and exposed to LKT or inactive pro-LKT protein from a lktC(-) mutant strai n. After exposure, DNA fragmentation in lymphocytes was quantified colorime trically by diphenylamine assay and visualized by agarose gel electrophores is. At high LKT concentrations, bovine lymphocytes were lysed, but at low c oncentrations, LKT caused DNA fragmentation characteristic of apoptosis. Ma ximal DNA fragmentation in bovine lymphocytes was induced by 0.1 TU ml(-1) LKT following 3 h exposure, but only background level of DNA fragmentation was observed with the inactive pro-LKT. Equine lymphocytes that are resista nt to LKT intoxication did not show DNA fragmentation following exposure to LKT. Preincubation of LKT with a neutralizing anti-LKT monoclonal antibody inhibited LKT-induced DNA fragmentation. Electrophoresis of DNA from bovin e lymphocytes treated with 0.1 TU ml(-1) LKT demonstrated the typical 'ladd er' pattern of internucleosomal DNA cleavage, the hallmark of apoptosis ass ociated with activation of endonucleases. LKT-induced DNA fragmentation was inhibited by 0.5 mM ZnCl2, an endonuclease inhibitor. The results indicate d that LKT at low concentrations induced apoptotic cell death of bovine lym phocytes, which may play a role in initiation and persistence of P. haemoly tica infection. (C) 1999 Elsevier Science B.V. All rights reserved.