Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils

Objective-To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosi s and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. Sample Population-Neutrophils isolated from blood samples obtained from hea lthy calves. Procedure-Neutrophil suspensions were exposed to lytic or sublytic dilution s of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis, Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control so lutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N, N-tetraacetic acid (EGTA) p rior to diphenyl amine analysis. Results-Examination by TEM revealed that bovine neutrophils exposed to lyti c dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apopt osis, had changes consistent with apoptosis, Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of e xtracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributabl e to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. Conclusions and Clinical Relevance-The ability of LKT to cause apoptosis in stead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, dependin g on the LKT concentration in pneumonic lesions. Furthermore, although Ca2 promotes A23187-induced apoptosis, it is apparently not an essential secon d messenger for LKT-induced apoptosis.