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

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  • 1 Department of Physiological Sciences College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 2 1904 SE 14th, Moore, OK 73160.
  • | 3 Department of Physiological Sciences College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 4 Department of Physiological Sciences College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 5 Department of Veterinary Pathobiology College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 6 Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, 4200 E 9th Ave, Denver, CO 80262.
  • | 7 Department of Veterinary Pathobiology College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.

Abstract

Objective—To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis 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 healthy calves.

Procedure—Neutrophil suspensions were exposed to lytic or sublytic dilutions 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 solutions 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) prior to diphenyl amine analysis.

Results—Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable 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 instead of oncosis is concentration- dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis. ( Am J Vet Res 2001;62:136–141)

Abstract

Objective—To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis 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 healthy calves.

Procedure—Neutrophil suspensions were exposed to lytic or sublytic dilutions 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 solutions 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) prior to diphenyl amine analysis.

Results—Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable 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 instead of oncosis is concentration- dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis. ( Am J Vet Res 2001;62:136–141)