Effects of Pasteurella haemolytica leukotoxin on isolated bovine neutrophils

P. haemolytica leukotoxin caused rapid leakage of intracellular K+ (greater than 90% in 30 sec) from and cell swelling (approximately 100% increase in 15 min) of isolated bovine neutrophils. Incubation media made hypertonic by the addition of raffinose, dextran or inulin (carbohydrates with mol. wts of greater than or equal to 505) prevented leukotoxin-induced cell swelling, but not K+ leakage. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the leukotoxin-induced functional transmembrane pore size in bovine neutrophil plasma membranes is slightly smaller than the molecular size of raffinose, i.e. 1.2 nm. Morphologically, leukotoxin caused bovine neutrophils to swell, lose their membrane ruffling, develop a finely porous surface, and form large plasma membrane defects. Exposure of neutrophils to leukotoxin caused slower (5-50 min) leakage of 80% of the cellular L-lactate: NAD oxidoreductase (E.C. 1.1.27, lactate dehydrogenase, LDH). Leukotoxin-induced K+ leakage and cell swelling developed in Ca2+-free medium, whereas leakage of lactate dehydrogenase develop only in medium containing Ca2+ and was inhibited by the addition of ethylene glycol-bis(B-aminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA). This sequence of leukotoxin-induced changes in neutrophils is compatible with the mechanism of action of pore-forming cytolysins.