Time and temperature dependence of granulocyte damage by leucotoxic supernatants from Pasteurella haemolytica A1

Bacterial exotoxins may contribute to the pathogenic potential of micro-organisms through interactions with cells of the host defence system as well as by directly damaging host tissue. The present studies were designed to explore mechanisms of interaction between bovine granulocytes and the leucotoxin produced by Pasteurella haemolytica, a major cause of bovine respiratory disease. Leucotoxin-containing supernatant from P. haemolytica A1 caused rapid cell death in isolated bovine granulocytes that was close to half-maximal by 5 min and nearly 90% complete after 30 min at 37 degrees C. Maintaining granulocytes at ice-water temperature markedly attenuated or prevented the toxic effect; furthermore, if exposed to supernatants at ice-water temperature and then washed, most cells remained viable even after rewarming to room temperature. However, even a very brief exposure (about 5 s) at 37 degrees C led to extensive cell death even after immediate cold dilution and washing. Granule enzymes such as arylsulphatase were released far more slowly than cytosol contents. Leucotoxin purified by column chromatography showed temperature dependence and divergence between cytosol and granule marker release similar to those observed with the crude supernatant preparation. These findings indicate that irreversible interaction between P. haemolytica leucotoxin and bovine granulocytes is initiated very rapidly at 37 degrees C but markedly impeded at low temperature, while granule enzyme release follows cytosol marker release over a much longer period. The results suggest either a requirement for target cell metabolic activity to initiate toxin effects or a temperature-dependent receptor conformation, with granule enzyme release following as a secondary consequence of granulocyte death.