We obtained biologically active purified leukotoxin (Lkt) from Pasteurella haemolytica serotypel, strain 12296 using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three species of Lkt of molecular masses 95, 100, and 104 kDa were obtained. Purity of all three species of Lkt was confirmed by analytical SDS-PAGE and Western blot (immunoblot) analysis. Results from the chromogenic Limulus amebocyte lysate assay and silver staining of SDS-PAGE patterns indicated that the preparations were free of contaminating lipopolysaccharide. We then studied the kinetics of TNFα and IL-1β mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNFα and IL-1β gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNFα and IL-1β mRNA expression were detectable at 1 h after stimulation with 1 leukotoxin unit/ml. The expression peaked at 2 h, steadily declining up to 6 h, and was undetectable by 10 h. Secreted TNFα measured by bioassay peaked at 4-6 h and accumulated at a lesser concentration after 6 h. By contrast, secreted IL-1 peaked at 6 h and decreased significantly by 10 h. The ability of purified Lkt to induce TNFα and IL-1β gene expression and secretion of bioactive proteins was suppressed by Ca2+ chelating agents, 5 mM EDTA and 5 mM EGTA, but not polymyxin B. Heat-inactivation of the purified Lkt that had lost its cytocidal property completely abrogated induction of TNFα and IL-1β gene expression and secretion in bovine AMs. To the best of our knowledge, this is the first published report that describes a procedure to obtain a biologically active form of purified exotoxic leukotoxin from P. haemolytica, and demonstrates a role for Lkt in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.
- Inflammatory cytokines
- Pasteurella haemolytica