Purified pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages

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.