title: Purified Pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages.
abstract: 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 TNF alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNF alpha and IL-1 beta gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNF alpha and IL-1 beta 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 TNF alpha 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 TNF alpha and IL-1 beta 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 TNF alpha and IL-1 beta gene expression and secretion in bovine AMs.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D005098:Exotoxins / Q000302:isolation & purification / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000302:isolation & purification / Q000494:pharmacology*; D016676:Macrophages, Alveolar / Q000378:metabolism*; D016978:Mannheimia haemolytica; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D001427:Bacterial Toxins; D016207:Cytokines; D005098:Exotoxins; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; C018579:leukotoxin