Induction of inflammatory cytokines in bovine alveolar macrophages following stimulation with Pasteurella haemolytica lipopolysaccharide

Bovine tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) cDNAs were generated by reverse transcription and then by PCR amplification from lipopolysaccharide (LPS)-stimulated alveolar macrophage RNA. The amplified cDNAs were cloned into pPow and expressed in Escherichia coli DH5α. The expressed proteins were confirmed as TNF-α and IL-1β by Western blot (immunoblot) analysis and bioassays. We then used the cloned genes as probes in Northern (RNA) blots and investigated the kinetics of TNF-α and IL-1β mRNA expression in bovine alveolar macrophages stimulated with purified LPS from Pasteurella haemolytica 12296. The effect of LPS an TNF-α and IL-1β gene expression was dose dependent, and induction was observed at a concentration of 0.01 μg/ml. Both TNF-α and IL-1β mRNA expression were detectable within 0.5 h after stimulation with 1 μg of LPS per ml, peaked at 1 to 2 h, steadily declined up to 16 h, and were undetectable by 24 h. Secreted TNF-α measured by bioassay peaked at 4 h and accumulated at a lesser concentration in conditioned medium throughout the 24 h. By contrast, secreted IL-1β was induced at 8 h and reached a maximal concentration at 24 h after stimulation. The ability of LPS to induce TNF-α and IL-1β gone expression and secretion of bioactive proteins were suppressed by polymyxin B. Our findings support a role LPS from P. haemolytica in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.