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

Sang Yoo Han Sang Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, T. R. Ames

Research output: Contribution to journalArticlepeer-review

71 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)381-388
Number of pages8
JournalInfection and immunity
Volume63
Issue number2
DOIs
StatePublished - 1995

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