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

• Published in: Infection and Immunity Vol. 63; no. 2; pp. 381 - 388
• Main Authors: Yoo, H S, Maheswaran, S K, Lin, G, Townsend, E L, Ames, T R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-02-1995
• Subjects: Analysis of the immune response. Humoral and cellular immunity; Animals; Bacterial diseases; Bacterial diseases of the respiratory system; Base Sequence; Biological and medical sciences; bovin; Cattle; Cloning, Molecular; DNA Primers - chemistry; DNA, Complementary - genetics; facteur immunologique; factores inmunologicos; Fundamental and applied biological sciences. Psychology; Fundamental immunology; ganado bovino; Gene Expression; Human bacterial diseases; Immunobiology; immunological factors; In Vitro Techniques; Infectious diseases; Interleukin-1 - genetics; Interleukin-1 - metabolism; interleukine; interleukins; interleuquinas; lipopolisacaridos; lipopolysaccharide; lipopolysaccharides; Lipopolysaccharides - immunology; Lymphokines, interleukins ( function, expression); macrofagos; macrophage; Macrophage Activation; macrophages; Macrophages, Alveolar - immunology; Mannheimia haemolytica - immunology; Medical sciences; Molecular Sequence Data; pasteurella haemolytica; Pasteurellosis, Pneumonic - immunology; Polymyxin B - pharmacology; Regulatory factors and their cellular receptors; RNA, Messenger - genetics; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - metabolism; Bovine; Respiratory disease; Cytokine; Interleukin 1β; Gene expression; Pasteurellosis; Infection; Pasteurellaceae; Northern blotting; Polymerase chain reaction; Vertebrata; Mammalia; Bacteriosis; Lipopolysaccharide; Bacteria; Artiodactyla; Ungulata; Tumor necrosis factor α; Pasteurella haemolytica; Macrophage
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.63.2.381-388.1995

Bovine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) 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 DH5-alpha. The expressed proteins were confirmed as TNF-alpha and IL-1 beta 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-alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with purified LPS from Pasteurella haemolytica 12296. The effect of LPS on TNF-alpha and IL-1 beta gene expression was dose dependent, and induction was observed at a concentration of 0.01 microgram/ml. Both TNF-alpha and IL-1 beta mRNA expression were detectable within 0.5 h after stimulation with 1 microgram of LPS per ml, peaked at 1 to 2 h, steadily declined up to 16 h, and were undetectable by 24 h. Secreted TNF-alpha measured by bioassay peaked at 4 h and accumulated at a lesser concentration in conditioned medium throughout the 24 h. By contrast, secreted IL-1 beta was induced at 8 h and reached a maximal concentration at 24 h after stimulation. The ability of LPS to induce TNF-alpha and IL-1 beta gene expression and secretion of bioactive proteins were suppressed by polymyxin B. Our findings support a role for LPS from P. haemolytica in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.