Induction of inflammatory cytokines in bovine alveolar macrophages following stimulation with Pasteurella haemolytica lipopolysaccharide.
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.
Yoo
H S
HS
Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul 55108.
Maheswaran
S K
SK
Lin
G
G
Townsend
E L
EL
Ames
T R
TR
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology*; D008262:Macrophage Activation; D016676:Macrophages, Alveolar / Q000276:immunology*; D016978:Mannheimia haemolytica / Q000276:immunology*; D008969:Molecular Sequence Data; D012766:Pasteurellosis, Pneumonic / Q000276:immunology; D011112:Polymyxin B / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D011112:Polymyxin B