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 ma crophage RNA. The amplified cDNAs were cloned into pPow and expressed in Escherichia coli DH5 alpha. The expressed proteins were confirmed a s TNF-alpha and IL-1 beta by Western blot (immunoblot) analysis and bi oassays. We then used the cloned genes as probes in Northern (RNA) blo ts and investigated the kinetics of TNF-alpha and IL-1 beta mRNA expre ssion 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 mu g/ml. Both TNF-alpha and IL-1 beta mRNA expression were detectable within 0.5 h after stimulation with 1 mu g of LPS per ml, peaked at 1 to 2 h, steadily declined up to 16 h, and w ere undetectable by 24 h. Secreted TNF-alpha measured by bioassay peak ed at 4 h and accumulated at a lesser concentration in conditioned med ium throughout the 24 h. By contrast, secreted IL-1 beta was induced a t 8 h and reached a maximal concentration at 24 h after stimulation. T he ability of LPS to induce TNF-alpha and IL-1 beta gene expression an d secretion of bioactive proteins were suppressed by polymyxin B. Our findings support a role for LPS from P. haemolytica in the induction o f inflammatory cytokines in bovine pneumonic pasteurellosis.