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We assessed the kinetics of inducible nitric oxide synthase (iNOS) mRNA expression and production of nitric oxide (NO) in bovine alveolar macrophages (AMs) stimulated with purified lipopolysaccharide (LPS) from Pasteurella haemolytica strain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 micrograms/ml of LPS. Production of NO measured as secreted nitrite in supernatants took place in a time and dose-dependent manner with peak production at 24 h after LPS stimulation. Recombinant bovine gamma interferon (rb gamma IFN) augmented the LPS-induced iNOS gene expression and production of NO. The ability of LPS to induce iNOS gene expression and NO production either alone or in combination with rb gamma IFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor NG-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rb gamma IFN + LPS induced NO production. Our results also demonstrated that NO produced from an exogenous NO donor sodium nitroprusside (SNP), and NO generated from LPS-stimulated AMs (endogenous) caused cytotoxic injury to bovine pulmonary artery endothelial cells in a dose-dependent manner. The cytotoxic injury caused by NO generated from LPS stimulated AMs was inhibited by polymyxin B or L-NMMA. There was a markedly increased concentration of nitrite in the lung lavage fluids of calves following P. haemolytica infection. These findings support a role for NO in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

We obtained biologically active purified leukotoxin (Lkt) from Pasteurella haemolytica serotypel, strain 12296 using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three species of Lkt of molecular masses 95, 100, and 104 kDa were obtained. Purity of all three species of Lkt was confirmed by analytical SDS-PAGE and Western blot (immunoblot) analysis. Results from the chromogenic Limulus amebocyte lysate assay and silver staining of SDS-PAGE patterns indicated that the preparations were free of contaminating lipopolysaccharide. We then studied the kinetics of TNF alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNF alpha and IL-1 beta gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNF alpha and IL-1 beta mRNA expression were detectable at 1 h after stimulation with 1 leukotoxin unit/ml. The expression peaked at 2 h, steadily declining up to 6 h, and was undetectable by 10 h. Secreted TNF alpha measured by bioassay peaked at 4-6 h and accumulated at a lesser concentration after 6 h. By contrast, secreted IL-1 peaked at 6 h and decreased significantly by 10 h. The ability of purified Lkt to induce TNF alpha and IL-1 beta gene expression and secretion of bioactive proteins was suppressed by Ca2+ chelating agents, 5 mM EDTA and 5 mM EGTA, but not polymyxin B. Heat-inactivation of the purified Lkt that had lost its cytocidal property completely abrogated induction of TNF alpha and IL-1 beta gene expression and secretion in bovine AMs.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The effect of bovine respiratory syncytial virus (BRSV) upon alveolar macrophage (AM) function was investigated using an in vivo calf inoculation model. Alveolar macrophages were collected sequentially from live calves at multiple time points during the 14 day period following viral inoculation. Alveolar macrophages from bronchoalveolar lavage fluids were purified by density gradient centrifugation (> 95% AM) prior to in vitro evaluation of cell functions. There were significant but variable and inconsistent differences in the functions of AM from the BRSV inoculated calves compared to the control calves. Fc-receptor mediated phagocytosis was either increased or unchanged by BRSV inoculation. Nonopsonized phagocytosis was decreased during the early postinoculation period and later increased. There was a variable effect on AM phagosome lysosome fusion with increased fusion activity on postinoculation days 2 through 5, 7 and 12 but reduced activity on days 6 and 10. The AM respiratory burst, as measured by nitroblue tetrazolium dye reduction, was essentially unaffected with a reduction in activity on day 10 only. In this model, BRSV inoculation of calves primarily resulted in an alteration of the membrane associated phagocytic functions of the alveolar macrophages (p < 0.05).