Regular PaperInduction of nitric oxide production by bovine alveolar macrophages in response toPasteurella haemolyticaA1
Abstract
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) fromPasteurella haemolyticastrain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 μg/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 (rbγIFN) 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 rbγIFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor NG-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rbγIFN+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 followingP. haemolyticainfection. These findings support a role for NO in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.
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Characterisation of bovine inducible nitric oxide synthase
2007, Veterinary Immunology and ImmunopathologyInducible nitric oxide (iNOS) is an enzyme that catalyzes the production of the reactive nitrogen intermediate nitric oxide (NO). NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of cell death (apoptosis). NO plays a major role in the killing of intracellular pathogens as part of the innate immune response. iNOS is known to be induced by a number of stimuli including cytokines as well as pathogens and their components. As yet, a full-length bovine iNOS sequence has only been predicted from the genome, although partial sequences from cDNA are available. Here, we have identified a 3471 bp transcript for bovine iNOS, isolated from RNA from bovine alveolar macrophages stimulated with the intracellular pathogen Mycobacterium bovis. When translated this gives a protein of 1156 amino acids. Bovine iNOS shows a high degree of similarity to iNOS from other species, and also shares a common protein domain structure.
Contribution of respiratory burst activity to innate immune function and the effects of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro
2006, Veterinary Immunology and ImmunopathologyThe mechanisms of interaction between phagocytes and different bacteria that help resolve lung infections or contribute to lung pathology are poorly defined. Alveolar phagocytes (resident macrophages and recruited neutrophils) make a major contribution to innate immunity by mounting a respiratory burst that helps kill internalised bacteria. However, this ability may be altered during or after exposure to infection. This review considers the application and limitations of a variety of analytical methods for oxygen-dependent mechanisms of respiratory burst in phagocytes initiated by soluble and particulate activators. Particular reference is given to the study in vitro of phagocytes from healthy and diseased ruminants during either natural infection with Mycobacterium avium paratuberculosis or experimental infection with Pasteurella multocida or Mannheimia haemolytica.
Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries
2006, Comparative Immunology, Microbiology and Infectious DiseasesBighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (iNOS) inhibitor, NG-monomethyl-l-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.
Les moutons ‘Bighorn’ (Ovis canadensis) sont plus sensibles à l'infection respiratoire par Mannheimia haemolytica que les moutons domestiques. En présence de cette bactérie, les macrophages produisent un certain nombre de molécules qui jouent des rôles important dans la réponse inflammatoire, y compris les intermédiaires de pazote hautement tels que l'oxyde nitrique (NO). Les surnageants des macrophages ont été cultivés en présence de lipopolisaccharide de M. haemolytica pour mesurer la présence de poxyde nitrique. En réponse à la stimulation de lipopolisaccharide, les macrophages de moutons ‘Bighorn’ ont sécrété des niveaux d'oxyde nitrique sensiblement plus élevés que ceux obtenus à partir des macrophages non-stimulés. En revanche, les niveaux d'oxyde nitrique produits par des macrophages de moutons domestiques en réponse aux lipopolisaccharide de M. haemolytica n'ont pas différé par rapport aux niveaux détectés dans les cultures de cellules non-stimulées. Les niveaux de nitrite détectés dans les surnageants de culture de macrophage de ‘Bighorn’ stimulés par les lipopolisaccharide et traités avec un inhibiteur induisant la synthase d'oxyde nitrique, NG-monométhyle-l-arginine, étaient semblables à ceux observés dans les cultures non-stimulées.
Effects of the mycotoxin ochratoxin A and some of its metabolites on the human cell line THP-1
2003, ToxicologyThe immunomodulatory effects of ochratoxin A (OTA) and some of its metabolites on the human monocyte/macrophage line THP-1 are described. Metabolic activity, cell proliferation, cell membrane integrity, cell differentiation, phagocytic behaviour, nitrogen oxide synthesis and cell surface markers were largely suppressed by these mycotoxins at concentrations between 10 and 1000 ng/ml, in individual cases already at 1 ng/ml. After analysis of a crude toxin, a substance designated RE2 could be isolated besides OTA, which was identified as ochratoxin C (OTC). The latter showed a stronger suppressive effect on most functions studied than all other metabolites of OTA. Because of the immunomodulatory effects of OTA and OTC, more attention should be paid to their immunopathogenic importance in addition to their known cytotoxic and genotoxic effects. The occurrence and importance of the mycotoxin OTC should be more closely examined in this context.
Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin
2001, Microbial PathogenesisPasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80–200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2–5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-α and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-α. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.
Differential induction of NO synthesis by Gram-positive and Gram-negative bacteria and their components in bovine monocyte-derived macrophages
1999, Microbial PathogenesisThe ability of Gram-positive and Gram-negative bacteria to promote the induction of NO synthesis in bovine monocyte-derived macrophages (MDM) was tested. Heat-killed Gram-negative organisms induced NO synthesis at low concentrations (optimum 0.2 to 2 μg/ml wet weight), regardless of the strain, and the response was only moderately enhanced by co-administration of recombinant bovine interferon-γ (rboIFN-γ). The activity was largely, but not exclusively, due to lipopolysaccharide (LPS), since it was largely abrogated by co-incubation with polymyxin-B. Diphosphoryl-lipid-A and rough-strain LPS were two orders of magnitude more active than monophosphoryl-lipid A, but two orders of magnitude less active than smooth-strain LPS, suggesting that O side chains contribute to increasing the affinity of LPS or to act as a costimulus. Gram-positive bacteria as single stimuli were four orders of magnitude less potent in inducing NO synthesis than Gram-negative organisms, but upon costimulation with rboIFN-γ, some of them were excellent inducers of NO synthesis. A similar rboIFN-γ-enhanced NO synthesis induction was also observed for zymosan, muramyl dipeptide, lipoteichoic acid and lipoarabinomannan, although to a lesser extent than for the whole heat-inactivated prototypic organisms. Thus, bovine macrophages exposed to rboIFN-γ have mechanisms by which they universally react to bacterial compounds distinct from LPS by induction of NO synthesis.
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Author to whom correspondence should be addressed at: Department of Veterinary Pathobiology, University of Minnesota, 1971, Commonwealth Avenue, St. Paul, MN 55108, U.S.A.