Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS.
Abstract: Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes. In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP. Objective: The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS. In particular, neutrophil elastase (NE), a potent proteolytic enzyme, and its major inhibitor, alpha-1 proteinase inhibitor, were investigated. Methods: Neutrophils were isolated from equine peripheral blood (n = 5) by discontinuous Percoll gradient preparative centrifugation and primed with LPS prior to stimulation with fMLP. ROI were measured by lucigenin dependent chemiluminescence (LDCL). Concentrations of NE and API were determined by ELISA on cell free supernatants taken at 0, 2, 10, 30, 60 and 90 mins post stimulus. Data was analysed by Kruskal-Wallis and Mann-Whitney Tests. Results: Sequential exposure of Percoll purified equine blood neutrophils in vitro to LPS followed by fMLP resulted in the greatest release of NE from equine neutrophils and was required for ROI generation. However, LPS or fMLP stimulation alone resulted in an increase in NE release compared to unstimulated control cells. In contrast, significant API release was only induced by LPS stimulation or fMLP stimulation only after LPS priming, not fMLP on its own. Conclusions: These results suggest that different stimuli (fMLP or LPS) are capable of invoking similar responses from equine neutrophils with respect to NE release yet different ones with respect to API release. Conclusions: In addition, demonstration of elastase release induced by LPS and/or fMLP suggests that monitoring serum elastase levels is a potential diagnostic tool for detecting the early onset of endotoxaemia in the horse.
Publication Date: 2003-01-30 PubMed ID: 12553460DOI: 10.2746/042516403775467496Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article explores how two particular bacterial cell wall components, Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), can activate leukocytes in horses, potentially causing endotoxaemia, or the presence of endotoxins in the blood. The researchers found that these components may also stimulate the release of neutrophil elastase (NE), a protein-digesting enzyme, and its major inhibitor, alpha-1 proteinase inhibitor (API), which are critical to the horse’s immune response.
Objective and Methodology
- The main goal of the study was to determine if the release of NE and API from neutrophils depends on priming with LPS.
- To test this, the researchers isolated neutrophils from horse peripheral blood and primed them with LPS before stimulation with fMLP.
- The generation of reactive oxygen intermediates (ROI), which plays a crucial role in inflammatory and immune responses, were measured by lucigenin dependent chemiluminescence (LDCL).
- They then monitored the concentrations of NE and API through an ELISA test on cell-free supernatants at several intervals post stimulus.
Results
- The results demonstrated that exposure of equine neutrophils to LPS followed by fMLP resulted in the largest release of NE and was required for ROI generation.
- However, stimulation with LPS or fMLP alone resulted in an increased NE release compared to unstimulated control cells.
- On the other hand, significant API release was only triggered by either LPS stimulation or fMLP stimulation after LPS priming, and not by fMLP alone.
Conclusions
- The findings suggest that different triggers (fMLP or LPS) can elicit similar responses from equine neutrophils in terms of NE release, but they trigger different responses in terms of API release.
- The study also indicates that the observation of elastase release stimulated by LPS and/or fMLP implies that assessing serum elastase levels might be an effective diagnostic method for the early detection of endotoxaemia in horses.
Cite This Article
APA
Dagleish MP, Brazil TJ, Scudamore CL.
(2003).
Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS.
Equine Vet J, 35(1), 35-39.
https://doi.org/10.2746/042516403775467496 Publication
Researcher Affiliations
- The Wellcome Trust Centre for Research in Comparative Respiratory Medicine and Roslin, Midlothian EH25 9RG, UK.
MeSH Terms
- Animals
- Centrifugation, Density Gradient
- Endotoxemia / diagnosis
- Endotoxemia / veterinary
- Horse Diseases / blood
- Horse Diseases / diagnosis
- Horses / immunology
- Leukocyte Elastase / metabolism
- Lipopolysaccharides / pharmacology
- Luminescent Measurements
- N-Formylmethionine Leucyl-Phenylalanine / pharmacology
- Neutrophil Activation
- Neutrophils / drug effects
- Neutrophils / enzymology
- Neutrophils / metabolism
- Reactive Oxygen Species / metabolism
- Serine Proteinase Inhibitors / physiology
- Time Factors
- alpha 1-Antitrypsin / metabolism
- alpha 1-Antitrypsin / physiology
Citations
This article has been cited 2 times.- Long Y, Wang G, Li K, Zhang Z, Zhang P, Zhang J, Zhang X, Bao Y, Yang X, Wang P. Oxidative stress and NF-κB signaling are involved in LPS induced pulmonary dysplasia in chick embryos. Cell Cycle 2018;17(14):1757-1771.
- Grulke S, Franck T, Gangl M, Péters F, Salciccia A, Deby-Dupont G, Serteyn D. Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease. Can J Vet Res 2008 Jan;72(1):37-42.
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