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.
BACKGROUND
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.
Dagleish
M P
MP
The Wellcome Trust Centre for Research in Comparative Respiratory Medicine and Roslin, Midlothian EH25 9RG, UK.
Brazil
T J
TJ
Scudamore
C L
CL
D000818:Animals; D002499:Centrifugation, Density Gradient; D019446:Endotoxemia / Q000175:diagnosis / Q000662:veterinary; D006734:Horse Diseases / Q000097:blood / Q000175:diagnosis; D006736:Horses / Q000276:immunology*; D019272:Leukocyte Elastase / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000502:physiology; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000378:metabolism* / Q000502:physiology
D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019272:Leukocyte Elastase