Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.021 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.