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 howe ver there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to hum an respiratory disease it is not a major feature of the common equine neutr ophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain les s elastase than equivalent human cells and therefore there is a reduced ris k of excess and/or uninhibited elastase activity, which is considered the m ajor cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by me asuring elastinolytic activity whereas human neutrophil elastase content ha s been determined using immunological techniques, Neutrophils contain sever al intracellular protease inhibitors therefore measurement of elastase acti vity may underestimate the total NE content. The aim of the current study w as to develop immunological techniques to allow investigation of the cellul ar content, distribution and release of ENE from purified equine neutrophil s. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in eq uine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main i nhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.0 21 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, re spectively. This represents twice as much elastase as previously found in t he equine neutrophil and a comparable amount to that reported in human neut rophils. Immunolocalisation demonstrated that ENE 2A has a granular distrib ution 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 fro m 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 ge neration and total ENE 2A release during the initial 90 min post-ZAS stimul ation and the dependence of both events on extracellular cations. In conclu sion these studies have shown that horse and human neutrophil elastase cont ent and mediator release functions are more closely matched than was previo usly thought. This: suggests that the species differences in pathology resu lting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and ext ra-cellular protease inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.