Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation.

BACKGROUND: Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation. Objective To determine the kinetics of neutrophil priming, recruitment, activation and subsequent clearance in a naturally occurring equine disease model of neutrophilic pulmonary inflammation. METHODS AND RESULTS: A 5 h mouldy hay/straw challenge in hypersensitive horses induced transient pulmonary dysfunction lasting 4 days. At 24 h circulating neutrophils were primed and displayed delayed rates of spontaneous apoptosis in vitro. Neutrophil numbers in the airspaces peaked at 5 h and then fell abruptly, returning to pre-challenge levels by 4 days. Airspace neutrophils demonstrated increased respiratory burst activity compared with circulating cells and equine neutrophil elastase 2A concentrations increased in parallel with neutrophil numbers indicating in vivo priming and degranulation. The number of apoptotic neutrophils and proportion of alveolar macrophages containing phagocytosed apoptotic neutrophils increased significantly at 24 h and 4 days post-challenge corresponding to the period of most rapid neutrophil clearance. CONCLUSION: This is the first demonstration of spontaneous neutrophil apoptosis and phagocytic removal in a natural disease model of airway inflammation and provides critical kinetic data to support the hypothesis that this clearance pathway plays a central role in the resolution of neutrophilic inflammation.

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.

REASONS FOR PERFORMING STUDY: 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. OBJECTIVES: 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. POTENTIAL RELEVANCE: 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.

Chemoattractant properties of conditioned medium from equine corpora lutea collected at various stages of the oestrous cycle.

This study investigated the chemotactic activity of equine CL at different stages of the oestrous cycle. The purpose of this was to ascertain whether luteal tissue itself contributes to the massive influx of leucocytes around the time of natural and induced luteal regression. Corpora lutea were collected at different stages of dioestrus and after treatment with PGF2alpha. Culture medium harvested after incubation of luteal tissue for 20 h was chemotactic for both polymorphonuclear and mononuclear cells in late dioestrus (before functional regression) as well as after natural and induced luteal regression. By contrast, midluteal tissue showed no chemotactic activity. This is the first report of the ability of equine luteal tissue actively to recruit inflammatory cells in vitro and supports our earlier findings that this infiltration starts prior to functional luteolysis. We hypothesise that this early influx of inflammatory cells may play an active role in luteal regression. Further research is needed to identify the specific chemotactic factor(s).

Inhalation of organic dusts and lipopolysaccharide increases gelatinolytic matrix metalloproteinases (MMPs) in the lungs of heaves horses.

We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the majority of gelatinolytic activity comprising pro- and active MMP-9. The 5 h duration hay/straw challenge increased BALF gelatinolytic MMP activity in heaves horses at 5 and 24 h after the start of this challenge, with activity returning to baseline by Day 4. In contrast to hay/straw and HDS challenges, LPS inhalation increased BALF gelatinolytic MMP activity in both groups. For all challenges, absolute BALF neutrophil counts were highly significantly correlated (P<0.0001) with levels of proMMP-9 and active MMP-9, but not with levels of MMP-2 (P>0.05). As gelatinolytic MMPs are pro-inflammatory agents, they may contribute to lung dysfunction and tissue destruction in heaves horses exposed to airborne organic stable dusts.

Endoscopic and bacteriological findings in a chronic outbreak of strangles.

Recently there has been increased awareness of the role of the carrier state in propagating Streptococcus equi var equi (S equi) infections (strangles), although the anatomical location of the organisms in chronic carriers has not been consistently established. This case report describes a chronic strangles outbreak in a riding school, that was monitored over six months by repeated clinical and endoscopic guttural pouch examinations. All asymptomatic horses that had positive S equi cultures on nasal swabs or guttural pouch lavages were found to have lesions in their guttural pouches. These lesions included empyema, chondroids and previously undescribed chronic discharging lesions on the floor of the medical compartment of the guttural pouches. These observations further support previous studies indicating the importance of investigating the guttural pouches in horses suspected to be asymptomatic carriers of this organism.

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.

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils.

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-alpha, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.

Aorto-cardiac fistulas in seven horses.

This report describes the history, clinical, electrocardiographic and echocardiographic findings, treatment, outcome and post-mortem findings in seven horses with aorto-cardiac fistula. Affected horses included 5 stallions, one gelding and one mare; 2 each of the Thoroughbred, Arabian and Standardbred breeds and one Thoroughbred-cross with a mean +/- s.d. age of 12 +/- 4 years, range 6-18 years. The presenting signs were acute distress (four horses), exercise intolerance (two horses) and the lesion was detected during a routine examination in one horse. Five horses had monomorphic ventricular tachycardia on admission and one other had a history of this arrhythmia. Five horses had a characteristic continuous murmur loudest in the right fourth intercostal space. Echocardiography (six horses) and/or post-mortem examination (four horses) revealed the horses had aorto-cardiac fistulas arising from the right aortic sinus in all five horses in which the site was recorded. Two horses had ruptured aneurysmal dilatations of the aortic wall at this site. Fistulas extended into the right ventricle in four horses; the right atrium in two horses, the left ventricle in one horse, and five horses had dissecting tracts in the septal myocardium. Horses survived for periods ranging from 24 h to 4 years. Aorto-cardiac fistula should be considered in the differential diagnosis for horses presenting with acute distress, bounding arterial pulse, a right-sided continuous murmur and/or monomorphic ventricular tachycardia, particularly in middle-aged or older stallions. Echocardiography is the technique of choice for confirming the diagnosis and demonstrating accompanying cardiac changes.

Humane destruction of horses with a mixture of quinalbarbitone and cinchocaine.

One hundred and-two horses requiring to be euthanased for a variety of reasons were killed by the intravenous injection of a mixture of quinalbarbitone sodium (400 mg/ml) and cinchocaine hydrochloride (25 mg/ml). The dose rates used were 1 ml/10, 15, 20 and 30 kg bodyweight, and the time of injection was varied between 5 and 25 seconds. The average time to collapse from the start of the injection was 34 seconds and the average time to clinical death was 230 seconds. Slow injection (particularly of the low dose rates) and premedication with detomidine resulted in a longer time to collapse (median 46 seconds). Premedication with xylazine and low dose rates of the mixture resulted in an unacceptable degree of muscular activity and agonal gasping and death was delayed. Premedication with romifidine and butorphanol resulted in an apparent (but insignificant) reduction in the time to collapse and death but was also accompanied by significant agonal gasping. Without premedication quinalbarbitone and cinchocaine resulted in a smooth and quiet collapse with the cessation of cardiac and respiratory functions within three minutes in all cases, but the palpebral reflex of the horses was prolonged significantly beyond the time when all other reflex activity was lost. Occasional gasping and muscular tremors, particularly of the upper forelimb, occurred particularly when lower dose rates and either very slow or very fast rates of injection were used. One horse which was premedicated with xylazine and received a very low dose at a slow rate showed unacceptably violent muscular activity. At no other time was the procedure regarded as violent or unacceptable.(ABSTRACT TRUNCATED AT 250 WORDS)