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Notes: Background Mould extract inhalation challenges have been used extensively in the investigation of heaves. Such challenges have induced pulmonary neutrophilic inflammation and dysfunction, consistent with, but less severe than the natural disease. However, the method of mould extract production is likely to result in endotoxin contamination.Objective To investigate whether insufficient dose delivery was responsible for the shortfall in response to inhaled extract compared with natural disease, and whether endotoxin contamination of mould extract contributed to the pulmonary inflammation and dysfunction.Methods We measured the response of six heaves horses following inhalation of saline (placebo) and three doses of Aspergillus fumigatus extract. We then compared the response of six heaves horses to A. fumigatus extract inhalation before and after lipopolysaccharide (LPS) depletion.Results Inhalation challenge with 0.5, 1.6 and 5 mg of A. fumigatus extract resulted in a significant increase in bronchoalveolar lavage fluid (BALF) neutrophil ratio when compared with saline inhalation. Only 1.6 and 5 mg extract inhalation resulted in significant lung dysfunction compared with saline. There was no significant difference between 1.6 and 5 mg extract inhalation with respect to airway neutrophil numbers or lung function, suggesting a plateau in both measured responses. LPS depletion of 1.6 mg A. fumigatus extract resulted in a significant reduction in airway neutrophil numbers and increase in arterial oxygen tension. There was no significant difference between saline and the LPS-depleted A. fumigatus extract challenges with respect to neutrophil count and lung function. The reduction in airway neutrophil numbers was greater than would be predicted by extrapolation from previously reported soluble LPS dose–response inhalation experiments.Conclusion This study supports a role for other inhalants, in addition to soluble components of A. fumigatus, in the aetiopathogenesis of heaves. Also the amplification in response to LPS when inhaled with A. fumigatus extract, suggests that the role of inhaled endotoxin in the pulmonary inflammation and dysfunction in naturally occurring heaves may currently be underestimated.

Notes: 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.

Notes: Background Equine heaves is a naturally occurring organic dust-induced asthma characterized by airway neutrophilia, mucus hypersecretion and obstructive lung dysfunction. However, the relative role of different dust components in disease severity remains unclear.Objective This study investigated the relative contribution of inhaled endotoxin and organic dust particulates (mainly mould spores) in inducing heaves in heaves-susceptible horses.Methods Control and heaves-susceptible horses received inhalation challenges with hay dust suspension (HDS) before and after lipopolysaccharide (LPS) depletion. Heaves-susceptible horses also received inhalation challenge with HDS particulates with and without the addition of LPS and were housed in two separate dusty environments during which mould and endotoxin exposure was measured. The airway inflammatory and functional response to each challenge was measured.Results Depletion of endotoxin from HDS attenuated the airway neutrophilia and abrogated the airway dysfunction induced in heaves horses by inhaled HDS. The airway response was re-established by adding back LPS to the depleted HDS, confirming that the attenuation in airway response was due specifically to endotoxin depletion. Interestingly, the magnitude of alteration in airway response following endotoxin depletion and add-back was greater than that which could be attributed solely to endotoxin per se, indicating that the LPS activity was enhanced by the other dust components. Consistent with this possibility, washed particulates harvested from HDS enhanced the airway response to inhaled LPS in heaves horses. Heaves horses given two different hay/straw challenges had a significantly different severity of airway inflammation and dysfunction, despite airborne dust and endotoxin concentrations in the horses' breathing zones being similar.Conclusion Although inhaled endotoxin appears not to be the only determinant of disease severity in heaves, it does contribute significantly to the induction of airway inflammation and dysfunction. This contribution is largely via the synergistic action of inhaled endotoxin and organic dust particulates, although other soluble dust components also contribute to a lesser degree.