Evaluation of an in vitro degranulation challenge procedure for equine pulmonary mast cells

Pulmonary mast cells (PMC) are important components of the inflammatory process in equine allergic lung diseases such as heaves. Very little, however, is known of the degranulation kinetics of these cells and thus, their pathophysiologic role remains largely speculative. The purpose of this study was to develop a repeatable protocol for in vitro equine PMC degranulation. Five mature horses (sex: 2 M, 3 F; age: 8.8 +/- 6.5 y), historically free of pulmonary disease and normal on clinical respiratory examination, arterial blood gas analysis, pulmonary mechanics testing and histamine inhalation challenge, were studied. Bronchoalveolar lavage was performed on 4 separate occasions, at least 2 d apart, in a different lung lobe on each occasion. The lavage fluid was concentrated by centrifugation. Cells were resuspended in modified HEPES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC concentration determined. Cell inocula containing 30,000 PMC were incubated with 10(-8) to 6 x 10(-5) M A23187. Cells were then separated by centrifugation and histamine release (HR) was determined by fluorometric assay. The procedure was readily performed and yielded sufficient PMC for 30 to 60 inocula per lavage. Maximal HR (34.4 +/- 16.1%) was obtained with 10(-5) M A23187. The degranulation process was largely complete by 20 min but cell lysis was negligible. The challenge was repeatable within horse and produced a mean coefficient of variability of 23.0% following 20 min incubation with 10(-5) M A23187. We conclude that equine PMC degranulation can be repeatably performed in vitro and speculate that this protocol may be useful in further studies on the pathophysiology and treatment of equine allergic lung diseases.