EVALUATION OF AN IN-VITRO DEGRANULATION CHALLENGE PROCEDURE FOR EQUINE PULMONARY MAST-CELLS

Pulmonary mast cells (PMC) are important components of the inflammator y process in equine allergic lung diseases such as heaves, Very little , however, is known of the degranulation kinetics of these cells and t hus, their pathophysiologic role remains largely speculative, The purp ose of this study was to develop a repeatable protocol for in vitro eq uine PMC degranulation, Five mature horses (sex: 2 M, 3 F; age: 8.8 +/ - 6.5 y), historically free of pulmonary disease and normal on clinica l respiratory examination, arterial blood gas analysis, pulmonary mech anics testing and histamine inhalation challenge, were studied, Bronch oalveolar lavage was performed on 4 separate occasions, at least 2 d a part, in a different lung lobe on each occasion, The lavage fluid was concentrated by centrifugation, Cells were resuspended in modified HEP ES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC co ncentration determined, Cell inocula containing 30 000 PMC were incuba ted with 10(-8) to 6 X 10(-5) M A23187. Cells were then separated by c entrifugation and histamine release (HR) was determined by fluorometri c assay, The procedure was readily performed and yielded sufficient PM C for 30 to 60 inocula per lavage, Maximal HR (34.4 +/- 16.1%) was obt ained with 10(-5) M A23187. The degranulation process was largely comp lete by 20 min but cell lysis was negligible, The challenge was repeat able 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 spe culate that this protocol may be useful in further studies on the path ophysiology and treatment of equine allergic lung diseases.