FUNCTIONAL-CHARACTERIZATION OF EQUINE NEUTROPHILS IN RESPONSE TO CALCIUM IONOPHORE A23187 AND PHORBOL-MYRISTATE ACETATE EX-VIVO

Equine neutrophils (PMN) play a critical role in inflammatory processe s in horses. The objective of this study was to characterize equine PM N function ex vivo following stimulation with calcium ionophore A23187 (A23187) and phorbol myristate acetate (PMA). These stimulants trigge r different branches of the PMN activation process that occurs in vivo . Equine PMN were isolated from the whole blood of six clinically norm al geldings using a one-step discontinuous Percoll gradient technique. Neutrophil aggregation, degranulation, and superoxide anion productio n were evaluated in assay systems which had previously been establishe d to quantitate PMN function. Dose-response curves for A23187 and PMA were derived for the three functions. Results indicate that equine PMN aggregation and superoxide anion production are more responsive to ac tivation by PMA as the maximum change in percent transmittance and max imum nanomoles of superoxide anion produced following PMA stimulation (60.8% and 10.4 nmol per 10(6) cells, respectively) were greater than those values stimulated by A23187 (41.5% and 5.2 nmol per 10(6) cells, respectively). However, degranulation was found to be more responsive to A23187 stimulation (maximum percent degranulation: 56.1%) than to PMA stimulation (maximum percent degranulation: 30.7%). Dose-response curves following A23187 and PMA stimulation revealed that superoxide a nion production had the lowest threshold concentration among the three functions. Degranulation had the highest threshold concentration amon g the three functions for both stimulants. Results indicate that equin e PMN functions differ in their dependence on second messengers in the activation pathway. These functions also occur in a dose-dependent ma nner and differ in the threshold concentrations required for their sti mulation.