Functional characterization of equine neutrophils in response to calcium ionophore A23187 and phorbol myristate acetate
Equine neutrophils (PMN) play a critical role in inflammatory processes in horses. The objective of this study was to characterize equine PMN function ex vivo following stimulation with calcium ionophore A23187 (A23187) and phorbol myristate acetate (PMA). These stimulants trigger different branches of the PMN activation process that occurs in vivo. Equine PMN were isolated from the whole blood of six clinically normal geldings using a one-step discontinuous Percoll gradient technique. Neutrophil aggregation, degranulation, and superoxide anion production were evaluated in assay systems which had previously been established to guantitate 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 activation by PMA as the maximum change in percent transmittance and maximum nanomoles of superoxide anion produced following PMA stimulation (60.82% and 10.4nmols/10⁶cells, respectively) were greater than those values stimulated by A23187 (41.5% and 5.2nmols/10⁶cells, 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 anion production had the lowest threshold concentration among the three functions. Degranulation had the highest threshold concentration among the three functions for both stimulants.
Results indicate that equine PMN functions differ in their dependence on second messengers in the activation pathway. These functions also occur in a dose-dependent manner and differ in the threshold concentrations required for their stimulation.