Studies on the effects of adenosine A₃ receptor stimulation on human eosinophils isolated from non-asthmatic or asthmatic donors

Abstract.

Objective: This study was designed to demonstrate the presence of adenosine A₃ receptors on human peripheral blood eosinophils, and to investigate the effect of A₃ receptor stimulation on eosinophil function.¶Material: Eosinophils from either non-asthmatic or asthmatic donors.¶Methods: Eosinophils were isolated from peripheral venous blood by discontinuous gradient centrifugation and negative immunoselection. Receptor localisation was investigated by immunoblotting and by immunocytochemistry using a novel antibody specific for the human A₃ receptor. Two pharmacological responses were studied: elevation of intracellular calcium in single eosinophils, measured by microfluorimetry, and hydrogen peroxide generation in cell suspensions.¶Results: The expression of A₃ receptors by eosinophils was confirmed using the selective antibody. Addition of the A₃ receptor selective agonist, IB-MECA (100 nM), produced increases in intracellular calcium in less than 10% of the eosinophils isolated from non-asthmatic donors. These responses were only partially attenuated with the A₃ receptor antagonist, I-ABOPX. IB-MECA (0.001-1000 nM) did not stimulate hydrogen peroxide (H₂O₂) generation, nor did it enhance fMLP- or C5a-stimulated generation of H₂O₂. In fact high concentrations of IB-MECA inhibited the generation of H₂O₂ (when stimulated by fMLP or C5a), an effect probably mediated by A₂ receptors. Similar results were obtained using eosinophils from asthmatic donors.¶Conclusions: Stimulation of adenosine A₃ receptors does not appear to be a prime mechanism for free radical generation by human peripheral blood eosinophils.