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

Objective: This study was designed to demonstrate the presence of adenosine A, receptors on human peripheral blood eosinophils, and to investigate the effect of A(3) 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 A, 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 A(3) receptors by eosinophils was confirmed using the selective antibody. Addition of the A, 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 A, 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 A, receptors. Similar results were obtained using eosinophils from asthmatic donors. Conclusions: Stimulation of adenosine A(3) receptors does not appear to be a prime mechanism for free radical generation by human peripheral blood eosinophils.