Studies on the effects of adenosine A 3 receptor stimulation on human eosinophils isolated from non-asthmatic or asthmatic donors

Abstract

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. Eosinophils from either non-asthmatic or asthmatic donors. 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. 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. Stimulation of adenosine A3 receptors does not appear to be a prime mechanism for free radical generation by human peripheral blood eosinophils.