CC chemokine receptor-3 undergoes prolonged ligand-induced internalization

CC chemokine receptor-3 (CCR-3) is a major receptor involved in regulating eosinophil trafficking; therefore, elucidation of ligand-induced CCR-3 even ts has important implications in understanding the biological and pathologi cal properties of eosinophils. Previous studies have demonstrated that uniq ue receptor events occur in different cell types supporting investigation o f CCR-3-mediated events in eosinophilic cells. We now report biochemical ch aracterization of CCR-3 internalization following exposure of eosinophils t o CCR-3 ligands, Treatment of freshly isolated human eosinophils with CCR-3 ligands resulted in marked and differential internalization of CCR-3 in a dose-dependent manner. Exposure to 100 ng/ml eotaxin reduced surface expres sion to 43, 43, and 76% at 15 min, 1 h, and 3 h, respectively. RANTES (redu ced on activation T cell expressed and secreted) treatment induced more sig nificant and prolonged internalization of CCR-3 than eotaxin; following 100 ng/ml of RANTES, 29, 24, and 47% of the receptor was expressed at 15 min, 3 h, and 18 h, respectively. Confocal microscopy demonstrated that receptor modulation involved receptor internalization by an endocytic pathway share d with the transferrin receptor. Receptor internalization was accompanied b y partial degradation of CCR-3, and reexpression of CCR-3 was dependent in part upon de novo protein synthesis. Internalization was not blocked by pre treatment of eosinophils with pertussis toxin. Furthermore, staurosporine d id not inhibit internalization although it blocked phorbol la-myristate 13- acetate-induced CCR-3 down-modulation. These results demonstrate that CCR-3 ligands induce differential receptor internalization that is not dependent upon G(i)-protein coupling, calcium transients, or protein kinase C.