Persistence of Transferrin Receptors on RBCs Produced during Stress Erythropoiesis

Reticulocytes (Retics) are immature RBCs that are detected by staining of RNA in the internal organelles, mainly the ribosomes, which remain in mammalian erythroid cells following enucleation. Along with other internal organelles, ribosomes are degraded during maturation of Retics, resulting in complete loss of Retic staining in mature erythrocytes. At the same time as internal organelle degradation, plasma membranes of Retics mature with loss of specific proteins including transferrin receptors (TfR) and the alpha-4 component of several integrins (α4). Retic plasma membrane maturation involves an intrinsic mechanism, exocytosis, as well as an extrinsic mechanism that occurs, at least partially, in the spleen. TfR and α4 expressions on Retic plasma membranes and thiazole orange (TO) staining of RNA were examined by flow cytometry during murine Retic maturation under normal and stress conditions. During their 4 days of maturation in vitro, nascent reticulocytes derived from cultured erythroblasts stably expressed TfR in nearly 80% of cells and α4 in 40% of cells on their plasma membranes, while TO staining was completely lost over the 4 days. To compare in vivo and in vitro Retic maturation, mice were bled causing an anemia with reticulocytosis. Retic maturation in vitro was examined in cultures of Retic-rich blood removed from these bled mice, while Retic maturation in vivo was examined after the bled mice were hypertransfused to cease further erythroblast production. Surface TfR and α4 expressions disappeared during Retic maturation in vivo, but not in vitro. As the Retics matured in vivo, a population of erythrocytes with surface TfR expression, but without TO staining, accumulated to a maximum of 6% of the RBCs. These TfR+/TO− RBCs then disappeared gradually in vivo over several days indicating that they were developmentally between Retics and mature erythrocytes. These TfR+/TO− RBCs were not found in unbled, control mice (