Persistence of Transferrin Receptors on RBCs Produced during Stress Erythropoiesis
Authors: Rhodes Melissa , Kopsombut Prapaporn , Price James O , Koury Mark J. 
Published: April 2020

Abstract
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 (&lt;0.1% of RBCs). TfR+/TO− RBCs in vitro accumulated to 23% of RBCs and persisted for many more days, which suggested that maturation of stress Retics to erythrocytes is enhanced by extrinsic effects in vivo. To determine the potential role of the spleen in the extrinsic enhancement of stress Retic maturation, splenectomized mice were bled to about one-third of their normal Hct/RBC numbers, and their RBC populations monitored during recovery from this phlebotomy-induced anemia. Although recovery was prolonged in splenectomized mice compared to controls, the Hcts, total RBCs, and Retic (TO+ RBCs) numbers were similar to pre-phlebotomy values in both groups by day 17 post-phlebotomy. However, total TfR+ RBCs and TfR+/TO− RBCs remained very increased for more than 5 weeks post-phlebotomy, with splenectomized mice having significantly greater increases in total TfR+ and TfR+/TO− RBCs than controls. These results indicate that the spleen enhanced TfR loss without affecting RNA degradation during maturation of stress Retics. To investigate whether macrophages play a role in Retic maturation, bled mice were treated on day 4 post-phlebotomy with liposomal clodronate that selectively kills macrophages, an effect that lasts approximately 7 days. As expected from the erythropoietic-promoting role of macrophages in erythroblastic islands, liposomal clodronate-treated mice had decreased Retic production, but they also had significantly increased TfR+/TO− RBCs compared to control mice, indicating that macrophages enhance loss of TfR, without affecting RNA degradation in maturing stress Retics. These results indicate that the spleen and macrophages enhance TfR loss during maturation of stress reticulocytes and that TfR+/TO− RBCs have potential use in demonstrating previous erythropoietic stress, even after Hcts, total RBCs, and Retics have returned to normal.