Erythropoietin Induction by Anemia Is Required for CFU-E Expansion During Erythroid Recovery From Sublethal Radiation Injury

Abstract 3218

The massive steady-state output of the erythron makes the erythroid lineage exquisitely sensitive to clastogenic injury. While the rapid loss of reticulocytes is well-described, the response of bone marrow progenitors and precursors to sublethal irradiation and the mechanisms underlying recovery of the erythron remain poorly defined. Following 4 Gy total body irradiation (TBI) in C57Bl/6 mice, functional colony assays were utilized to study the erythroid progenitor compartment, consisting of immature day 7 erythroid burst-forming units (BFU-E) and more mature day 3 BFU-E and erythroid colony-forming units (CFU-E). Multispectral imaging flow cytometry was used to quantify the erythroid precursor compartment, consisting of proerythroblasts and progressively more mature basophilic, polychromatophilic, and orthochromatic erythroblasts. At 2 days after 4 Gy TBI, greater than 95% of erythroid progenitors and precursors in the marrow were lost. A significant decrease in peripheral reticulocyte output and a gradual 9% drop in hematocrit accompanied this marrow loss over the first 3–4 days after radiation exposure. Following this initial injury and mild radiation-induced anemia, a robust recovery of the erythron began with a significant increase in day 3 BFU-E at 5 days post-radiation immediately followed by a rapid expansion of CFU-E at 5–6 days post-radiation to 200% of unirradiated control marrow. In contrast, day 7 BFU-E only partially recovered in a gradual linear fashion. Subsequent maturation of CFU-E resulted in progressive waves of erythroid precursors, reticulocytes, and mature red cells, creating a “ripple effect” following sublethal radiation injury. These results indicate that erythroid repopulation following radiation damage is centered on specific expansion and maturation of later erythroid progenitors (day 3 BFU-E and CFU-E). Erythropoietin (EPO) is known to be the primary regulator of the erythroid lineage, and day 3 BFU-E and CFU-E form the EPO-responsive compartment of the erythron. Therefore, we hypothesized that EPO may be the primary driving force underlying day 3 BFU-E/CFU-E expansion and subsequent erythroid recovery from radiation injury. Endogenous plasma EPO levels increased 13-fold above steady-state levels at 4 days post-radiation. This spike in EPO levels preceded the CFU-E expansion seen at 5–6 days post-radiation. To specifically determine both the etiology of the endogenous EPO spike and the necessity of supra-physiologic levels of EPO for erythroid recovery from radiation injury, we performed loss-of-function studies in which mice were transfused with packed red cells post-radiation to maintain a normal hematocrit. Prevention of the mild radiation-induced anemia by transfusion also prevented the increase in endogenous EPO and significantly abrogated day 3 BFU-E/CFU-E recovery. These findings directly link radiation-induced anemia with EPO induction and erythroid lineage reconstitution. Gain-of-function studies were performed to determine whether EPO is sufficient to drive erythroid expansion after radiation injury. Administration of exogenous EPO at 1 hour post-radiation significantly advanced the timing of CFU-E expansion and subsequent recovery of the erythron. In addition, the accelerated synchronous wave of recovery following exogenous EPO very closely mirrored the physiological wave of recovery during the endogenous EPO response, indicating that the previously observed “ripple effect” is an inherent component of EPO-induced erythroid recovery from radiation injury. Finally, administration of EPO at 4 days post-radiation, at the peak of the endogenous EPO response, further enhanced CFU-E recovery to over 330% of unirradiated control levels, providing additional evidence that EPO drives expansion of irradiated CFU-E. These studies, taken together, indicate that the anemia-induced EPO response following radiation injury is both necessary and sufficient for CFU-E expansion that leads to recovery of the erythron. A better understanding of the response of the erythroid lineage to clastogenic injury will ultimately lead to improved therapies to protect and mitigate the hematopoietic system from radiation and chemotherapy damage.