We investigated the acute response of the erythron to radiation injury and found that a wide spectrum of the maturing erythron in the bone marrow is extremely sensitive in a dose-dependent manner to radiation injury. Recent studies have found similar depletions of hematopoietic stem cell (HSC) populations following sublethal TBI, indicating HSC are also very radiosensitive [43
]. Dose-response studies of the bone marrow indicate that granulocyte-macrophage progenitors and stromal cell precursors are most radioresistant, followed by multilineage hematopoietic progenitors and HSC-enriched populations (CAFC-28), and finally by highly radiosensitive erythroid progenitors [22
]. Our findings confirm the marked radiosensitivity of erythroid progenitors and provide new data indicating that maturing erythroid precursors are also very rapidly injured and depleted.
These findings provide an explanation of the downturn in MN-RET frequency seen at higher sublethal doses of radiation [14
]. Despite the significant loss of maturing erythroblasts 24 hours after 1 Gy TBI, there is persistence and recovery of erythroblast numbers by 2 days post-irradiation (, 1 Gy). These cells, which are moderately damaged but still able to undergo terminal erythroid maturation, are the likely source of MN-RET that appear as a cohort in the bloodstream 43–50 hours post-irradiation [14
]. However, higher sublethal irradiation doses lead to chromosomal damage and possibly other injuries that preclude effective repair, thereby inducing extensive erythroblast cell death (, 4 Gy). Although the proportion of erythroblast precursors that form micronuclei following 4 Gy TBI are likely even higher than at 1 Gy TBI, the vast majority of these severely damaged cells are lost before terminal erythroid maturation can occur, leading to a marked decrease in the frequency of MN-RET at higher doses of sublethal irradiation.
Our investigation using IFC allows for a new perspective of erythroid maturation by quantitative analysis of highly specific and previously difficult-to-access erythroid subpopulations. CFU-E were defined more than 30 years ago by their ability to give rise to small colonies of mature erythroid cells in vitro
]. Although this methodology provides quantitative, functional data, the actual colony-forming cell cannot be directly investigated by this approach. Building on recent studies describing the immunophenotype of CFU-E [30
], we identified CFU-E using a novel three-stain IFC panel. This was possible because IFC can detect Ter119 antibody binding at levels below standard flow cytometry detection by restricting measurements to cell areas and decreasing background. One benefit of this novel phenotypic accessibility is that despite the presence of phenotypic CFU-E at 1 hour following 4 Gy TBI (), colony assay analysis at this same time-point showed no evidence of CFU-E (data not shown). This lack of functional CFU-E is likely due to the severe injury and apoptotic induction of CFU-E following 4 Gy TBI (), preventing damaged CFU-E from forming colonies in vitro
. As a result, these injured CFU-E can only be analyzed by phenotypic classification methods. Thus, our IFC gating method allows, for the first time, access to CFU-E along with all of the downstream erythroid precursor subpopulations.
The rapid kinetics of cell loss and the marked induction of apoptosis in response to radiation injury indicate that CFU-E and ProE behave similarly to each other and very differently from later-stage erythroblast precursors. In addition, steady-state gene regulation underlies a switch from a pro-apoptotic phenotype in ProE to an anti-apoptotic phenotype in later erythroblasts. This erythroid compartmentalization does not correlate with changes in cell cycle during erythroid maturation, as significant downregulation of cellular proliferation does not occur until the final OrthoE stage of erythroblast maturation. Although cellular proliferation rates contribute to radiosensitivity following sublethal TBI [34
], our cell cycle data demonstrate that observed differences in erythroid loss and apoptosis at the ProE to BasoE transition are not primarily due to cell cycle-related induction of checkpoint repair and apoptosis pathways. Thus, erythroid maturation can be viewed not only as a progression of colony-forming progenitors to phenotypic precursors, but also as a progression of immature, pro-apoptotic erythroblasts (CFU-E/ProE) to maturing, anti-apoptotic erythroblasts (BasoE/PolyE/OrthoE) based on the functional response of these cell types to radiation injury ().
Functional compartmentalization model of erythropoiesis
The functional transition from ProE to late-stage erythroblasts is supported by the response of the erythron to other proapoptotic stimuli. For instance, interaction of Fas on immature erythroblasts with FasL on later erythroblasts has been shown to be crucial for erythropoietic regulation by inducing apoptosis in early Fas-expressing erythroid precursors [45
]. A similar negative feedback of erythropoiesis occurs through the apoptotic TRAIL pathway [45
]. Furthermore, significant evidence indicates that steady-state erythropoiesis is regulated by erythropoietin (EPO)-mediated survival of EPO-responsive cells, which encompass CFU-E and ProE [49
]. These data, taken together, support the concept that EPO mediates survival and proliferation of pro-apoptotic, EPO-responsive cells until the switch in apoptotic phenotype occurs during later erythroid maturation. Our findings of the response of the erythron to sublethal radiation injury, in combination with steady-state apoptotic gene expression studies, uncover a critical transition as proerythroblasts progress to later-stage erythroid cells.