Direct Interaction with Macrophages Increases Proerythroblast Proliferation While Preserving Erythropoietin (EPO) Dependence.
Authors: Rhodes Melissa M. , Kopsombut Prapaporn , Bondurant Maurice , Price James O. , Koury Mark J. 
Published: April 2020

Abstract
INTRODUCTION: EPO regulates erythropoiesis by preventing apoptosis at the relatively late developmental stages of CFU-E and proerythroblasts. Cells in these EPO-dependent stages are actively dividing, but after several divisions they enter a G0 state from which they enucleate. In vivo these erythroid progenitor cells associate physically with macrophages in the bone marrow, forming erythroblastic islands. Erythroblastic islands appear to be necessary for proper development of erythroblasts into erythrocytes, but our current knowledge about erythroid progenitor-macrophage interactions in the erythroblastic islands is limited.
METHODS: Spleens of mice in the acute erythroblastic phase of Friend virus disease were used to reconstitute erythroblastic islands in a co-culture system that enabled study of interactions between macrophages and developmentally synchronized EPO-dependent erythroid progenitors. Proliferation and differentiation of these erythroid progenitors in macrophage co-cultures was compared to controls in which the same erythroid progenitors were cultured alone. Erythroblasts adherent to macrophages and non-adherent erythroblasts from co-cultures, as well as control erythroblasts cultured without macrophages were collected at 6, 20, 32, and 44 hrs after initial culture for cell counts, cytospin preparations for morphology, flow cytometry analyses for apoptosis (TUNEL), cell cycle phases, and expression of two surface molecules known to be expressed on differentiating erythroblasts, phosphatidylserine (PS) and α4 integrin. Experiments were also done with erythroblasts cultured in macrophage-conditioned media.
RESULTS: Splenic erythroblasts cultured alone proliferated 4.6 ± 0.7 fold over 44 h, while erythroblasts co-cultured with splenic macrophages proliferated 14.2 ± 2 fold (n=12). Control erythroblasts had the same proliferation in macrophage-conditioned medium as they did in normal medium. In EPO dose-response experiments, percentages of apoptosis were the same among adherent and non-adherent co-cultured erythroblasts and control erythroblasts. Cytospin preparations revealed no differences in morphology among non-adherent and adherent erythroblasts in co-cultures and control erythroblasts. No differences were found in enucleation percentages, extruded nuclei, or reticulocyte formation at 44 h. Likewise no differences were found in percentages of apoptotic cells, distribution of cell cycle phases, or surface expressions of PS or α4 integrin during the 44 h of differentiation.
CONCLUSIONS: Co-culture with macrophages in reconstituted erythroblastic islands dramatically increases the erythroblast proliferation, without affecting differentiation. The increase in proliferation is not due to decreased apoptosis, increased EPO responsiveness, or soluble factors released by the macrophages. Preservation of EPO-dependence during this expansion of erythroblasts mediated by direct interaction with macrophages indicates that erythropoietic regulation by EPO affects a larger population of erythroid progenitor cells in later stages of erythropoiesis and, thereby, accounts for relatively rapid increases or decreases in erythrocyte production following changes in EPO levels in vivo.