Mobilizing Doses Of G-CSF Stop Medullary Erythropoiesis By Depleting F4/80+ VCAM1+ ER-HR3+ CD169+ Erythroid-Island Macrophages

G-CSF mobilizes hematopoietic stem cells (HSCs) from the bone marrow (BM) into the blood by suppressing a subset of HSC niche supportive macrophages. As macrophages are the central component of erythropoietic islands in BM, spleen and liver, we examined the effect of G-CSF on erythropoiesis in C57BL/6 mice.

Mobilizing doses of G-CSF caused a marked whitening of the BM, a 15-fold decrease in the number of phenotypic erythroblasts, a 1.5-fold decrease in polychromatic and orthochromatic erythroblasts, and a 4.5-fold reduction in reticulocytes in the BM. Conversely, more immature pro-erythroblasts increased 4.4-fold. As the cell surface antigen ER-HR3 identifies erythroid island macrophages in mouse liver and spleen, while VCAM-1 and CD169 on macrophages have been independently reported to be critical for erythropoiesis, we followed the expression on these antigens on BM macrophages during mobilization. G-CSF treatment caused a 35-fold reduction in the number of CD11b⁺ F4/80⁺ VCAM1⁺ ER-HR3⁺ CD169⁺ Ly6G⁺ macrophages that paralleled the loss of erythroblasts. As a result, splenic erythropoiesis was up-regulated to compensate for the loss of medullary erythropoiesis with a 4-5 fold increase in pro-erythroblasts, all erythroblast subsets and reticulocytes. In another set of experiments, we quantified medullar erythropoiesis and macrophages during recovery after a 4 day G-CSF treatment. Erythroblasts and supportive macrophages significantly recovered as early as 24 hours after cessation of G-CSF but it took 7 days to normalize to pre-mobilization values. This suggests that mobilizing doses of G-CSF transiently block erythroblast differentiation specifically in the BM (but not the spleen) by affecting central macrophages in erythroid islands.

To confirm that CD11b⁺ F4/80⁺ VCAM1⁺ ER-HR3⁺ CD169⁺ Ly6G⁺ macrophages are critical to the maturation of pro-erythroblasts into erythroblasts, we next performed a broad macrophage depletion by injecting clodronate-loaded liposomes, or a selective depletion of CD169⁺ macrophages in mice knocked-in with diphtheria toxin receptor into the Siglec1 (CD169) gene. Both clodronate liposome treatment in wild-type mice, and diphtheria toxin treatment in Siglec1^DTR/+ mice caused a concomitant depletion of CD11b⁺ F4/80⁺ VCAM1⁺ ER-HR3⁺ CD169⁺ macrophages, loss of erythroblasts and accumulation of pro-erythroblasts. Unlike G-CSF, these two treatments also blocked splenic erythropoiesis.

In conclusion, we propose that 1) CD11b⁺ F4/80⁺ VCAM1⁺ ER-HR3⁺ CD169⁺ Ly6G⁺ macrophages include nursing macrophages at the centre of erythroid islands and are essential for the maturation of pro-erythroblasts to erythroblasts and 2) mobilizing doses of G-CSF transiently stop medullary erythropoiesis by depleting CD11b⁺ F4/80⁺ VCAM1⁺ ER-HR3⁺ CD169⁺ Ly6G⁺ macrophages in erythropoietic islands in the BM, but not in the spleen.