Role of Fas / FasL in Regulation of Basophillic Erythroblast Homeostasis.

Many studies have shown Fas- Fas Ligand (FasL) mediated apoptosis to be important in maturation and differentiation of erythroid precursors in vitro. To determine if there is a similar process regulating erythropoietic homeostasis in vivo, we studied erythropoiesis in Fas(lpr) and FasL (gld) deficient mice. We postulated that deficiency of Fas or FasL should result in changes in red blood cell (RBC) parameters and/or decreased levels of apoptosis of erythroblasts. To test this hypothesis under steady state conditions, blood and bone marrow were collected from 10-week old C57Bl/6 control mice, B6.MRL-Tnfrsf6 lpr /J CD95 deficient mice, and B6Smn.C3-Tnfsf6 gld /J CD95L deficient mice. Hematology was studied using a Bayer Advia 120 and femoral bone marrow was analyzed by 6-color flow cytometry using a Becton Dickinson FACSAria. Hematologic analysis revealed no differences in reticulocyte counts, RBC counts or hemoglobin (Hgb) in either lpr or gld mice compared to C57Bl/6 controls. Similarly, analysis of bone marrow revealed no differences in % of Ter-119_bright CD71_bright basophilic erythroblast (BEB), % apoptotic BEB (annexin V+, 7-AAD_dim) or % FasL+ BEB in either gld or lpr mice compared to control. As expected, lpr mice expressed 10 fold fewer Fas+ BEB while similar levels were observed in gld mice compared to controls. To test our hypothesis under stimulated conditions, control, lpr and gld mice received a single s.c. dose of 10,000 units of recombinat human erythropoietin (rhEPO). Bone marrow samples were collected 48 hours after dosing and blood samples 4, 8 and 16 days after dosing. Hematologic analysis revealed no differences in the erythropoietic response among the three strains of mice tested. Moreover, treatment with rhEPO had no effect on % Fas+ BEB in any strain, but induced a 2–5 fold increase in the % FasL+ BEB and a 2–3 fold increase in apoptotic BEB in all three strains. Based on our observations, we conclude Fas/FasL is unlikely to play a pivotal role in regulating erythroid homeostasis.