Targeted Disruption of GAS6-Mertk Pathway Leads to Defects in Physiological Clearance of Expelled Nuclei from Erythroblasts by Bone Marrow Macrophages.

Late in erythropoiesis, nuclei are expelled from erythroblasts and engulfed by macrophages located in the blood island. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as a signal for their engulfment by macrophages. PS opsonins milk-fat-globule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors a_vb₅ (and a_vb₃) and Axl/Mertk/Tyro3, are involved in the phagocytosis of apoptotic cells. Because fetal liver and bone marrow macrophages do not express MFG-E8, GAS6-Mertk pathway might constitute a major pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from the spleens of phlebotomized mice (500 μl), and tested the capacity of bone marrow-derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6^−/−), Axl (Axl^−/−), Mertk (mertk^kd) or Tyro3 (Tyro3^−/−) to internalize these nuclei. Spleen erythroblasts were isolated 4 days after phlebotomy and cultured for 5 hours. Nuclei were obtained after disconnection from reticulocytes by mechanical shaking. Released nuclei were then identified by flow cytometry according to their size and their positive staining for the erythroid lineage marker Ter119 and Annexin V for PS labelling. Purity of the preparation was double-checked by morphological examination of cytospin preparations. Primary BMDM isolated from wild-type (WT) controls and GAS6^−/−, Axl^−/−, Mertk^kd, Tyro3^−/−, Axl/Tyro3^−/−, Axl^−/−/Mertk^kd mice were incubated with nuclear preparation for 3 hours. BMDM were then washed to remove un-engulfed nuclei, analyzed in bright field and stained with May-Grünwald-Giemsa. Phagocytosis was determined by counting the number of BMDM with ingested nuclei and the phagocytosis index indicated the number of engulfed nuclei per macrophage. We found that GAS6^−/− BMDM cleared 30% less nuclei than WT BMDM (p<0.01). We observed a slight decrease of internalization capacity for Axl^−/− BMDM, whereas Tyro3^−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, Mertk deficiency nearly abolished the nuclei phagocytosis (p<0.001). Axl/Tyro3^−/− and Axl^−/−/Mertk^kd BMDM were tested in comparison with WT BMDM and single knockouts, and did not show any cumulative effects when compared to single knockouts. Thus, Mertk was critical for the phagocytosis of nuclei from erythroblasts whereas the role of Axl and Tyro3 appeared to be negligible. In conclusion, we have shown that GAS6 and its receptor Mertk were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island. Indeed, GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and Mertk receptor on BMDM, allowing their efficient clearance.