The Hematopoietic Reconstitution Defect of Mice Lacking CXCR4 Is Related to an Altered Retention of Hematopoietic Cells in the Bone Marrow.

Abstract

In steady-state hematopoiesis, most hematopoietic stem cells (HSC) reside within the bone marrow (BM) and are confined within specialized niches, whereas terminally differentiated cells leave the BM and migrate into the blood. Within the BM microenvironment (BMM), self-renewal, proliferation and differentiation of HSC occur through specific interactions with heterogeneous populations of stromal cells and extracellular matrix proteins. The BMM also regulates the retention, the migration and the homing of HSC. Some of these effects are mediated through soluble mediators such as chemokines. The chemokine SDF-1 and its receptor CXCR4 play an important role on HSC but the mechanisms are not fully understood. Here, we show that radioprotection of lethally irradiated mice injected with less than 2x106 fetal liver (FL) cells from E14.5 CXCR4−/− embryos was markedly impaired when compared to CXCR4+/+ counterparts (10% survival in CXCR4−/− mice versus 65% survival in CXCR4+/+), but this defect was rescued when hosts were engrafted with 5x106 cells. This quantitative defect contrasted with a similar content in hematopoietic colony forming cells (CFCs), CFU-S and Lin−/lowSca-1+c-kit+ cells in E14.5 CXCR4−/− and CXCR4+/+ FL. In addition, we show that the homing of CFCs was significantly altered in the spleen but not in the BM as detected with a CFSE-staining assay. Nevertheless, mice engrafted with 5x106 cells from either E14.5 CXCR4−/− and CXCR4+/+ FL shown similar levels of chimerism at both 5 and 16 weeks post transplant. Thus, no significant difference was observed either on the total number of circulating leukocytes and erythrocytes in both group of chimeras. Interestingly, CXCR4+/+ chimeras exhibited a thrombocytosis before week 12, which was not observed in CXCR4−/− engrafted mice. Later platelet count returned to normal. As reported in other studies, CXCR4−/− chimeras were characterized by a decrease in the absolute number of circulating lymphocytes and an elevated number of circulating granulocytes, from week 2 to week 16 post transplant. Also, using a CFCs assay, we observed a 30-fold increase in CFCs in the circulation of CXCR4−/− chimeras (8544 ± 249 CFCs per ml of blood versus 280 ± 42 CFCs per ml of blood in CXCR4 +/+ chimeras, p<0.001) and this increment was already observed before hematopoiesis had reached a steady state level at week 2 (4760 ± 480 CFCs per mL of blood in CXCR4−/− mice versus 140 ± 28 CFCs per mL of blood in CXCR4+/+ mice, p<0.001). Secondary transplantations were performed using irradiated Ly5.1 hosts injected with a mixture of either 100 ml CXCR4+/+ or 100 μl CXCR4−/− chimeras' PBLs and 1.5x105 BM Ly-5.1+ cells. At week 5 post transplant, all recipients engrafted with PBLs from CXCR4−/− donors exhibited a significant level of chimerism in contrast to those injected with PBLs from CXCR4+/+ donors (6.4 ± 3.1% versus 0.7 ± 0.6% of circulating Ly5.2+ cells in control mice). Altogether, our results demonstrate that the defect in hematopoietic reconstitution of CXCR4−/− fetal liver cells is more related to an altered anchorage in the BM than a real defect in the homing of primitive hematopoietic cells. Impaired retention in the BMM in concert with an altered homing in the spleen may, at least in short-term hematopoietic reconstitution, lead to a diminution in the number of available progenitors required for radioprotection.