The generation of neutrophils from hematopoietic precursors and their release to the peripheral circulation are highly regulated processes that ensure the maintenance of homeostatic neutrophil levels in the blood and their rise in response to bacterial infections and other signals. Defective neutrophil maturation/release are associated with various forms of neutropenia, which may precede and be pathogenetically linked to the development of myeloid leukemias. G-CSF has emerged a critical physiological regulator of granulopoiesis since mice carrying homozygous deletions of colony-stimulating factor (G-CSF) or its receptor are severely neutropenic, and dominant-negative mutations of G-CSFR have been linked to severe defects of granulopoiesis. Administration of G-CSF induces an expansion of myeloid lineage cells in the bone marrow, and promotes the release of mature myeloid cells and hematopoietic progenitor cells from the bone marrow to the peripheral blood. Based on these properties, G-CSF is widely used to induce granulopoiesis and to mobilize hematopoietic progenitors to the peripheral blood. More recently, a CXCR4 competitive inhibitor, AMD3100/Plerixafluor, has been approved by FDA and a mobilizing agent for hematopoitic precursors in conjunction with G-CSF. The biological activities of G-CSF are solely mediated by its activation of the G-CSF-receptor (R) that is expressed on myeloid lineage progenitor cells. Compelling evidence from genetic studies and other studies demonstrated that G-CSF indirectly promotes hematopoietic cell and neutrophil mobilization to the peripheral blood by modulating the activities of the chemokine SDF1 and/or its receptor CXCR4. WHIM, a genetic disorder associated with mutations in the intracellular domain of CXCR4 leading to increased CXCR4 function causes a retention of immature neutrophils into the bone marrow and severe peripheral neutropenia. AMD3100, a competitive inhibitor of SDF-1 binding to its receptor and a mutant form of SDF-1, which induces prolonged downregulation of the CXCR4 surface receptor, promotes the mobilization of neutrophils and hematopoietic cells to the peripheral blood. During stem cell mobilization with G-CSF, SDF-1 and CXCR4 protein levels decrease in the bone marrow. We have examined the mechanisms responsible for reduced CXCR4 expression. Initially, we found that G-CSF reduces CXCR4 expression in bone marrow Gr1+ myeloid cells, which express G-CSFR. In additional studies, we have obtained evidence that the transcriptional repressor Gfi-1 is involved in G-CSF-induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood: G-CSF promotes expression of Gfi-1, which reduces CXCR4 expression and function. In related experiments, we have generated mutants of CXCR4 that mimic mutations in the C-terminal domain found in patients with WHIM syndrome. We have examined the signaling mechanisms initiated by wild-type CXCR4 and compared with signaling from mutants CXCR4 receptors. Our results indicate that unlike the normal receptor, mutant CXCR4 fails to appropriately recruit beta arrestin2 to the receptor complex. As a consequence internalization of the mutant CXCR4 receptor from the cell surface to the cytoplasmic compartment is delayed, degradation is delayed, and signaling from the mutant receptor is also delayed. Since WHIM patients are heterozygotes for the mutant CXCR4 receptor and carry both the normal and the mutant allele, the net result is that CXCR4 signaling is extended in time, as it is the result of activation of both the normal and the mutant receptor. Thus, patients with WHIM have a super-functional CXCR4 receptor and presumably fail to release neutrophils from the bone marrow to the peripheral blood due to continuous signaling by the ligand SDF1, which holds the mature neutrophils in the bone marrow compartment. In addition to promoting the release of mature myeloid cells, G-CSF promotes the release of HSPC (hematopoietic stem/progenitor cells) from the bone marrow to the peripheral blood. Mobilization of hematopoietic progenitor cells (HPC) from the bone marrow to the peripheral blood by G-CSF is the primary means to acquire stem cell grafts for hematopoietic cell transplantation avoiding invasive bone marrow collection. Since HPC represent a small minority of all blood cells mobilized by G-CSF, there is a need for understanding the underlying mechanisms to develop selective drugs. We now found that G-CSF indirectly reduces expression of surface vascular cell adhesion molecule 1 (VCAM-1) on bone marrow HSPC, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles/exosomes in the bone marrow extracellular compartment. We find that HSPC, stromal cells and endothelial cells readily incorporate these exosomes, and that miR126 represses VCAM-1 expression on bone marrow HSPC, stromal cells and endothelial cells. In line with this, miR126-null mice display a reduced mobilization response to G-CSF. Altogether, our results implicate miR126 in the regulation of HPC trafficking between the bone marrow and peripheral sites, clarify the role of VCAM-1 in G-CSF-mediated mobilization, and have important implications for improved approaches to selective mobilization of HPC. Ongoing studies designed to further understanding of HSPC mobilization have detected an important role of the receptor/ligand pair EphrinB2/EphB4. We unveiled the mutually exclusive bone marrow distribution of EphB4 receptors in the sinusoids and EphrinB2 ligands in hematopoietic cells, and discovered an EphB4/EphrinB2-dependent pathway that controls HSPCs mobilization. Blockade of EphB4/EphrinB2 reduced HSPCs and other myeloid cells mobilization to the circulation. In murine cancer models, in which hematopoietic cells derived from the bone marrow promote tumor growth, EphB4/EphrinB2 blockade reduced tumor infiltration with HSPCs and tumor progression. These results identify EphB4/EphrinB2 signaling as critical to hematopoietic cells mobilization from bone marrow and provide a new strategy for reducing cancer progression by targeting the bone marrow. Other ongoing studies have focused on the generation of hematopoietic cells from aortic endothelium, and the characterization of the biochemical requirements underlying this critical developmental step. Adult-type intraembryonic hematopoiesis arises from specialized endothelial cells of the dorsal aorta (DA). Despite the critical importance of this specialized endothelium for establishment of hematopoietic stem cells and adult hematopoietic lineages, the mechanisms regulating its emergence are incompletely understood. We show that EphrinB2, a principal regulator of endothelial cell function, controls the development of endothelium producing adult-type hematopoiesis. The absence of EphrinB2 impairs DA-derived hematopoiesis. Transmembrane EphrinB2 and its EphB4 receptor interact in the emerging DA, which transiently harbors EphrinB2+ and EphB4+ endothelial cells, thereby providing an opportunity for bi-directional cell-to-cell signaling to control the emergence of the hemogenic endothelium. Embryonic Stem (ES) cell-derived EphrinB2+ cells are enriched with hemogenic endothelial precursors. EphrinB2 silencing impairs ES generation of hematopoietic cells but not generation of endothelial cells. The identification of EphrinB2 as an essential regulator of adult hematopoiesis provides important insight in the regulation of early hematopoietic commitment.
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