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. G-CSF has emerged a critical 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 neutrophils 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. 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 SDF-1 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 mmature 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, promote the mobilization of neutrophils and hematopoietic cells to the peripheral blood. Osteoblasts, stromal cells and endothelial cells constitutively express SDF-1 in the bone marrow; hematopoietic cells express CXCR4. 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 Gr-1+ myeloid cells, which express G-CSFR. This is consistent with earlier observations that CXCR4 levels are reduced on neutrophils and CD34+ hematopoietic cells found in the circulation after G-CSF-induced mobilization. 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. We found that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also likely promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing 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 from wild-type CXCR4 and compared with signaling from mutants CXCR4 receptors. Our preliminary results indicate that unlike the normal receptor, mutant CXCR4 fails to appropriately recruit beta arrestin to the receptor complex. Recently, a new receptor for SDF-1 has been identified, named RDC-1/CXCR7. The role of this receptor in the release of neutrophils from the bone marrow has not been investigated. In addition, GFI-1 null mice have been generated, showing severe neutropenia and defective neutrophil maturation. Ongoing studies are focused on the study of RDC-1/CXCR7 function and on GFI-1 regulation of myeloid differentiation.
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