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IntroductionThe majority of hematopoietic progenitor cells (HPCs) reside in the bone marrow surrounded by a complex, highly organized microenvironment. Under normal conditions, a small number of HPCs are released into the peripheral blood. Agents with distinct cellular targets and biologic activities can induce the mobilization of HPCs into blood, including hematopoietic growth factors, chemotherapeutic agents, and chemokines. 1,2 Recently, mobilized peripheral blood HPCs have become the principal cellular source for reconstitution of the hematopoietic system following myeloablative therapy. Currently, granulocyte colony-stimulating factor (G-CSF) is the most widely used agent to induce HPC mobilization due to its potency, predictability, and safety. 3 However, the mechanisms responsible for G-CSF-induced HPC mobilization have not been defined.We previously showed that G-CSF receptor (G-CSFR) expression on HPCs is not required for their mobilization by G-CSF, suggesting that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. 4 The nature of the transacting signals that mediate G-CSF-induced HPC mobilization is unknown; however, accumulating evidence suggests that interaction of CXCL12 (stromal-derived factor 1 [SDF-1]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), may play an important role in regulating G-CSF-induced HPC mobilization. CXCL12 is a CXC chemokine constitutively produced in the bone marrow by stromal cells. 5 Studies of CXCL12-or CXCR4-deficient mice have established that these genes are necessary for the normal migration of HPCs from the fetal liver to the bone marrow and in the efficient retention of myeloid precursors in the adult bone marrow. 6,7 Moreover, treatment with AMD-3100, a specific antagonist of CXCR4, induces rapid and robust HPC mobilization in both humans and mice. 8,9 Finally, we and others showed that CXCL12 protein expression in the bone marrow is significantly decreased following G-CSF treatment. [10][11][12] Collectively, these data suggest a model in which disruption of CXCL12/CXCR4 signaling is a key step in G-CSF-induced HPC mobilization.The mechanisms mediating the G-CSF-induced decrease in CXCL12 protein expression in the bone marrow have not been For personal use only. on May 12, 2018. by guest www.bloodjournal.org From defined. Previous reports suggested that neutrophil elastase (NE) and cathepsin G (CG) might regulate CXCL12 protein expression in the bone marrow through proteolytic cleavage of CXCL12. 10,11 However, mice genetically lacking NE and CG display normal G-CSF-induced HPC mobilization, and the expected decrease in bone marrow CXCL12 protein was observed. 13 Thus, the G-CSFinduced decrease in CXCL12 protein expression in the bone marrow does not require these proteases. It is possible that other proteases can compensate for the loss of NE and CG. Alternatively, nonproteolytic mechanisms may regulate CXCL12 expression in the bone marrow during G-CSF-induced HPC mobilization.In this study, we characterize G-CSF...

We have generated mice carrying a homozygous null mutation in the granulocyte colony-stimulating factor receptor (G-CSFR) gene. G-CSFR-deficient mice have decreased numbers of phenotypically normal circulating neutrophils. Hematopoietic progenitors are decreased in the bone marrow, and the expansion and terminal differentiation of these progenitors into granulocytes is impaired. Neutrophils isolated from G-CSFR-deficient mice have an increased susceptibility to apoptosis, suggesting that the G-CSFR may also regulate neutrophil survival. These data confirm a role for the G-CSFR as a major regulator of granulopoiesis in vivo and provide evidence that the G-CSFR may regulate granulopoiesis by several mechanisms. However, the data also suggest that G-CSFR-independent mechanisms of granulopoiesis must exist.

Neutrophils are released from the bone marrow in a regulated fashion to maintain homeostatic levels in the blood and to respond to physiological stresses, including infection. We show that under basal conditions granulocyte colony-stimulating factor (G-CSF) is an essential regulator of neutrophil release from the bone marrow. Nonredundant signals generated by the membrane-proximal 87 amino acids of the G-CSF receptor (G-CSFR) are sufficient to mediate this response. Surprisingly, G-CSFR expression on neutrophils is neither necessary nor sufficient for their mobilization from the bone marrow, suggesting that G-CSF induces neutrophil mobilization indirectly through the generation of trans-acting signals. Evidence is provided suggesting that downregulation of stromal cell-derived factor 1 expression in the bone marrow may represent such a signal.

Expression of the G-CSF receptor on bone marrow monocytes is sufficient to trigger HSC mobilization in response to G-CSF, in part via effects on osteoblast lineage cells.

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE ؋ CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE ؋ CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12␣ protein expression in the bone marrow of Ne ؋ CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization. IntroductionThe use of hematopoietic progenitor cells (HPCs) to reconstitute hematopoiesis following myeloablative therapy has significantly improved the clinical outcome for patients with a variety of diseases. Recently, mobilized peripheral blood HPCs instead of bone marrow-derived HPCs have been used because of reduced engraftment times and relative ease of collection. Although the great majority of HPCs reside within the bone marrow, a small number of HPCs also circulate in the peripheral blood. The number of circulating HPCs can be dramatically increased, or mobilized, by a wide variety of stimuli including hematopoietic growth factors, chemotherapy, and chemokines. 1,2 Currently, granulocyte colony-stimulating factor (G-CSF) is the most commonly used agent to mobilize HPCs because of its potency and lack of serious toxicity. However, the mechanisms that mediate G-CSF-induced HPC mobilization are incompletely understood.We previously showed that expression of the G-CSF receptor (G-CSFR) on HPCs is not required for their mobilization into the blood in response to G-CSF. 3 This observation suggests that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. Recent studies suggest that hematopoietic proteases released by neutrophils into the bone marrow microenvironment may represent such a signal. A highly proteolytic microenvironment is induced in the bone marrow during HPC mobilization by G-CSF. 4 In particular, matrix metalloproteinase-9 (MMP-9 or gelatinase B), neutrophil elastase (NE), and cathepsin G (CG) accumulate...

Current evidence suggests that hematopoietic stem/progenitor cell (HSPC) mobilization by granulocyte colonystimulating factor (G-CSF) is mediated by induction of bone marrow proteases, attenuation of adhesion molecule function, and disruption of CXCL12/CXCR4 signaling in the bone marrow. The relative importance and extent to which these pathways overlap or function independently are uncertain. Despite evidence of protease activation in the bone marrow, HSPC mobilization by G-CSF or the chemokine Gro␤ was abrogated in CXCR4 Ϫ/Ϫ bone marrow chimeras. In contrast, HSPC mobilization by a VLA-4 antagonist was intact. To determine whether other mobilizing cytokines disrupt CXCR4 signaling, we characterized CXCR4 and CXCL12 expression after HSPC mobilization with Flt3 ligand (Flt3L) and stem cell factor (SCF). Indeed, treatment with Flt3L or SCF resulted in a marked decrease in CXCL12 expression in the bone marrow and a loss of surface expression of CXCR4 on HSPCs. RNA in situ and sorting experiments suggested that the decreased CXCL12 expression is secondary to a loss of osteoblast lineage cells. Collectively, these data suggest that disruption of CXCR4 signaling and attenuation of VLA-4 function are independent mechanisms of mobilization by G-CSF. IntroductionUnder basal conditions, the great majority of hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow. In response to injury or after administration of a wide range of pharmacologic agents, HSPCs are mobilized into the circulation. Clinically, HSPC mobilization has been exploited as a means to obtain hematopoietic stem cells for therapeutic transplantation. In fact in current practice, mobilized blood has essentially replaced bone marrow as a source of HSPCs for stem cell transplantation. 1,2 The most widely used mobilizing agent in the clinical setting is granulocyte-colony-stimulating factor (G-CSF). Treatment with G-CSF mobilizes sufficient numbers of HSPCs for transplantation in most donors with a minimum of toxicity. Nevertheless, development of more rapid and effective mobilization protocols would be of great clinical interest. 2 This could be achieved, in theory, by identifying downstream mechanisms important in HSPC mobilization and targeting those mechanisms directly, either individually or in combination.Although the mechanisms by which G-CSF induces HSPC mobilization are not fully understood, there is evidence that 3 general pathways are in involved: activation of proteases, attenuation of adhesion molecule function, and disruption of CXCL12/CXCR4 signaling (reviewed in Levesque et al 3 and Papayannopoulou 4 ). G-CSF administration is associated with the activation of several proteases and down-regulation of serpin family protease inhibitors in the bone marrow. [5][6][7][8] This results in the induction of a proteolytic bone marrow environment that may contribute to HSPC mobilization by degrading extracellular matrix and cleaving key signaling molecules. 5,7,9 However, mice genetically deficient in several implicated proteases mobil...

To investigate the role of signal transducer and activator of transcription (STAT) proteins in granulocyte colony-stimulating factor (G-CSF)-regulated biological responses, we generated transgenic mice with a targeted mutation of their G-CSF receptor (termed d715F) that abolishes G-CSF-dependent STAT-3 activation and attenuates STAT-5 activation. Homozygous mutant mice are severely neutropenic with an accumulation of immature myeloid precursors in their bone marrow. G-CSF-induced proliferation and granulocytic differentiation of hematopoietic progenitors is severely impaired. Expression of a constitutively active form of STAT-3 in d715F progenitors nearly completely rescued these defects. Conversely, expression of a dominant-negative form of STAT-3 in wild-type progenitors results in impaired G-CSF-induced proliferation and differentiation. These data suggest that STAT-3 activation by the G-CSFR is critical for the transduction of normal proliferative signals and contributes to differentiative signals.