Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTPepsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine SDF-1 to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady-state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, while β2-adrenergic antagonist inhibited both steady-state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1 ؉ /c-Kit ؉ /Lin ؊ (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P 1 , or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit ؉ /Lin ؊ cells expressing surface S1P 1 in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin ؉ mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P 1 axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility. (Blood. 2012;119(11):2478-2488) IntroductionMotility is a key feature of hematopoietic stem and progenitor cells (HSPCs). These cells are continuously released at basal levels from the bone marrow (BM) reservoir to the circulation during steady state homeostasis together with maturing leukocytes, and at increased rates on stress situations, such as bleeding or inflammation. 1,2 The complex process of HSPC trafficking is orchestrated by various cytokines, chemokines, proteolytic enzymes, and adhesion molecules 3-5 through a dynamic interplay between the immune and nervous systems within the bone microenvironment. 1,2,6-8 HSPC mobilization can be clinically induced by a variety of cytokines and drugs, such as granulocyte colony stimulating factor (G-CSF, the most commonly used agent), 9,10 sulfated polysaccharides, 11,12 and recently also by AMD3100. 13,14 Repetitive G-CSF administrations cause mobilization by inducing proliferation and differentiation of HSPC, thus increasing their pool size, accompanied by reduced retention in the BM microenvironment. 15 The chemokine stromal cell-derived factor-1 (SDF-1, also termed CXCL12), which is the most powerful chemoattractant of both human and murine HSPCs, 16,17 and its major receptor CXCR4 are key players in HSPC mobilization. 10,12,13,[18][19][20][21] SDF-1 is transiently increased in the murine BM during G-CSF stimulation followed by its downregulation at both protein 18,22 and mRNA 23 levels, reaching a nadir at the peak of HSPC mobilization. 18 The intensified SDF-1/CXCR4 interactions induce enhanced production of reactive oxygen species (ROS) through activation of the HGF/c-Met path...

Abstract.We develop an optical whole-body imaging technique for monitoring normal and leukemic hematopoietic cell homing in vivo. A recently developed nearinfrared ͑NIR͒ lipophilic carbocyanine dye 1,1Ј-dioctadecyl-3 ,3,3Ј ,3Ј-tetramethylindotricarbocyanine iodide ͑DiR͒ is used to safely and directly label the membranes of human leukemic Pre-B ALL G2 cell lines as well as primary murine lymphocytes and erythrocytes. DiR has absorption and fluorescence maxima at 750 and 782 nm, respectively, which corresponds to low light absorption and autofluorescence in living tissues. This allows us to obtain a significant signal with very low background level. A charge-coupled device ͑CCD͒-based imager is used for noninvasive whole-body imaging of DiR-labeled cell homing in intact animals. This powerful technique can potentially visualize any cell type without use of specific antibodies conjugated with NIR fluorescent tag or loading cells with transporter-delivered NIR fluorophores. Thus, in vivo imaging based on NIR lipophilic carbocyanine dyes in combination with advanced optical techniques may serve as a powerful alternative or complementation to other small animal imaging methods. Noninvasive, in vivo imaging of targeted cells is a promising field for studying cell behavior and movement in animal models. Intravital microscopy and whole-body fluorescent optical imaging are among the most widely used approaches for visualizing naive or genetically modified cells in vivo.

The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor B ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by infl uencing both the hematopoietic and nonhematopoietic compartments.