ALBERT

Description: Hematopoietic stem cells reside in the bone marrow (BM) niches in the endosteum region, in close proximity to bone-forming osteoblasts and bone-resorbing osteoclasts (Ocl). Tightly regulated SDF-1/CXCR4 interactions retain stem and progenitor cells within their stromal microenvironment. G-CSF induced mobilization impairs this balance by inducing SDF-1 secretion, its proteolytic degradation, and CXCR4 upregulation. Increased Ocl activity in the BM of G-CSF treated mice was documented, however, no active role for Ocl in mobilization was revealed. Ocl secrete the mobilizing chemokines IL-8 and SDF-1, the cytokine HGF, and the metalloprotease MMP-9, which participate in stress-induced mobilization. We hypothesized that in addition to their role in physiological bone remodeling, Ocl are also involved in stem cell mobilization. Ten daily injections of either G-CSF, Pamidronate (Ocl apoptotic drug used to treat osteoporotic patients) or both, led to mobilization of murine progenitor cells, accompanied by Ocl activation in the endosteum region (assayed by TRAP staining for activated Ocl). Interestingly, Pamidronate treatment induced two waves of mobilization on days 5 and 10, correlating with increased levels of TRAP+ multinucleated Ocl in the endosteum and increase in SDF-1 and HGF mRNA in the BM. Stress-inducing conditions, which are also known to mediate bone remodeling, namely LPS stimulation (mimicking bacterial infection) and controlled bleeding (mimicking injury), also triggered progenitor mobilization and CXCR4 upregulation. Of note, LPS-induced mobilization was dependent on functional CXCR4 upregulation and MMP2/9 secretion, was accompanied by Ocl activation and reduction in BM SDF-1 levels. Treatment of primary murine osteoblast/ Ocl precursor cultures with G-CSF, SDF-1 and HGF, increased dose dependently the formation of TRAP+ multinucleated Ocl, suggesting that SDF-1 and HGF are important regulators of the cross talk between Ocl activation and stem cell mobilization. Mobilization was documented in mice receiving five daily injections of either SDF-1 (10ug), or HGF (1.5ug) together with increased levels of Ocl precursors in the blood and BM, as well as number and size of TRAP+ Ocl in the endosteum region. Unexpectedly, Pamidronate also activated SDF-1 expression in cultures of primary mouse osteoblasts, suggesting Ocl activation prior to apoptosis by this drug. Finally, the effect of the major Ocl proteinase, cathepsin K (CTK) as a candidate regulator of stem cell mobilization was investigated. Unexpectedly, recombinant human CTK inactivated human SDF-1 and abolished its chemotactic activity in vitro. This process was completely abrogated by a broad range proteinase inhibitor. We identified N-terminal cleavage and a dose dependent degradation of SDF-1 by CTK. These results suggest that CTK produced and secreted by activated Ocl also participates in the impairment of the steady state homeostatic balance of SDF-1/CXCR4 interactions, inducing local degradation of SDF-1 in the endosteum, thus facilitating stem cell mobilization. Our findings indicate that molecules involved in stem and progenitor cell anchorage, migration, and mobilization are also involved in Ocl activation. In summary, our results add mechanistic insight to the osteoblast/Ocl endosteal interactions and molecular pathways that regulate stem cell mobilization, which is of relevance for pathological stem cell malignancies and clinical mobilization, particularly for patients with poor mobilization.

Description: The phosphatase CD45 is a key regulator of antigen receptor signaling in lymphocytes. Still, CD45 is highly expressed in all hematopoietic lineages at most stages of development, suggesting that this phosphatase also regulates other cells and processes. During development, as well as in clinical transplantation, hematopoietic stem cells (HSCs) migrate through the circulation to the bone marrow (BM) and repopulate it. Migration and development of HSCs are multi-step processes, which are tightly regulated by interplays between cytokines, chemokines, adhesion molecules and proteolytic enzymes; however, not all the related key players have been fully identified. In this study we explored the involvement of CD45 in hematopoietic cell motility and development, its role in cytokine signaling and adhesion interactions. The roles of CD45 were tested by either blocking the function of CD45 expressed on human and murine HSCs by neutralizing antibodies, or by utilizing CD45 knockout (KO) mice. Our results show that blocking CD45 completely prevented homing of human CD34+ enriched progenitors to the murine BM, consequently abrogating repopulation in transplanted NOD/SCID mice. In addition, CD45 neutralization impaired the capacity of human progenitors to migrate in-vitro towards a gradient of SDF-1, suggesting a cross-talk between SDF-1 and CD45 signaling. Furthermore, blocking CD45 on human G2 cells (pre-B ALL line) activated signaling pathways, including an increase in phosphorylation of MAP kinase and the tyrosine kinase Pyk2, which are involved in cell adhesion and migration. This activation enhanced cell adhesion to stromal and endothelial cell lines in-vitro. Importantly, blockage of CD45 in human progenitors resulted in cell aggregation, which inhibited cell proliferation and impaired the capacity to form colonies in-vitro. In an additional set of experiments we tested the role of CD45 in cell mobilization. In-vivo studies in normal mice demonstrated that neutralization of CD45 function inhibited the release of mature leukocytes and progenitor cells from the BM to the circulation both under steady state conditions and in stress-induced recruitment by stimulation with G-CSF or LPS. More importantly, BM derived mononuclear cells from CD45KO mice displayed a significant reduction in in-vivo homing and in-vitro migration compared to their wild type counterparts. Furthermore, G-CSF induced mobilization was impaired in CD45KO mice, and accompanied by a reduction in MMP-9 secretion from blood-derived leukocytes. Unexpectedly, the ability of CD45KO progenitors to form colonies in-vitro was impaired in the absence of in-vivo BM environment, documenting a crucial role for phosphatases such as CD45 in stem cell differentiation. Taken together, our findings demonstrate that functional CD45 is essential for human and murine hematopoietic cell migration and development (both homing and mobilization) by the regulation of adhesion and cytokine-induced signaling machineries. We suggest that in these cells CD45 may act as a negative regulator of major signaling pathways controlling adhesion properties and maintaining the balance between anchorage and release. We reveal a novel and dual role for CD45 in regulation of hematopoietic cell trafficking in general and progenitor cell motility and development in particular.

Description: Abstract 553 Egress of hematopoietic stem and progenitor cells (HSPC) from the bone marrow (BM) reservoir to the circulation in steady state conditions is a key requirement for normal hematopoiesis. It is dramatically enhanced by G-CSF-induced mobilization, which is widely used for clinical HSPC transplantation. An interplay between cytokines, chemokines (mainly SDF-1 (CXCL12) and its major receptor CXCR4), adhesion molecules, matrix metalloproteinases and neurotransmitters, tightly regulate HSPC egress and mobilization. Recent observations indicate an essential role for sphingolipids, and particularly sphingosine-1-phosphate (S1P) and its major receptor S1P1 in leukocyte trafficking in vivo. Furthermore, several pharmacological agents that target S1P and S1P1 attenuate development of autoimmune and cardiovascular diseases as well as cancer. Based on these findings, we hypothesized that HSPC motility, both in steady state and in stress-induced conditions, is regulated by S1P/S1P1 signaling. We found that cells expressing S1P1 receptor are mainly located near sinusoids in the murine BM, suggesting involvement of S1P/S1P1 axis in HSPC steady state egress. To identify the role of S1P1 in HSPC homeostatic release, we injected mice with the inhibitor FTY720 and discovered a significant decrease in primitive Sca-1+/c-Kit+/Lineage- (SKL) cell numbers in the peripheral blood along with their accumulation in the BM, 24 hr post a single i.p injection. To examine the S1P/S1P1 axis involvement in stress induced mobilization, we tested S1P levels following G-CSF administration. S1P concentrations were decreased in BM supernatants and increased in the peripheral blood, suggesting the formation of a gradient towards the blood, with a potential HSPC mobilization capacity. Accordingly, a 5-fold decreased transcription level of sphingosine kinase 1 (Sphk1, S1P producing enzyme) and a milder increased transcription level of sphingosine phosphatase 1 (SPP1, S1P degrading enzyme) were observed in the BM of G-CSF treated mice. These changes in both S1P modulating enzymes expression levels were mediated by mTOR signaling, independent of the PI3K pathway. Another effect of G-CSF mobilization was enhancing the percentage of BM HSPC expressing surface S1P1 receptor, which was abolished upon inhibition of mTOR by Rapamycin. These findings imply that the reduction in S1P BM levels enabled increased S1P1 receptor expression and HSPC recruitment to the blood. Co-injections of FTY720 with G-CSF revealed decreased numbers of primitive SKL and immature colony-forming cells in the blood, indicating reduced HSPC mobilization. Accordingly, administration of G-CSF to Sphk1 KO mice, which have low S1P plasma concentrations, led to decreased mobilization of primitive SKL cells and progenitors to the blood. We also investigated the cross talk between S1P/S1P1 and SDF-1/CXCR4 axes. Disruption of the S1P/S1P1 axis during G-CSF administration (by co-injections of FTY720 or by using Sphk1 KO mice) reduced HSPC mobilization however, BM mononuclear cells obtained from these mice exhibited enhanced migration to a gradient of SDF-1 in vitro. These results imply that SDF-1/CXCR4 activation is not sufficient for HSPC mobilization. Previously, we have shown that CXCR4 neutralizing antibodies co-administrated on days 4 and 5 of G-CSF treatment, significantly but not completely inhibited HSPC mobilization (Petit et al., Nat Immunol, 2002). Interestingly, such treatment in Sphk1 KO mice completely inhibited mobilization and increased primitive SKL cells in the BM. These results suggest that S1P/S1P1 axis has an important role in parallel to SDF-1/CXCR4 axis during stress-induced mobilization since inactivation of both pathways resulted in total abrogation of HSPC recruitment to the blood. Finally, we show that S1P can inhibit SDF-1 transcription in murine BM stromal cells via activation of the p38/Akt/mTOR signaling pathway. Since SDF-1 reduction in the BM is essential for HSPC mobilization, S1P-induced inhibition of its transcription allows the progenitor cells to detach and migrate. Taken together, our findings reveal involvement of S1P and its major receptor S1P1 in HSPC egress and stress-induced mobilization. These findings may help broaden our understanding regarding the mechanisms behind HSPC motility and thus improve clinical mobilization protocols and drug development based on targeting the S1P/S1P1 axis. Disclosures: No relevant conflicts of interest to declare.

Description: 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 S1P1, 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 S1P1 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/S1P1 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.

Description: Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

Description: A major limitation to clinical stem cell–mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34+ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1–mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34+ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1–induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34+/CD38-/low cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34+ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors. (Blood. 2004;103:2942-2949)

Description: Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor–induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell–derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1+/c-Kit+/Lin− cells and interfered with their enhanced chemotactic migration to stromal cell–derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met–mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.