Mechanisms of G-CSF-Mediated Hematopoietic Stem and Progenitor Mobilization

Mechanisms of G-CSF-mediated hematopoietic stem and progenitor mobilization

AM Greenbaum and DC Link

Department of Medicine, Washington University School of Medicine, St Louis, MO, USA

Under normal conditions, the great majority of hematopoietic G-CSF mobilizes HSPCs through a hematopoietic stem/progenitors cells (HSPCs) reside in the bone marrow. The intermediate number of HSPCs in the circulation can be markedly increased in response to a number of stimuli, including hematopoietic growth factors, myeloablative agents and environmental stres- In addition to mature neutrophils and monocytes, the G-CSF ses such as infection. The ability to ‘mobilize’ HSPCs from receptor (G-CSFR) is expressed on a broad range of HSPCs the bone marrow to the blood has been exploited clinically including HSCs.8 There are also reports of G-CSFR expression to obtain HSPCs for stem cell transplantation and, more on endothelial cells.9 To determine the cellular target(s) of recently, to stimulate therapeutic angiogenesis at sites of G-CSF required for HSPC mobilization, a series of bone marrow tissue ischemia. Moreover, there is recent interest in the use chimeras were generated (Figure 1). Wild-type mice reconsti- of mobilizing agents to sensitize leukemia and other hemato- À/À poietic malignancies to cytotoxic agents. Key to optimizing tuted with G-CSFR (Csf3r) bone marrow cells failed to À/À clinical mobilizing regimens is an understanding of the mobilize HSPCs in response to G-CSF. In contrast, G-CSFR fundamental mechanisms of HSPC mobilization. In this review, mice reconstituted with wild-type bone marrow displayed we discuss recent advances in our understanding of the normal HSPC mobilization by G-CSF. This experiment showed mechanisms by which granulocyte colony-stimulating factor that G-CSFR signaling in hematopoietic cells but not stromal (G-CSF), the prototypical mobilizing agent, induces HSPC cells is required for HSPC mobilization by G-CSF. As the mobilization. Leukemia (2011) 25, 211–217; doi:10.1038/leu.2010.248; G-CSFR is expressed on HSPCs, the simplest model suggests that published online 16 November 2010 G-CSF directly acts on HSPC to induce their mobilization. Keywords: stem cell mobilization, hematopoietic stem and However, strongly arguing against this model, in mixed bone progenitor cell, G-CSF, CXCL12, CXCR4, monocytes marrow chimeras containing both wild-type and G-CSFRÀ/À HSPCs, both types of cells were mobilized equally after G-CSF Introduction treatment. Collectively, these studies support a model in which G-CSF acts on a hematopoietic intermediary that, in Granulocyte colony-stimulating factor (G-CSF) is the most turn, generates trans-acting signal(s) that lead to HSPC commonly used mobilizing agent for stem cell transplantation. mobilization.10 The nature of these trans-acting signals is still G-CSF-mobilized hematopoietic stem/progenitors cells (HSPCs) an open question. F are associated with more rapid engraftment and in some The hematopoietic cell type(s) that mediate HSPC mobiliza- F circumstances superior overall survival in comparison to tion by G-CSF have not been defined. The G-CSFR is expressed unmanipulated bone marrow,1 although they may be associated 2,3 on neutrophils, monocytes/macrophages, HSPCs, and a subset with increased rates of relapse in autologous grafts. There are of B lymphocytes and NK cells. Although mobilization in several features of G-CSF-induced HSPC mobilization that need lymphocyte-deficient mouse models is normal,11 depletion of to be considered when developing a model of mobilization. neutrophils using an antibody against Gr1 (Ly6C/G) results in First, HSPC mobilization by G-CSF is delayed, with peak levels reduced HSPC mobilization by G-CSF.12 However, Gr1 is of circulating HSPCs achieved after 5–7 days of treatment. expressed on both neutrophils and a subset of monocytes; thus, Second, a broad spectrum of HSPCs are mobilized including a role for reduced monocytes in this phenotype is possible. To hematopoietic stem cells (HSCs) as well as committed myeloid, more directly address this issue, we generated transgenic mice SPOTLIGHT megakaryocytic and erythroid progenitors. Finally, mobilized that express the G-CSFR only in monocyte lineage cells HSPCs have characteristic phenotypic features that are distinct by driving G-CSFR expression using the CD68 promoter on a from HSPCs that reside in the bone marrow under steady-state G-CSFRÀ/À background (unpublished data). Despite being conditions. Most notably, relative to bone marrow HSPCs, neutropenic at baseline, these mice display normal HSPC a higher percentage of mobilized blood cells are in the G0 or G1 mobilization by G-CSF, demonstrating that G-CSFR signals in phase of the cell cycle,4 and the expression of VLA-4, c-kit5 and 6 monocyte lineage cells are sufficient to induce the mobilization CXCR4 on their cell surface is reduced. HSPCs are selectively cascade while normal neutrophil counts are not necessary. mobilized after the M phase of the cell cycle, providing a Moreover, a recent report from Winkler et al.13 demonstrate that potential explanation for the preponderance of HSPCs in the G0 7 depletion of monocyte lineage cells using a suicide gene or or G1 phase of the cell cycle in the blood. clodronate-loaded liposomes results in mobilization of HSPCs. Monocyte lineage cells in the bone marrow include mono- Correspondence: Dr DC Link, Division of Oncology, Washington cytes/macrophages, osteoclasts and myeloid dendritic cells. University School of Medicine, Washington University in St Louis, Kollet et al.14 reported that activation of osteoclasts by injection Campus Box 8007, 660 South Euclid Avenue, St Louis, MO 63110, of RANK ligand was associated with moderate HSPC mobiliza- USA. E-mail: [email protected] tion. Conversely, inhibition of osteoclasts, either genetically by Received 25 June 2010; revised 2 September 2010; accepted 14 knocking out PTPe or by injecting mice with calcitonin, blunts September 2010; published online 16 November 2010 the mobilization response to G-CSF. Of note, osteoclasts Mechanisms of G-CSF mediated HSPC mobilization AM Greenbaum and DC Link 212

SPOTLIGHT Figure 1 G-CSF mobilizes HSPCs through a hematopoietic intermediate. A series of bone marrow transplants were conducted to determine if the target of G-CSF was derived from hematopoietic cells or the bone marrow stroma. Wild-type HSPCs are represented in black; G-CSFR-deficient HSPCs are represented in white. (a) Wild-type mice mobilize normally, but (b) G-CSFR-deficient mice do not mobilize with G-CSF. (c) Wild-type mice transplanted with G-CSFR-deficient marrow fail to mobilize with G-CSF while (d) G-CSFR-deficient mice transplanted with wild-type marrow mobilize normally, suggesting that expression of G-CSFR on the hematopoietic compartment is necessary for HSPCs to mobilize while stromal expression of the G-CSFR is not. (e) Expression of the G-CSFR on HSPCs is not necessary for their mobilization. Wild-type mice were transplanted with a mixture of wild-type and G-CSFR-deficient marrow to create a mixed chimera with approximately 50% wild-type and 50% G-CSFR- deficient marrow. When mobilized with G-CSF, both the wild-type and G-CSFR-deficient HSPCs mobilize equally.

produce the protease cathepsin K, which can cleave CXCL12 (VCAM-1) has a major role in anchoring HSPCs to bone marrow in vitro. Together, these data suggest that osteoclasts may be the stromal cells31 and regulating HSPC trafficking between the target cell that initiates HSPC mobilization by G-CSF. However, marrow and peripheral sites.32 VLA-4 on HSPCs tethers them to other studies indicate that osteoclasts may actually inhibit VCAM-1-expressing cells, such as sinusoid endothelium and mobilization, as mice administered bisphosphonates exhibit stromal reticular cells33 and also fibronectin34 in the extra- increased mobilization in response to G-CSF,15 even with cellular matrix. Conditional deletion of VLA-4 shows that this newer generation bisphosphonates, which deplete osteoclast integrin is required for normal HSPC activity.35 Similarly, numbers.13 genetic ablation of VCAM-1 leads to constitutive mobilization, that is enhanced with G-CSF.36 Moreover, antibodies to VCAM-1 or VLA-4 mobilize HSPCs in both mice37 and C-kit/KitL axis humans.38 Of potential clinical relevance, treatment with a small molecule inhibitor of VLA-4 (BIO5192) strongly mobilizes C-kit receptor (often simply called ‘c-kit’)Ftogether with kit HPSC in mice.39 G-CSF has been reported to induce proteases ligand (kitL, also known as stem cell factor or steel factor)Fis an that cleave VCAM-1.40-41 However, studies in protease-deficient important molecule that regulates HSPCs. All HSCs express high mice demonstrated that this cleavage is not necessary levels of c-kit,16 and kitL has a crucial role in promoting their for mobilization.27 Thus, while the VCAM-1/VLA-4 axis has a quiescence17 and self-renewal.18 G-CSF induces the production major role in HSPC homeostasis, its role in G-CSF-mediated of proteases that cleave c-kit19 and kitL,20 releasing both as mobilization is unclear. soluble forms. The importance of the disruption of the c-kit/kitL axis to G-CSF-induced mobilization is controversial. In support of an important role, mobilized HSPC express lower levels of CXCR4/CXCL12 axis c-kit than bone marrow resident HPSC,21 and soluble levels of c-kit correlate with HSPC yield from the peripheral blood.22 Together with its major receptor CXCR4, the chemokine Moreover, disruption of c-kit signaling by the administration of CXCL12, also known as stromal-derived growth factor-1 (SDF-1), soluble c-kit induces modest HPSC mobilization and also has a crucial role in regulating HSPC trafficking, homing42 and augments mobilization in response to G-CSF.23 On the other maintenance.43–44 Its major receptor CXCR4 is expressed hand, stimulation of c-kit signaling by administration of kitL throughout the hematopoietic lineage from HSCs to mature induces robust HPSC mobilization24 and c-kit-deficient mice cells. CXCL12 also has an additional receptor, CXCR7,45 which have reduced G-CSF-induced mobilization.25,26 In addition, is believed to act as a decoy receptor to sequester the mice deficient in the protease matrix metalloproteinase 9 ligand;46,47 however, CXCR7 does not appear to have a major (MMP-9), which is thought to mediate kitL cleavage, mobilize role in hematopoiesis.48 Additionally, there are several normally in most strains of mice.27–30 low-affinity ligands of CXCR4, such as trefoil factor 249 and defensin50 but their significance in hematopoiesis is unclear. There is considerable evidence showing that CXCR4 signaling VCAM-1/VLA-4 axis provides a key retention signal for HSPCs in the bone marrow. CXCL12 is constitutively expressed at high levels in the bone Together with its major ligand very late antigen 4 (VLA-4, also marrow and is a potent chemoattractant for HSPCs.51,52 In mice known as a4b1 integrin), vascular cell adhesion molecule 1 lacking CXCL12.53 or CXCR4,54 there is a failure of the

Leukemia Mechanisms of G-CSF mediated HSPC mobilization AM Greenbaum and DC Link 213 migration of HSPC from the fetal liver to the bone marrow, and with the death of half of all osteoblasts because of apoptosis.80 CXCR4À/À bone marrow chimeras exhibit constitutive mobiliza- Moreover, the surviving osteoblasts reduce their expression of tion.55–56 Finally, treatment of humans or mice with a CXCR4 CXCL12 mRNA by approximately one-half.60 Interestingly, antagonist results in rapid HSPC mobilization.57–59 osteoblast suppression is a common feature of HSPC mobiliza- Numerous lines of evidence suggest that the domi- tion by other cytokines, including Flt3 ligand and kitL.56 Though nant mechanism by which G-CSF induces HSPC mobilization alterations in osteoblast lineage cells are likely to be a key step is through suppression of the CXCL12/CXCR4 axis. While in G-CSF-induced mobilization, the mechanisms by which this G-CSF treatment leads to an early, transient increase in leads to HSPC egress from the bone marrow remain to be CXCL12 production,59 prolonged treatment causes a elucidated. progressive decrease in CXCL12 mRNA60 in the bone marrow with a highly correlated concurrent drop in CXCL12 protein.59–61 Indeed, the level of CXCL12 is inversely correlated Neutrophil-derived proteases are induced after G-CSF with the degree of mobilization, with the lowest levels of treatment CXCL12 mRNA or protein correlating with maximal mobilization.11,59,60 G-CSF administration also is associated with After G-CSF treatment, bone marrow myeloid cells decreased CXCR4 expression on mobilized HSPC.6,61 Direct release a wide number of proteases such as MMP-9, evidence for the requirement for CXCR4 signaling in HSPC cathepsin G and neutrophil elastase,41 thus shifting the bone mobilization comes from the study of CXCR4À/À bone marrow marrow to a more proteolytic environment. These proteases chimeras. These mice, while displaying constitutive mobiliza- were suggested to cause mobilization by cleaving a variety of tion, do not further mobilize HSPC in response to G-CSF HSPC supporting molecules, such as kitL,81 VCAM-1,40 but maintain the ability to further mobilize with a VLA-4 CXCL1261,64 and its receptor CXCR4.61 Serine protease antagonist.56 inhibitors known as serpins have also been implicated in Numerous pathways have been shown to modulate mobiliza- mobilization. After G-CSF, the levels of serpin1 and serpin3 are tion either by altering baseline CXCL12 production or modulat- reduced in the marrow,82 thus making the marrow environment ing CXCR4 responsiveness. Pharmacologically inhibiting or more proteolytic. In fact, administration of serpin1a inhibits genetically reducing bone marrow synthesis of catecholamines interleukin (IL)-8-mediated mobilization,83 although the role blunts mobilization by G-CSF,11 most likely by regulating bone serpins play in G-CSF-mediated mobilization has yet to be marrow stromal cell production of CXCL12.62 Cell surface defined. molecules can also modulate CXCL12 responsiveness. For The contribution of neutrophil proteases to G-CSF-induced example, CD26 (DPP4, dipeptidylpeptidase 4) and carboxypepti- HSPC mobilization is controversial and is confounded by dase M are proteases present on the surface HSPCs that can different groups using different strains of mice. Depletion of cleave CXCL12, thereby inhibiting CXCR4 signaling.63,64 neutrophils84 or administration of anti-MMP-9 antibodies85 Pharmacologic or genetic inhibition of CD26 is associated with inhibits the rapid mobilization seen in response to the increased CXCR4 signaling and impaired G-CSF-induced HSPC chemokine IL-8. Moreover, one report suggested that MMP-9- mobilization.65 Similarly, platelet endothelial cell adhesion deficient mice have a mobilization defect in response to molecule-1 (PECAM-1/CD31) is an adhesion molecule expressed G-CSF.81 Similarly, systemic administration of inhibitors to MMP-9 on both stromal and hematopoietic cells. Mice lacking PECAM-1 or neutrophil elastase inhibits G-CSF-mediated mobilization.12,59 exhibit constitutive mobilization, perhaps because of reduced However, the role that proteases have in mobilization was HSPC responsiveness to CXCL12.66 Finally, a recent report from thrown into question when it was shown that MMP-9-deficient Tesio et al.67 showed that G-CSF induces hepatocyte growth mice mobilize normally in response to IL-8,27 and other groups factor/c-Met signaling in HSPC, potentially contributing to were unable to reproduce the mobilization defect previously mobilization by inhibiting the ability of HSPC to chemotax in seen in these mice using G-CSF.27–30 Moreover, mice lacking response to CXCL12. combinations of neutrophil elastase, cathepsin G, or MMP-9 mobilize normally even in the presence of a broad-spectrum metalloproteinase inhibitor.27 Collectively, these data suggest G-CSF results in a marked suppression of osteoblast lineage that while not absolutely required, neutrophil proteases may cells in the bone marrow augment G-CSF-induced HSPC mobilization in the appropriate genetic background. SPOTLIGHT The bone marrow microenvironment has a critical role in the maintenance of HSPCs. Primitive HSPCs preferentially localize in the bone marrow either to a perivascular localization68,69 or Regulation of HSPC mobilization by uPAR near the endosteum.70–73 There is strong evidence that osteoblast lineage cells are required to maintain the ‘endosteal The urokinase-type plasminogen activator receptor (uPAR) is stem cell niche’. Expansion of osteoblast lineage cells by genetic most well known for binding and activating urokinase plasmi- or pharmacologic means results in a concurrent expansion of nogen activator (uPA, commonly known as urokinase), HSCs.74–75 Conversely, ablation of mature osteoblasts using a which cleaves plasminogen to plasmin during the clotting suicide gene results in a loss of HSCs.76 Relevant to G-CSF- cascade. However, uPAR also binds to integrins and G-protein- induced HSPC mobilization, osteoblast lineage cells are a coupled receptors and initiates downstream signaling source of CXCL12 in the bone marrow.59,60,77 Fluorescence78 cascades to regulate cellular processes, such as cell adhesion and RNA in situ56 experiments suggest that, at baseline, CXCL12 and migration.86 A subset of HSPCs expresses uPAR on their is expressed by cells near the endosteum and scattered cells surface, which helps tether them to the niche via inter- throughout the bone marrow. However, on G-CSF treatment, actions with VLA-4.87 On G-CSF treatment, uPAR is cleaved expression of CXCL12 in the bone marrow decreases dramati- from the cell surface by plasmin87 and released into the cally.56,59,61 Strikingly, G-CSF also results in a profound extracellular space, wherein it is subsequently proteolytically suppression of mature osteoblast number and function,11,13,60,79 cleaved into smaller fragments.88 Selleri et al.89 suggest that

Leukemia Mechanisms of G-CSF mediated HSPC mobilization AM Greenbaum and DC Link 214 uPAR cleavage might contribute to HSPC mobilization by: (1) G-CSF mobilization model disrupting uPAR’s interaction with integrins; (2) inhibiting CXCR4 signaling by soluble uPAR fragments; and (3) inducing Figure 2 illustrates our working model for G-CSF-induced HSPC HPSC migration by soluble uPAR fragments. Consistent with mobilization. At baseline, osteoblast lineage cells express key this model, genetic or pharmacologic approaches leading to molecules that regulate HSPC function and retention in the bone increased plasmin activation (resulting in increased uPAR marrow, including CXCL12, VCAM-1 and kitL. In this model, cleavage) are associated with enhanced G-CSF-induced HPSC the mobilization cascade is initiated by G-CSF signaling in mobilization.90 monocyte lineage cells in the bone marrow. This results in the production or suppression of, as yet undeﬁned, transacting signals that result in the suppression of osteoblast lineage cells. The loss of osteoblast lineage cells disrupts key interactions Complement regulates mobilization regulating HSPC function, including CXCR4, VLA-4 and c-kit signaling. In particular, disruption of the CXCR4/CXCL12 Recent studies implicate the complement system in the axis appears to have a dominant role in HSPC mobilization by regulation of HSPC trafﬁcking in the bone marrow. The G-CSF. A number of other pathways are activated during G-CSF complement component C3 is synthesized by bone marrow administration that may augment HSPC mobilization by stromal cells,91 and on G-CSF administration, C3 is cleaved into modulating CXCR4 signaling, including complement and uPAR C3a and C3b.92 HSPCs express the receptor for C3a, and activation. binding of C3a augments their chemotaxis to CXCL12. This

SPOTLIGHT suggests that C3a may negatively regulate mobilization. Indeed, mice lacking either C3a or its receptor have enhanced mobilization in response to G-CSF, as do mice treated with an Open questions/future directions inhibitor of C3a receptor.93 In addition to C3, other complement components such as C5 are also acted on by G-CSF adminis- The precise subtype of monocyte lineage cells in the bone tration. In contrast to C3, C5 likely positively regulates marrow and the nature of the trans-acting signals that regulate mobilization, as C5-deficient mice mobilize poorly,93 and osteoblast lineage cells are currently unknown. The identifica- activation of C5 correlates with better mobilization in humans.94 tion of these trans-acting factors may provide novel targets to This is not a direct effect of C5 on HSPCs, as they do not express modulate HSC function and/or bone metabolism. The subtype of C5a receptor, but it may be related to its effect on neutrophil osteoblast lineage cells that regulates HSPC trafficking also has activation.95 In addition, formation of the membrane attack not been defined. Indeed, there is evidence that mature complex is believed to lead to the lysis of red blood cells, which osteoblasts may not be the major source of CXCL12 protein in releases the lipid sphingosine-1-phosphate, which is a potent the marrow. Sugiyama et al.69 reported the presence of CXCL12- chemoattractant for HSPCs.96 How complement is activated abundant reticular cells that were scattered throughout the bone during G-CSF is not entirely clear, but evidence suggests that it marrow and were in direct contact with most HSCs.69 Moreover may be via antibody activation of complement. Immunoglobu- Meńdez-Ferrer et al.97 recently reported a novel population of lin-deficient mice strains including RAG2À/À, SCID or Jh mice nestin-expressing mesenchymal stem cells, which regulate mobilize poorly in response to G-CSF, a phenotype rescued by HSCs, produce CXCL12 and are regulated by G-CSF. Studies injected immunoglobulin G.93 However, this mechanism is not to characterize the effect of G-CSF administration on different entirely proven, as others have shown that immunoglobulin- bone marrow stromal cell populations may be informative. deficient RAG1À/À and IL-7RaÀ/À mice mobilize normally with It is also unclear how disruption of CXCR4 signaling in HPSCs G-CSF.11 leads to their egress from the bone marrow. We recently

Figure 2 Model of G-CSF-induced HSPC mobilization. At baseline (left panel), osteoblast lineage cells produce key molecules such as CXCL12, VLA-4 and c-kit that retain HSPCs in the bone marrow. G-CSF signaling in monocytic cells results in the production (or suppression) of currently undeﬁned transacting signals that, in turn, result in the suppression of osteoblast lineage cells (right panel). The net effect of this signaling cascade is the disruption of key interactions that regulate HSPC function, most notably CXCL4/CXCL12 signaling. Mobilization is also augmented by other pathways which alter CXCR4 signaling, such as complement and uPAR activation.

Leukemia Mechanisms of G-CSF mediated HSPC mobilization AM Greenbaum and DC Link 215 reported that expression of the CXCL2 chemokine by bone 10 Liu F, Poursine-Laurent J, Link D. Expression of the G-CSF receptor marrow endothelial cells may provide a second signal directing on hematopoietic progenitor cells is not required for their neutrophil migration from the bone marrow into the circula- mobilization by G-CSF. Blood 2000; 95: 3025–3031. tion.98 This suggested a ‘tug-of-war’ model in which CXCL2 11 Katayama Y, Battista M, Kao W, Hidalgo A, Peired A, Thomas S et al. Signals from the sympathetic nervous system regulate expression by endothelial cells (directing release) and CXCL12 hematopoietic stem cell egress from bone marrow. Cell 2006; expression by endosteal osteoblasts (directing retention) anta- 124: 407–421. gonistically regulate neutrophil release from the bone marrow. 12 Pelus LM, Bian H, King AG, Fukuda S. Neutrophil-derived MMP-9 Whether similar signals direct HPSC egress is currently mediates synergistic mobilization of hematopoietic stem and unknown. Of note, Ratajczak et al.96 recently reported that progenitor cells. Blood 2004; 103: 110–119. high levels of sphingosine-1-phosphate generated in the blood 13 Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F et al. Bone marrow macrophages maintain hematopoietic stem during G-CSF administration by complement activation may cell (HSC) niches and their depletion mobilizes HSC. Blood 2010, contribute to HPSC migration into the blood. doi: 10.1182/blood-2009-11-253534. In summary, though our understanding of the molecular 14 Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y mechanisms contributing to HSPC mobilization by G-CSF has et al. Osteoclasts degrade endosteal components and promote improved, there remain many important unanswered questions. mobilization of hematopoietic progenitor cells. Nat Med 2006; 12: Future research in this area has the potential of advancing 657–664. 15 Takamatsu Y, Simmons PJ, Moore RJ, Morris HA, To LB, Levesque J. our understanding of the stem cell niche, regulation of HSC Osteoclast-mediated bone resorption is stimulated during short-term function/trafficking and bone metabolism and may lead to novel administration of granulocyte colony-stimulating factor but is not strategies to increase HSPC mobilization yields and enhance responsible for hematopoietic progenitor cell mobilization. Blood HSPC homing and engraftment following transplantation. 1998; 92: 3465–3473. 16 Orlic D, Fischer R, Nishikawa S, Nienhuis AW, Bodine DM. Purification and characterization of heterogeneous pluripotent Conflict of interest hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 1993; 82: 762–770. The authors declare no conflict of interest. 17 Thoren LA, Liuba K, Bryder D, Nygren JM, Jensen CT, Qian H et al. Kit regulates maintenance of quiescent hematopoietic stem cells. J Immunol 2008; 180: 2045–2053. 18 Miller CL, Rebel VI, Helgason CD, Lansdorp PM, Eaves CJ. Acknowledgements Impaired steel factor responsiveness differentially affects the detection and long-term maintenance of fetal liver hematopoietic We would like to thank Mahil Rao for his generous contribution of stem cells in vivo. Blood 1997; 89: 1214–1223. Figure 1. 19 Le´vesque J, Hendy J, Winkler IG, Takamatsu Y, Simmons PJ. Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from References the surface of hematopoietic progenitor cells. Exp Hematol 2003; 31: 109–117. 1 Stem Cell Trialists’ Collaborative Group. Allogeneic peripheral 20 Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR blood stem-cell compared with bone marrow transplantation in the et al. Recruitment of stem and progenitor cells from the bone management of hematologic malignancies: an individual patient marrow niche requires MMP-9 Mediated release of kit-ligand. data meta-analysis of nine randomized trials. J Clin Oncol 2005; Cell 2002; 109: 625–637. 23: 5074–5087. 21 Roberts MM, Swart BW, Simmons PJ, Basser RL, Begley CG, To LB. 2 Gorin NC, Labopin M, Reiffers J, Milpied N, Blaise D, Witz F et al. Prolonged release and c-kit expression of haemopoietic precursor Higher incidence of relapse for acute myelocytic leukemia patients cells mobilized by stem cell factor and granulocyte colony infused with higher doses of CD34+ cells from leukapheresis stimulating factor. Br J Haematol 1999; 104: 778–784. products autografted during the first remission. Blood 2010; 116: 22 Ishiga K, Kawatani T, Tajima F, Omura H, Nanba E, Kawasaki H. 3157–3162. Serum-soluble c-kit levels during mobilization of peripheral blood 3 Gorin N, Labopin M, Blaise D, Reiffers J, Meloni G, Michallet M stem cells correlate with stem cell yield. Int J Hematol 2000; 72: et al. Higher incidence of relapse with peripheral blood rather than 186–193. marrow as a source of stem cells in adults with acute myelocytic 23 Nakamura Y, Tajima F, Ishiga K, Yamazaki H, Oshimura M, leukemia autografted during the first remission. J Clin Oncol 2009; Shiota G et al. Soluble c-kit receptor mobilizes hematopoietic 27: 3987–3993. stem cells to peripheral blood in mice. Exp Hematol 2004; 32: 4 Uchida N, He D, Friera AM, Reitsma M, Sasaki D, Chen B et al. 390–396. SPOTLIGHT The unexpected G0/G1 cell cycle status of mobilized hemato- 24 Molineux G, Migdalska A, Szmitkowski M, Zsebo K, Dexter TM. poietic stem cells from peripheral blood. Blood 1997; 89: The effects on hematopoiesis of recombinant stem cell factor 465–472. (ligand for c-kit) administered in vivo to mice either alone or in 5 Bellucci R, De Propris MS, Buccisano F, Lisci A, Leone G, Tabilio combination with granulocyte colony-stimulating factor. Blood A et al. Modulation of VLA-4 and L-selectin expression on normal 1991; 78: 961–966. CD34+ cells during mobilization with G-CSF. Bone Marrow 25 Papayannopoulou T, Priestley GV, Nakamoto B. Anti-VLA4/ Transplant 1999; 23: 1–8. VCAM-1-induced mobilization requires cooperative signaling 6 Dlubek D, Drabczak-Skrzypek D, Lange A. Low CXCR4 mem- through the kit/mkit ligand pathway. Blood 1998; 91: 2231–2239. brane expression on CD34+ cells characterizes cells mobilized to 26 Roberts AW, Foote S, Alexander WS, Scott C, Robb L, Metcalf D. blood. Bone Marrow Transplant 2005; 37: 19–23. Genetic influences determining progenitor cell mobilization and 7 Wright DE, Cheshier SH, Wagers AJ, Randall TD, Christensen JL, leukocytosis induced by granulocyte colony-stimulating factor. Weissman IL. Cyclophosphamide/granulocyte colony-stimulating Blood 1997; 89: 2736–2744. factor causes selective mobilization of bone marrow hematopoie- 27 Levesque J, Liu F, Simmons P, Betsuyaku T, Senior R, Pham C et al. tic stem cells into the blood after M phase of the cell cycle. Blood Characterization of hematopoietic progenitor mobilization in 2001; 97: 2278–2285. protease-deficient mice. Blood 2004; 104: 65–72. 8 Demetri GD, Griffin JD. Granulocyte colony-stimulating factor and 28 Robinson SN, Pisarev VM, Chavez JM, Singh RK, Talmadge JE. Use its receptor. Blood 1991; 78: 2791–2808. of matrix metalloproteinase (MMP)-9 knockout mice demonstrates 9 Bussolino F, Ziche M, Wang JM, Alessi D, Morbidelli L, Cremona that MMP-9 activity is not absolutely required for G-CSF or Flt-3 O et al. In vitro and in vivo activation of endothelial cells by ligand-induced hematopoietic progenitor cell mobilization or colony-stimulating factors. J Clin Invest 1991; 87: 986–995. engraftment. Stem Cells 2003; 21: 417–427.

Leukemia Mechanisms of G-CSF mediated HSPC mobilization AM Greenbaum and DC Link 216 29 Papayannopoulou T, Priestley GV, Bonig H, Nakamoto B. The role poiesis in mice deﬁcient in the second CXCL12/SDF-1 receptor, of G-protein signaling in hematopoietic stem/progenitor cell CXCR7. Proc Natl Acad Sci USA 2007; 104: 14759–14764. mobilization. Blood 2003; 101: 4739–4747. 49 Dubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. 30 Robinson SN, Seina SM, Gohr JC, Sharp JG. Hematopoietic Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial progenitor cell mobilization by granulocyte colony-stimulating and lymphocytic cancer cell lines. J Biol Chem 2009; 284: factor and erythropoietin in the absence of matrix metalloprotei- 3650–3662. nase-9. Stem Cells Dev 2005; 14: 317–328. 50 Feng Z, Dubyak GR, Lederman MM, Weinberg A. Cutting edge: 31 Jung Y, Wang J, Havens A, Sun Y, Wang J, Jin T et al. Cell-to-cell human beta defensin 3Fa novel antagonist of the HIV-1 contact is critical for the survival of hematopoietic progenitor cells coreceptor CXCR4. J Immunol 2006; 177: 782–786. on osteoblasts. Cytokine 2005; 32: 155–162. 51 Wright D, Bowman E, Wagers A, Butcher E, Weissman I. 32 Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf Hematopoietic stem cells are uniquely selective in their migratory NS. The VLA4/VCAM-1 adhesion pathway deﬁnes contrasting response to chemokines. J Exp Med 2002; 195: 1145–1154. mechanisms of lodgement of transplanted murine hemopoietic 52 Aiuti A, Webb I, Bleul C, Springer T, Gutierrez-Ramos J. The progenitors between bone marrow and spleen. Proc Natl Acad chemokine SDF-1 is a chemoattractant for human CD34+ Sci USA 1995; 92: 9647–9651. hematopoietic progenitor cells and provides a new mechanism 33 Jacobsen K, Kravitz J, Kincade PW, Osmond DG. Adhesion to explain the mobilization of CD34+ progenitors to peripheral receptors on bone marrow stromal cells: in vivo expression of blood. J Exp Med 1997; 185: 111–120. vascular cell adhesion molecule-1 by reticular cells and sinusoidal 53 Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, endothelium in normal and gamma-irradiated mice. Blood 1996; Kitamura Y et al. Defects of B-cell lymphopoiesis and bone- 87: 73–82. marrow myelopoiesis in mice lacking the CXC chemokine PBSF/ 34 Williams DA, Rios M, Stephens C, Patel VP. Fibronectin and VLA- SDF-1. Nature 1996; 382: 635–638.

SPOTLIGHT 4 in haematopoietic stem cell-microenvironment interactions. 54 Zou Y, Kottmann A, Kuroda M, Taniuchi I, Littman D. Function of Nature 1991; 352: 438–441. the chemokine receptor CXCR4 in haematopoiesis and in 35 Priestley GV, Scott LM, Ulyanova T, Papayannopoulou T. cerebellar development. Nature 1998; 393: 595–599. Lack of alpha4 integrin expression in stem cells restricts 55 Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is competitive function and self-renewal activity. Blood 2006; 107: required for the retention of B lineage and granulocytic precursors 2959–2967. within the bone marrow microenvironment. Immunity 1999; 10: 36 Ulyanova T, Scott L, Priestley G, Jiang Y, Nakamoto B, Koni P et al. 463–471. VCAM-1 expression in adult hematopoietic and nonhematopoietic 56 Christopher MJ, Liu F, Hilton MJ, Long F, Link DC. Suppression of cells is controlled by tissue-inductive signals and reflects their CXCL12 production by bone marrow osteoblasts is a common and developmental origin. Blood 2005; 106: 86–94. critical pathway for cytokine-induced mobilization. Blood 2009; 37 Papayannopoulou T, Nakamoto B. Peripheralization of hemo- 114: 1331–1339. poietic progenitors in primates treated with anti-VLA4 integrin. 57 Liles WC, Broxmeyer HE, Rodger E, Wood B, Hu¨bel K, Cooper S Proc Natl Acad Sci USA 1993; 90: 9374–9378. et al. Mobilization of hematopoietic progenitor cells in healthy 38 Zohren F, Toutzaris D, Kla¨rner V, Hartung H, Kieseier B, Haas R. volunteers by AMD3100, a CXCR4 antagonist. Blood 2003; 102: The monoclonal anti-VLA-4 antibody natalizumab mobilizes 2728–2730. CD34+ hematopoietic progenitor cells in humans. Blood 2008; 58 Broxmeyer H, Orschell C, Clapp D, Hangoc G, Cooper S, Plett P 111: 3893–3895. et al. Rapid mobilization of murine and human hematopoietic 39 Ramirez P, Rettig MP, Uy GL, Deych E, Holt MS, Ritchey JK et al. stem and progenitor cells with AMD3100, a CXCR4 antagonist. BIO5192, a small molecule inhibitor of VLA-4, mobilizes J Exp Med 2005; 201: 1307–1318. hematopoietic stem and progenitor cells. Blood 2009; 114: 59 Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L 1340–1343. et al. G-CSF induces stem cell mobilization by decreasing bone 40 Levesque J, Takamatsu Y, Nilsson SK, Haylock DN, Simmons PJ. marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002; 3: Vascular cell adhesion molecule-1 (CD106) is cleaved by 687–694. neutrophil proteases in the bone marrow following hematopoietic 60 Semerad C, Christopher M, Liu F, Short B, Simmons P, Winkler I progenitor cell mobilization by granulocyte colony-stimulating et al. G-CSF potently inhibits osteoblast activity and CXCL12 factor. Blood 2001; 98: 1289–1297. mRNA expression in the bone marrow. Blood 2005; 106: 41 Le´vesque J, Hendy J, Takamatsu Y, Williams B, Winkler IG, 3020–3027. Simmons PJ. Mobilization by either cyclophosphamide or granu- 61 Levesque J, Hendy J, Takamatsu Y, Simmons P, Bendall L. locyte colony-stimulating factor transforms the bone marrow into a Disruption of the CXCR4/CXCL12 chemotactic interaction during highly proteolytic environment. Exp Hematol 2002; 30: 440–449. hematopoietic stem cell mobilization induced by GCSF or 42 Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T et al. cyclophosphamide. J Clin Invest 2003; 111: 187–196. Dependence of human stem cell engraftment and repopulation of 62 Meńdez-Ferrer S, Lucas D, Battista M, Frenette PS. Haematopoietic NOD/SCID mice on CXCR4. Science 1999; 283: 845–848. stem cell release is regulated by circadian oscillations. Nature 43 Kawabata K, Ujikawa M, Egawa T, Kawamoto H, Tachibana K, 2008; 452: 442–447. Iizasa H et al. A cell-autonomous requirement for CXCR4 in long- 63 Marquez-Curtis L, Jalili A, Deiteren K, Shirvaikar N, Lambeir A, term lymphoid and myeloid reconstitution. Proc Natl Acad Janowska-Wieczorek A. Carboxypeptidase M expressed by human Sci USA 1999; 96: 5663–5667. bone marrow cells cleaves the C-terminal lysine of stromal cell- 44 Cashman J, Clark-Lewis I, Eaves A, Eaves C. Stromal-derived factor derived factor-1alpha: another player in hematopoietic stem/ 1 inhibits the cycling of very primitive human hematopoietic cells progenitor cell mobilization? Stem Cells 2008; 26: 1211–1220. in vitro and in NOD/SCID mice. Blood 2002; 99: 792–799. 64 Christopherson KW, Cooper S, Broxmeyer HE. Cell surface 45 Balabanian K, Lagane B, Infantino S, Chow KYC, Harriague J, peptidase CD26/DPPIV mediates G-CSF mobilization of mouse Moepps B et al. The chemokine SDF-1/CXCL12 binds to and progenitor cells. Blood 2003; 101: 4680–4686. signals through the orphan receptor RDC1 in T lymphocytes. J Biol 65 Christopherson KW, Cooper S, Hangoc G, Broxmeyer HE. CD26 is Chem 2005; 280: 35760–35766. essential for normal G-CSF-induced progenitor cell mobilization 46 Hartmann TN, Grabovsky V, Pasvolsky R, Shulman Z, Buss EC, as determined by CD26À/À mice. Exp Hematol 2003; 31: Spiegel A et al. A crosstalk between intracellular CXCR7 and 1126–1134. CXCR4 involved in rapid CXCL12-triggered integrin activation but 66 Ross E, Freeman S, Zhao Y, Dhanjal T, Ross E, Lax S et al. A novel not in chemokine-triggered motility of human T lymphocytes and role for PECAM-1 (CD31) in regulating haematopoietic progenitor CD34+ cells. J Leukoc Biol 2008; 84: 1130–1140. cell compartmentalization between the peripheral blood and bone 47 Boldajipour B, Mahabaleshwar H, Kardash E, Reichman-Fried M, marrow. PLoS ONE 2008; 3: e2338. Blaser H, Minina S et al. Control of chemokine-guided cell 67 Tesio M, Golan K, Corso S, Giordano S, Schajnovitz A, Vagima Y migration by ligand sequestration. Cell 2008; 132: 463–473. et al. Enhanced c-Met activity promotes G-CSF induced mobiliza- 48 Sierro F, Biben C, Martıńez-Munõz L, Mellado M, Ransohoff RM, tion of hematopoietic progenitor cells via ROS signaling. Blood Li M et al. Disrupted cardiac development but normal hemato- 2010, doi:10.1182/blood-2009-06-230359.

Leukemia Mechanisms of G-CSF mediated HSPC mobilization AM Greenbaum and DC Link 217 68 Kiel M, Yilmaz O, Iwashita T, Terhorst C, Morrison S. SLAM family 85 Pruijt JFM, Fibbe WE, Laterveer L, Pieters RA, Lindley IJD, Paemen receptors distinguish hematopoietic stem and progenitor cells and L et al. Prevention of interleukin-8-induced mobilization of reveal endothelial niches for stem cells. Cell 2005; 121: 1109–1121. hematopoietic progenitor cells in rhesus monkeys by inhibitory 69 Sugiyama T, Kohara H, Noda M, Nagasawa T. Maintenance of the antibodies against the metalloproteinase gelatinase B (MMP-9). hematopoietic stem cell pool by CXCL12-CXCR4 chemokine Proc Natl Acad Sci USA 1999; 96: 10863–10868. signaling in bone marrow stromal cell niches. Immunity 2006; 25: 86 Blasi F, Carmeliet P. uPAR: a versatile signalling orchestrator. 977–988. Nat Rev Mol Cell Biol 2002; 3: 932–943. 70 Yoshimoto M, Shinohara T, Heike T, Shiota M, Kanatsu-Shinohara 87 Tjwa M, Sidenius N, Moura R, Jansen S, Theunissen K, Andolfo A M, Nakahata T. Direct visualization of transplanted hematopoietic et al. Membrane-anchored uPAR regulates the proliferation, marrow cell reconstitution in intact mouse organs indicates the presence of pool size, engraftment, and mobilization of mouse hematopoietic a niche. Exp Hematol 2003; 31: 733–740. stem/progenitor cells. J Clin Invest 2009; 119: 1008–1018. 71 Nilsson S, Johnston H, Coverdale J. Spatial localization of 88 Selleri C, Montuori N, Ricci P, Visconte V, Carriero MV, Sidenius transplanted hemopoietic stem cells: inferences for the localization N et al. Involvement of the urokinase-type plasminogen activator of stem cell niches. Blood 2001; 97: 2293–2299. receptor in hematopoietic stem cell mobilization. Blood 2005; 72 Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J 105: 2198–2205. et al. Live-animal tracking of individual haematopoietic stem/ 89 Selleri C, Montuori N, Ricci P, Visconte V, Baiano A, Carriero MV progenitor cells in their niche. Nature 2009; 457: 92–96. et al. In vivo activity of the cleaved form of soluble urokinase 73 Xie Y, Yin T, Wiegraebe W, He XC, Miller D, Stark D et al. receptor: a new hematopoietic stem/progenitor cell mobilizer. Detection of functional haematopoietic stem cell niche using Cancer Res 2006; 66: 10885–10890. real-time imaging. Nature 2009; 457: 97–101. 90 Tjwa M, Janssens S, Carmeliet P. Plasmin therapy enhances 74 Zhang J, Niu C, Ye L, Huang H, He X, Tong W et al. Identification mobilization of HPCs after G-CSF. Blood 2008; 112: 4048–4050. of the haematopoietic stem cell niche and control of the niche 91 Reca R, Mastellos D, Majka M, Marquez L, Ratajczak J, Franchini S size. Nature 2003; 425: 836–841. et al. Functional receptor for C3a anaphylatoxin is expressed 75 Calvi L, Adams G, Weibrecht K, Weber J, Olson D, Knight M et al. by normal hematopoietic stem/progenitor cells, and C3a Osteoblastic cells regulate the haematopoietic stem cell niche. enhances their homing-related responses to SDF-1. Blood 2003; Nature 2003; 425: 841–846. 101: 3784–3793. 76 Visnjic D, Kalajzic Z, Rowe D, Katavic V, Lorenzo J, Aguila H. 92 Ratajczak J, Reca R, Kucia M, Majka M, Allendorf DJ, Baran JT Hematopoiesis is severely altered in mice with an induced et al. Mobilization studies in mice deficient in either C3 or C3a osteoblast deficiency. Blood 2004; 103: 3258–3264. receptor (C3aR) reveal a novel role for complement in retention of 77 Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E hematopoietic stem/progenitor cells in bone marrow. Blood 2004; et al. Prospective identification, isolation, and systemic transplan- 103: 2071–2078. tation of multipotent mesenchymal stem cells in murine bone 93 Reca R, Cramer D, Yan J, Laughlin MJ, Janowska-Wieczorek A, marrow. J Exp Med 2009; 206: 2483–2496. Ratajczak J et al. A novel role of complement in mobilization: 78 Nagasawa T. The chemokine CXCL12 and regulation of HSC and immunodeficient mice are poor granulocyte-colony stimulating B lymphocyte development in the bone marrow niche. Adv Exp factor mobilizers because they lack complement-activating Med Biol 2007; 602: 69–75. immunoglobulins. Stem Cells 2007; 25: 3093–3100. 79 Froberg M, Garg U, Stroncek D, Geis M, McCullough J, Brown D. 94 Jalili A, Shirvaikar N, Marquez-Curtis L, Qiu Y, Korol C, Lee H Changes in serum osteocalcin and bone-specific alkaline phos- et al. Fifth complement cascade protein (C5) cleavage fragments phatase are associated with bone pain in donors receiving disrupt the SDF-1/CXCR4 axis: further evidence that innate granulocyte-colony-stimulating factor for peripheral blood stem immunity orchestrates the mobilization of hematopoietic stem/ and progenitor cell collection. Transfusion 1999; 39: 410–414. progenitor cells. Exp Hematol 2010; 38: 321–332. 80 Christopher MJ, Link DC. Granulocyte colony-stimulating factor 95 Lee HM, Wu W, Wysoczynski M, Liu R, Zuba-Surma EK, Kucia M induces osteoblast apoptosis and inhibits osteoblast differentiation. et al. Impaired mobilization of hematopoietic stem/progenitor cells J Bone Miner Res 2008; 23: 1765–1774. in C5-deficient mice supports the pivotal involvement of innate 81 Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR immunity in this process and reveals novel promobilization effects et al. Recruitment of stem and progenitor cells from the bone of granulocytes. Leukemia 2009; 23: 2052–2062. marrow niche requires MMP-9 mediated release of kit-ligand. 96 Ratajczak MZ, Lee H, Wysoczynski M, Wan W, Marlicz W, Cell 2002; 109: 625–637. Laughlin MJ et al. Novel insight into stem cell mobilization-plasma 82 Winkler IG, Hendy J, Coughlin P, Horvath A, Le´vesque J. Serine sphingosine-1-phosphate is a major chemoattractant that directs protease inhibitors serpina1 and serpina3 are down-regulated in the egress of hematopoietic stem progenitor cells from the bone bone marrow during hematopoietic progenitor mobilization. J Exp marrow and its level in peripheral blood increases during Med 2005; 201: 1077–1088. mobilization due to activation of complement cascade/membrane 83 van Pel M, van Os R, Velders GA, Hagoort H, Heegaard PMH, attack complex. Leukemia 2010; 24: 976–985. Lindley IJD et al. Serpina1 is a potent inhibitor of IL-8-induced 97 Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, hematopoietic stem cell mobilization. Proc Natl Acad Sci USA MacArthur BD, Lira SA et al. Mesenchymal and haematopoietic SPOTLIGHT 2006; 103: 1469–1474. stem cells form a unique bone marrow niche. Nature 2010; 466: 84 Pruijt JFM, Verzaal P, van Os R, de Kruijf EFM, van Schie MLJ, 829–834. Mantovani A et al. Neutrophils are indispensable for hematopoie- 98 Eash KJ, Greenbaum AM, Gopalan PK, Link DC. CXCR2 and tic stem cell mobilization induced by interleukin-8 in mice. CXCR4 antagonistically regulate neutrophil trafficking from murine Proc Natl Acad Sci USA 2002; 99: 6228–6233. bone marrow. J Clin Invest 2010; 120: 2423–2431.

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