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• W2005344557 abstract "The contribution of osteoclasts to hematopoietic stem/progenitor cell (HSPC) retention in the bone marrow is controversial. Studies of HSPC trafficking in osteoclast-deficient mice are limited by osteopetrosis. Here, we employed two non-osteopetrotic mouse models to assess the contribution of osteoclasts to basal and granulocyte colony-stimulating factor (G-CSF)-induced HSPC mobilization. We generated Rank−/− fetal liver chimeras using Csf3r−/− recipients to produce mice lacking G-CSF receptor expression in osteoclasts. Basal and G-CSF-induced HSPC mobilization was normal in these chimeras. We next acutely depleted osteoclasts in wild-type mice using the RANK ligand inhibitor osteoprotegerin. Marked suppression of osteoclasts was observed after a single injection of osteoprotegerin-Fc. Basal and G-CSF-induced HSPC mobilization in osteoprotegerin-Fc-treated mice was comparable to that in control mice. Together, these data indicate that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF. The contribution of osteoclasts to hematopoietic stem/progenitor cell (HSPC) retention in the bone marrow is controversial. Studies of HSPC trafficking in osteoclast-deficient mice are limited by osteopetrosis. Here, we employed two non-osteopetrotic mouse models to assess the contribution of osteoclasts to basal and granulocyte colony-stimulating factor (G-CSF)-induced HSPC mobilization. We generated Rank−/− fetal liver chimeras using Csf3r−/− recipients to produce mice lacking G-CSF receptor expression in osteoclasts. Basal and G-CSF-induced HSPC mobilization was normal in these chimeras. We next acutely depleted osteoclasts in wild-type mice using the RANK ligand inhibitor osteoprotegerin. Marked suppression of osteoclasts was observed after a single injection of osteoprotegerin-Fc. Basal and G-CSF-induced HSPC mobilization in osteoprotegerin-Fc-treated mice was comparable to that in control mice. Together, these data indicate that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF. Granulocyte-colony stimulating factor (G-CSF) is the agent most commonly used to mobilize hematopoietic stem/progenitor cells (HSPCs) from the bone marrow to blood. G-CSF induces HSPC mobilization, in large part, by suppressing CXCL12 production from bone marrow stromal cells [1Petit I. Szyper-Kravitz M. Nagler A. et al.G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4.Nat Immunol. 2002; 3 ([erratum appears in Nat Immunol. 2002;3:787]): 687-694Crossref PubMed Scopus (1137) Google Scholar, 2Levesque J.P. Hendy J. Takamatsu Y. Simmons P.J. Bendall L.J. Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide.J Clin Invest. 2003; 111: 187-196Crossref PubMed Scopus (635) Google Scholar, 3Semerad C.L. Christopher M.J. Liu F. et al.G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow.Blood. 2005; 106: 3020-3027Crossref PubMed Scopus (399) Google Scholar, 4Christopher M.J. Liu F. Hilton M.J. Long F. Link D.C. Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization.Blood. 2009; 114: 1331-1339Crossref PubMed Scopus (182) Google Scholar]. We recently reported that expression of the G-CSF receptor (G-CSFR) only in CD68+ monocytic-lineage cells is sufficient to induce HSPC mobilization by G-CSF [5Christopher M.J. Rao M. Liu F. Woloszynek J.R. Link D.C. Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice.J Exp Med. 2011; 208: 251-260Crossref PubMed Scopus (249) Google Scholar]. Within the monocytic lineage, the G-CSFR is expressed on monocytes, macrophage, myeloid dendritic cells, and osteoclasts. There is evidence linking macrophages with HSPC trafficking. Specifically, pharmacologic or genetic approaches that delete macrophages result in suppression of CXCL12 production from stromal cells and HSPC mobilization [6Chow A. Lucas D. Hidalgo A. et al.Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche.J Exp Med. 2011; 208: 261-271Crossref PubMed Scopus (578) Google Scholar, 7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar]. However, the contribution of osteoclasts to HSPC mobilization is less clear. Kollet and colleagues reported that activation of osteoclasts by injection of RANK ligand (RANKL) is associated with moderate HSPC mobilization [8Kollet O. Dar A. Shivtiel S. et al.Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells.Nat Med. 2006; 12: 657-664Crossref PubMed Scopus (620) Google Scholar]. Likewise, Cho et al. reported that activation of osteoclasts with RANKL enhanced mobilization in response to inflammatory stress [9Cho K.A. Joo S.Y. Han H.S. Ryu K.H. Woo S.Y. Osteoclast activation by receptor activator of NF-kappaB ligand enhances the mobilization of hematopoietic progenitor cells from the bone marrow in acute injury.Int J Mol Med. 2010; 26: 557-563PubMed Google Scholar]. These data suggest that osteoclasts promote HSPC egress from the bone marrow. Conversely, there are data suggesting that osteoclasts may contribute to HSPC retention in the bone marrow. Inhibition of osteoclast activity with bisphosphonate treatment is associated with a modest increase in HSPC mobilization by G-CSF [7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar, 10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar]. Moreover, Miyamoto and colleagues found that in several transgenic mouse lines with impaired osteoclast activity (specifically Csf1-, Fos-, or Rankl-deficient mice), G-CSF-induced HSPC mobilization is increased [10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar]. Each of these studies has certain limitations. Most notably, osteoclast deficiency is associated with osteopetrosis, extramedullary hematopoiesis, and constitutive HSPC mobilization, potentially confounding studies of HSPC trafficking. Here, we employed two non-osteopetrotic mouse models to assess the contribution of osteoclasts to basal and G-CSF-induced HSPC mobilization.MethodsMiceSex- and age-matched, wild-type Csf3r−/− mice [11Liu F. Wu H.Y. Wesselschmidt R. Komaga T. Link D.C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice.Immunity. 1996; 5: 491-501Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar] and Rank+/− mice [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar] on a C57BL/6 background were maintained under standard pathogen-free conditions according to methods approved by the Washington University Animal Studies Committee.Rank−/− fetal liver chimerasFetal liver cells were harvested from a Rank+/− mouse intercross at 13.5–14.5 days postconception. Genotyping was performed on fetal tissue to identify Rank−/− fetal livers [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]. Two million cells were injected retro-orbitally into lethally irradiated (1100 cGy) wild-type or Csf3r−/− 6- to 12-week-old mice.G-CSF and osteoprotegerin-FcSix-to eight-week-old male wild-type mice were used in these experiments. Recombinant human G-CSF (Amgen) was administered at a dose of 250 μg/kg/day subcutaneously for 7 days. Mice were analyzed 3 to 4 hours after the final cytokine dose. Recombinant osteoprotegerin-Fc (OPG-Fc) was a generous gift from Amgen. Mice were given a single subcutaneous injection of 100 μg of OPG-Fc or saline alone.Colony-forming cells assaysWe plated 10 μL blood, 5 × 104 nucleated spleen cells, or 2.0 × 104 nucleated bone marrow cells in 1.25 mL methylcellulose medium supplemented with a cocktail of recombinant cytokines (MethoCult 3434, StemCell Technologies, Vancouver, BC, Canada). Colonies were counted after 7 days of culture in a humidified chamber with 5% carbon dioxide at 37°C.Real-time quantitative reverse transcription polymerase chain reactionFemurs were flushed directly with TRIzol reagent (Invitrogen), and RNA was isolated according to the manufacturer's instructions. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the TaqMan One-step RT-PCR Master Mix Reagents Kit on a GeneAmp 5700 Sequence Detection System (Life Technologies, Grand Island, NY). TaqMan primer/probe mixes for cathepsin K (Mm00484039_m1), tartrate-resistant acid phosphatase (TRAP) (Mm00475698_m1), Csf3r (Mm00432735_m1), and β-actin (Mm00607939_s1) were obtained from Applied Biosystems.Flow cytometryThe following antibodies were used (all from eBiosciences, San Diego, CA, USA): Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3e (145-2C11), Ter-119 (TER-119), Sca-1 (D7), and c-kit (2B8). Cells were analyzed either on a FACScan or Gallios flow cytometer.TRAP stainingParaffin-embedded bone marrow sections were stained for TRAP and quantified by histomorphometry, as described previously [13Wu C.A. Pettit A.R. Toulson S. Grøndahl L. Mackie E.J. Cassady A.I. Responses in vivo to purified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) implanted in a murine tibial defect model.J Biomed Mater Res A. 2009; 91: 845-854Crossref PubMed Scopus (23) Google Scholar].StatisticsStatistical significance of differences was calculated using two-tailed Student t tests.Results and discussionTo generate adult mice with defective osteoclasts but without severe osteopetrosis, we first generated Rank−/− fetal liver chimeras (Fig. 1). RANK signaling is required for osteoclast development [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]; thus, we predicted that Rank−/− fetal liver chimeras would lack osteoclasts. However, osteoclast number was only modestly reduced in these mice (Fig. 1A–D). This is likely secondary to radio-resistant recipient osteoclasts and is consistent with a recent report indicating recipient macrophage expansion after transplantation, if donor macrophage development is compromised [14Hashimoto D. Chow A. Noizat C. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (1382) Google Scholar].To circumvent this problem, we transplanted Rank−/− (or Rank+/+) fetal liver cells into Csf3r−/− recipients (Fig. 1E). Because donor osteoclast development is blocked in the absence of RANK signaling, any remaining osteoclasts in these mice should lack the G-CSF receptor. Donor engraftment with Csf3r-sufficient cells was confirmed by measuring Csf3r mRNA expression in bone marrow cells 6–8 weeks after transplantation (Fig. 1F). Of note, the number of osteoclasts (as measured by bone marrow cathepsin K and TRAP expression) was comparable for Csf3r−/− recipients reconstituted with wild-type bone marrow and those reconstituted with Rank−/− bone marrow (Fig. 1G, H). As expected, these mice are not osteopetrotic, as evidenced by bone marrow histology (Supplementary Figure 1, online only, available at www.exphem.org) and normal bone marrow and spleen cellularity (Supplementary Figure 2, online only, available at www.exphem.org). At baseline, the numbers of bone marrow, peripheral blood, and spleen Kit+ Sca+ lineage− (KLS) cells and colony-forming units in cells (CFU-C) were similar in Rank−/− Csf3r−/− and Rank+/+ Csf3r−/− chimeras (Fig. 1I, J). Moreover, a similar robust mobilization response to G-CSF with both types of chimeras was observed.The Rank−/− Csf3r−/− chimera data indicate that G-CSF signaling in osteoclasts is not required for HSPC mobilization by G-CSF. However, it is possible that osteoclasts may regulate HSPC trafficking independent of the G-CSF receptor. To address this possibility, we acutely depleted osteoclasts by treating wild-type mice with OPG-Fc. OPG is a decoy receptor for RANKL [15Simonet W.S. Lacey D.L. Dunstan C.R. et al.Osteoprotegerin: A novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4299) Google Scholar]. Recombinant OPG-Fc is a chimeric molecule fusing the RANKL binding domain of OPG with the Fc portion of human immunoglobulin. OPG-Fc has a long circulating half-life, and treatment with OPG-Fc reversibly depletes osteoclasts in rats [16Ominsky M.S. Li X. Asuncion F.J. et al.RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.J Bone Miner Res. 2008; 23: 672-682Crossref PubMed Scopus (114) Google Scholar]. As expected, we observed a marked suppression of osteoclasts in wild-type mice after a single injection of OPG-Fc that persisted for at least 9 days. Cathepsin K and TRAP mRNA expression in the bone marrow decreased 50- and 8.4-fold, respectively (Fig. 2A, B ), and TRAP + cells were nearly absent (Fig. 2C, D and Supplementary Figure 3, online only, available at www.exphem.org) (number of osteoclasts per millimeter of bone surface: 3.5 ± 0.11 [saline treated] vs. 0.19 ± 0.02 [day 7 after OPG-Fc treatment], p < 0.001). Of note, OPG-Fc had no effect on the numbers of monocytes, macrophages, and myeloid dendritic cells (Supplementary Figure 4, online only, available at www.exphem.org). Osteoclast depletion by itself did not result in mobilization of KLS cells to blood or spleen (Fig. 2E). We next treated mice with OPG-Fc followed by G-CSF (Fig. 2F). Again, severe osteoclast suppression was observed in OPG-Fc-treated mice, whether or not G-CSF also was given (Fig. 2G). Importantly, G-CSF-induced mobilization of KLS cells and CFU-C to blood and spleen was similar in OPG-Fc treated and control mice (Fig. 2H, I).Figure 2Osteoclast ablation does not perturb basal or G-CSF-induced HSPC mobilization. (A, B) Mice were treated with a single dose of OPG-Fc at day 0. Osteoclast number was assessed by measuring bone marrow cathepsin K (A) or TRAP (B) mRNA expression relative to β-actin mRNA (n = 2 or 3 per time point). (C, D) Representative sections of trabecular bone stained for TRAP from untreated (C) or OPG-Fc treated (D) mice 7 days after treatment with a single dose of OPG-Fc. (E) Wild-type mice were given a single dose of OPG-Fc at day 0; the frequency of KLS cells in the peripheral blood, bone marrow, and spleen was assayed at the indicated time points. The last bar on each graph is based on mice treated with G-CSF alone for 5 days (n = 2–5 per time point). (F) Mice were treated with either a single dose of OPG-Fc, a 5-day course of G-CSF beginning 3 days after the OPG-Fc treatment, or a 5-day course of G-CSF alone. (G) Cathepsin K or TRAP mRNA levels in the bone marrow were measured on day 7. (H, I) Numbers of KLS cells (H) or CFU-C (I) in the peripheral blood, bone marrow, and spleen were measured at the conclusion of the G-CSF course (n = 2–5 per time point). CFU-C = colony-forming units in cells; G-CSF = granulocyte colony-stimulating factor; HSPC = hematopoietic stem/progenitor cell; ns = not significant; OPG = osteoprotegerin; TRAP = tartrate-resistant acid phosphatase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collectively, our data indicate that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF. Consequently, the increase in basal and G-CSF-induced HSPC mobilization observed in constitutive models of osteoclast deficiency is likely secondary to osteopetrosis. Of note, treatment with bisphosphonates augments HSPC mobilization by G-CSF in mice [7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar, 10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar], suggesting that off-target effects of these agents, rather than osteoclast suppression, are responsible for the enhanced mobilization response. Collectively, these data suggest that pharmacologic strategies to augment HSPC mobilization by disrupting osteoclast function are unlikely to be effective. Granulocyte-colony stimulating factor (G-CSF) is the agent most commonly used to mobilize hematopoietic stem/progenitor cells (HSPCs) from the bone marrow to blood. G-CSF induces HSPC mobilization, in large part, by suppressing CXCL12 production from bone marrow stromal cells [1Petit I. Szyper-Kravitz M. Nagler A. et al.G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4.Nat Immunol. 2002; 3 ([erratum appears in Nat Immunol. 2002;3:787]): 687-694Crossref PubMed Scopus (1137) Google Scholar, 2Levesque J.P. Hendy J. Takamatsu Y. Simmons P.J. Bendall L.J. Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide.J Clin Invest. 2003; 111: 187-196Crossref PubMed Scopus (635) Google Scholar, 3Semerad C.L. Christopher M.J. Liu F. et al.G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow.Blood. 2005; 106: 3020-3027Crossref PubMed Scopus (399) Google Scholar, 4Christopher M.J. Liu F. Hilton M.J. Long F. Link D.C. Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization.Blood. 2009; 114: 1331-1339Crossref PubMed Scopus (182) Google Scholar]. We recently reported that expression of the G-CSF receptor (G-CSFR) only in CD68+ monocytic-lineage cells is sufficient to induce HSPC mobilization by G-CSF [5Christopher M.J. Rao M. Liu F. Woloszynek J.R. Link D.C. Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice.J Exp Med. 2011; 208: 251-260Crossref PubMed Scopus (249) Google Scholar]. Within the monocytic lineage, the G-CSFR is expressed on monocytes, macrophage, myeloid dendritic cells, and osteoclasts. There is evidence linking macrophages with HSPC trafficking. Specifically, pharmacologic or genetic approaches that delete macrophages result in suppression of CXCL12 production from stromal cells and HSPC mobilization [6Chow A. Lucas D. Hidalgo A. et al.Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche.J Exp Med. 2011; 208: 261-271Crossref PubMed Scopus (578) Google Scholar, 7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar]. However, the contribution of osteoclasts to HSPC mobilization is less clear. Kollet and colleagues reported that activation of osteoclasts by injection of RANK ligand (RANKL) is associated with moderate HSPC mobilization [8Kollet O. Dar A. Shivtiel S. et al.Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells.Nat Med. 2006; 12: 657-664Crossref PubMed Scopus (620) Google Scholar]. Likewise, Cho et al. reported that activation of osteoclasts with RANKL enhanced mobilization in response to inflammatory stress [9Cho K.A. Joo S.Y. Han H.S. Ryu K.H. Woo S.Y. Osteoclast activation by receptor activator of NF-kappaB ligand enhances the mobilization of hematopoietic progenitor cells from the bone marrow in acute injury.Int J Mol Med. 2010; 26: 557-563PubMed Google Scholar]. These data suggest that osteoclasts promote HSPC egress from the bone marrow. Conversely, there are data suggesting that osteoclasts may contribute to HSPC retention in the bone marrow. Inhibition of osteoclast activity with bisphosphonate treatment is associated with a modest increase in HSPC mobilization by G-CSF [7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar, 10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar]. Moreover, Miyamoto and colleagues found that in several transgenic mouse lines with impaired osteoclast activity (specifically Csf1-, Fos-, or Rankl-deficient mice), G-CSF-induced HSPC mobilization is increased [10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar]. Each of these studies has certain limitations. Most notably, osteoclast deficiency is associated with osteopetrosis, extramedullary hematopoiesis, and constitutive HSPC mobilization, potentially confounding studies of HSPC trafficking. Here, we employed two non-osteopetrotic mouse models to assess the contribution of osteoclasts to basal and G-CSF-induced HSPC mobilization. MethodsMiceSex- and age-matched, wild-type Csf3r−/− mice [11Liu F. Wu H.Y. Wesselschmidt R. Komaga T. Link D.C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice.Immunity. 1996; 5: 491-501Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar] and Rank+/− mice [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar] on a C57BL/6 background were maintained under standard pathogen-free conditions according to methods approved by the Washington University Animal Studies Committee.Rank−/− fetal liver chimerasFetal liver cells were harvested from a Rank+/− mouse intercross at 13.5–14.5 days postconception. Genotyping was performed on fetal tissue to identify Rank−/− fetal livers [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]. Two million cells were injected retro-orbitally into lethally irradiated (1100 cGy) wild-type or Csf3r−/− 6- to 12-week-old mice.G-CSF and osteoprotegerin-FcSix-to eight-week-old male wild-type mice were used in these experiments. Recombinant human G-CSF (Amgen) was administered at a dose of 250 μg/kg/day subcutaneously for 7 days. Mice were analyzed 3 to 4 hours after the final cytokine dose. Recombinant osteoprotegerin-Fc (OPG-Fc) was a generous gift from Amgen. Mice were given a single subcutaneous injection of 100 μg of OPG-Fc or saline alone.Colony-forming cells assaysWe plated 10 μL blood, 5 × 104 nucleated spleen cells, or 2.0 × 104 nucleated bone marrow cells in 1.25 mL methylcellulose medium supplemented with a cocktail of recombinant cytokines (MethoCult 3434, StemCell Technologies, Vancouver, BC, Canada). Colonies were counted after 7 days of culture in a humidified chamber with 5% carbon dioxide at 37°C.Real-time quantitative reverse transcription polymerase chain reactionFemurs were flushed directly with TRIzol reagent (Invitrogen), and RNA was isolated according to the manufacturer's instructions. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the TaqMan One-step RT-PCR Master Mix Reagents Kit on a GeneAmp 5700 Sequence Detection System (Life Technologies, Grand Island, NY). TaqMan primer/probe mixes for cathepsin K (Mm00484039_m1), tartrate-resistant acid phosphatase (TRAP) (Mm00475698_m1), Csf3r (Mm00432735_m1), and β-actin (Mm00607939_s1) were obtained from Applied Biosystems.Flow cytometryThe following antibodies were used (all from eBiosciences, San Diego, CA, USA): Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3e (145-2C11), Ter-119 (TER-119), Sca-1 (D7), and c-kit (2B8). Cells were analyzed either on a FACScan or Gallios flow cytometer.TRAP stainingParaffin-embedded bone marrow sections were stained for TRAP and quantified by histomorphometry, as described previously [13Wu C.A. Pettit A.R. Toulson S. Grøndahl L. Mackie E.J. Cassady A.I. Responses in vivo to purified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) implanted in a murine tibial defect model.J Biomed Mater Res A. 2009; 91: 845-854Crossref PubMed Scopus (23) Google Scholar].StatisticsStatistical significance of differences was calculated using two-tailed Student t tests. MiceSex- and age-matched, wild-type Csf3r−/− mice [11Liu F. Wu H.Y. Wesselschmidt R. Komaga T. Link D.C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice.Immunity. 1996; 5: 491-501Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar] and Rank+/− mice [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar] on a C57BL/6 background were maintained under standard pathogen-free conditions according to methods approved by the Washington University Animal Studies Committee. Sex- and age-matched, wild-type Csf3r−/− mice [11Liu F. Wu H.Y. Wesselschmidt R. Komaga T. Link D.C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice.Immunity. 1996; 5: 491-501Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar] and Rank+/− mice [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar] on a C57BL/6 background were maintained under standard pathogen-free conditions according to methods approved by the Washington University Animal Studies Committee. Rank−/− fetal liver chimerasFetal liver cells were harvested from a Rank+/− mouse intercross at 13.5–14.5 days postconception. Genotyping was performed on fetal tissue to identify Rank−/− fetal livers [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]. Two million cells were injected retro-orbitally into lethally irradiated (1100 cGy) wild-type or Csf3r−/− 6- to 12-week-old mice. Fetal liver cells were harvested from a Rank+/− mouse intercross at 13.5–14.5 days postconception. Genotyping was performed on fetal tissue to identify Rank−/− fetal livers [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]. Two million cells were injected retro-orbitally into lethally irradiated (1100 cGy) wild-type or Csf3r−/− 6- to 12-week-old mice. G-CSF and osteoprotegerin-FcSix-to eight-week-old male wild-type mice were used in these experiments. Recombinant human G-CSF (Amgen) was administered at a dose of 250 μg/kg/day subcutaneously for 7 days. Mice were analyzed 3 to 4 hours after the final cytokine dose. Recombinant osteoprotegerin-Fc (OPG-Fc) was a generous gift from Amgen. Mice were given a single subcutaneous injection of 100 μg of OPG-Fc or saline alone. Six-to eight-week-old male wild-type mice were used in these experiments. Recombinant human G-CSF (Amgen) was administered at a dose of 250 μg/kg/day subcutaneously for 7 days. Mice were analyzed 3 to 4 hours after the final cytokine dose. Recombinant osteoprotegerin-Fc (OPG-Fc) was a generous gift from Amgen. Mice were given a single subcutaneous injection of 100 μg of OPG-Fc or saline alone. Colony-forming cells assaysWe plated 10 μL blood, 5 × 104 nucleated spleen cells, or 2.0 × 104 nucleated bone marrow cells in 1.25 mL methylcellulose medium supplemented with a cocktail of recombinant cytokines (MethoCult 3434, StemCell Technologies, Vancouver, BC, Canada). Colonies were counted after 7 days of culture in a humidified chamber with 5% carbon dioxide at 37°C. We plated 10 μL blood, 5 × 104 nucleated spleen cells, or 2.0 × 104 nucleated bone marrow cells in 1.25 mL methylcellulose medium supplemented with a cocktail of recombinant cytokines (MethoCult 3434, StemCell Technologies, Vancouver, BC, Canada). Colonies were counted after 7 days of culture in a humidified chamber with 5% carbon dioxide at 37°C. Real-time quantitative reverse transcription polymerase chain reactionFemurs were flushed directly with TRIzol reagent (Invitrogen), and RNA was isolated according to the manufacturer's instructions. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the TaqMan One-step RT-PCR Master Mix Reagents Kit on a GeneAmp 5700 Sequence Detection System (Life Technologies, Grand Island, NY). TaqMan primer/probe mixes for cathepsin K (Mm00484039_m1), tartrate-resistant acid phosphatase (TRAP) (Mm00475698_m1), Csf3r (Mm00432735_m1), and β-actin (Mm00607939_s1) were obtained from Applied Biosystems. Femurs were flushed directly with TRIzol reagent (Invitrogen), and RNA was isolated according to the manufacturer's instructions. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the TaqMan One-step RT-PCR Master Mix Reagents Kit on a GeneAmp 5700 Sequence Detection System (Life Technologies, Grand Island, NY). TaqMan primer/probe mixes for cathepsin K (Mm00484039_m1), tartrate-resistant acid phosphatase (TRAP) (Mm00475698_m1), Csf3r (Mm00432735_m1), and β-actin (Mm00607939_s1) were obtained from Applied Biosystems. Flow cytometryThe following antibodies were used (all from eBiosciences, San Diego, CA, USA): Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3e (145-2C11), Ter-119 (TER-119), Sca-1 (D7), and c-kit (2B8). Cells were analyzed either on a FACScan or Gallios flow cytometer. The following antibodies were used (all from eBiosciences, San Diego, CA, USA): Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3e (145-2C11), Ter-119 (TER-119), Sca-1 (D7), and c-kit (2B8). Cells were analyzed either on a FACScan or Gallios flow cytometer. TRAP stainingParaffin-embedded bone marrow sections were stained for TRAP and quantified by histomorphometry, as described previously [13Wu C.A. Pettit A.R. Toulson S. Grøndahl L. Mackie E.J. Cassady A.I. Responses in vivo to purified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) implanted in a murine tibial defect model.J Biomed Mater Res A. 2009; 91: 845-854Crossref PubMed Scopus (23) Google Scholar]. Paraffin-embedded bone marrow sections were stained for TRAP and quantified by histomorphometry, as described previously [13Wu C.A. Pettit A.R. Toulson S. Grøndahl L. Mackie E.J. Cassady A.I. Responses in vivo to purified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) implanted in a murine tibial defect model.J Biomed Mater Res A. 2009; 91: 845-854Crossref PubMed Scopus (23) Google Scholar]. StatisticsStatistical significance of differences was calculated using two-tailed Student t tests. Statistical significance of differences was calculated using two-tailed Student t tests. Results and discussionTo generate adult mice with defective osteoclasts but without severe osteopetrosis, we first generated Rank−/− fetal liver chimeras (Fig. 1). RANK signaling is required for osteoclast development [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]; thus, we predicted that Rank−/− fetal liver chimeras would lack osteoclasts. However, osteoclast number was only modestly reduced in these mice (Fig. 1A–D). This is likely secondary to radio-resistant recipient osteoclasts and is consistent with a recent report indicating recipient macrophage expansion after transplantation, if donor macrophage development is compromised [14Hashimoto D. Chow A. Noizat C. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (1382) Google Scholar].To circumvent this problem, we transplanted Rank−/− (or Rank+/+) fetal liver cells into Csf3r−/− recipients (Fig. 1E). Because donor osteoclast development is blocked in the absence of RANK signaling, any remaining osteoclasts in these mice should lack the G-CSF receptor. Donor engraftment with Csf3r-sufficient cells was confirmed by measuring Csf3r mRNA expression in bone marrow cells 6–8 weeks after transplantation (Fig. 1F). Of note, the number of osteoclasts (as measured by bone marrow cathepsin K and TRAP expression) was comparable for Csf3r−/− recipients reconstituted with wild-type bone marrow and those reconstituted with Rank−/− bone marrow (Fig. 1G, H). As expected, these mice are not osteopetrotic, as evidenced by bone marrow histology (Supplementary Figure 1, online only, available at www.exphem.org) and normal bone marrow and spleen cellularity (Supplementary Figure 2, online only, available at www.exphem.org). At baseline, the numbers of bone marrow, peripheral blood, and spleen Kit+ Sca+ lineage− (KLS) cells and colony-forming units in cells (CFU-C) were similar in Rank−/− Csf3r−/− and Rank+/+ Csf3r−/− chimeras (Fig. 1I, J). Moreover, a similar robust mobilization response to G-CSF with both types of chimeras was observed.The Rank−/− Csf3r−/− chimera data indicate that G-CSF signaling in osteoclasts is not required for HSPC mobilization by G-CSF. However, it is possible that osteoclasts may regulate HSPC trafficking independent of the G-CSF receptor. To address this possibility, we acutely depleted osteoclasts by treating wild-type mice with OPG-Fc. OPG is a decoy receptor for RANKL [15Simonet W.S. Lacey D.L. Dunstan C.R. et al.Osteoprotegerin: A novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4299) Google Scholar]. Recombinant OPG-Fc is a chimeric molecule fusing the RANKL binding domain of OPG with the Fc portion of human immunoglobulin. OPG-Fc has a long circulating half-life, and treatment with OPG-Fc reversibly depletes osteoclasts in rats [16Ominsky M.S. Li X. Asuncion F.J. et al.RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.J Bone Miner Res. 2008; 23: 672-682Crossref PubMed Scopus (114) Google Scholar]. As expected, we observed a marked suppression of osteoclasts in wild-type mice after a single injection of OPG-Fc that persisted for at least 9 days. Cathepsin K and TRAP mRNA expression in the bone marrow decreased 50- and 8.4-fold, respectively (Fig. 2A, B ), and TRAP + cells were nearly absent (Fig. 2C, D and Supplementary Figure 3, online only, available at www.exphem.org) (number of osteoclasts per millimeter of bone surface: 3.5 ± 0.11 [saline treated] vs. 0.19 ± 0.02 [day 7 after OPG-Fc treatment], p < 0.001). Of note, OPG-Fc had no effect on the numbers of monocytes, macrophages, and myeloid dendritic cells (Supplementary Figure 4, online only, available at www.exphem.org). Osteoclast depletion by itself did not result in mobilization of KLS cells to blood or spleen (Fig. 2E). We next treated mice with OPG-Fc followed by G-CSF (Fig. 2F). Again, severe osteoclast suppression was observed in OPG-Fc-treated mice, whether or not G-CSF also was given (Fig. 2G). Importantly, G-CSF-induced mobilization of KLS cells and CFU-C to blood and spleen was similar in OPG-Fc treated and control mice (Fig. 2H, I).Collectively, our data indicate that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF. Consequently, the increase in basal and G-CSF-induced HSPC mobilization observed in constitutive models of osteoclast deficiency is likely secondary to osteopetrosis. Of note, treatment with bisphosphonates augments HSPC mobilization by G-CSF in mice [7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar, 10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar], suggesting that off-target effects of these agents, rather than osteoclast suppression, are responsible for the enhanced mobilization response. Collectively, these data suggest that pharmacologic strategies to augment HSPC mobilization by disrupting osteoclast function are unlikely to be effective. To generate adult mice with defective osteoclasts but without severe osteopetrosis, we first generated Rank−/− fetal liver chimeras (Fig. 1). RANK signaling is required for osteoclast development [12Dougall W.C. Glaccum M. Charrier K. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google Scholar]; thus, we predicted that Rank−/− fetal liver chimeras would lack osteoclasts. However, osteoclast number was only modestly reduced in these mice (Fig. 1A–D). This is likely secondary to radio-resistant recipient osteoclasts and is consistent with a recent report indicating recipient macrophage expansion after transplantation, if donor macrophage development is compromised [14Hashimoto D. Chow A. Noizat C. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (1382) Google Scholar]. To circumvent this problem, we transplanted Rank−/− (or Rank+/+) fetal liver cells into Csf3r−/− recipients (Fig. 1E). Because donor osteoclast development is blocked in the absence of RANK signaling, any remaining osteoclasts in these mice should lack the G-CSF receptor. Donor engraftment with Csf3r-sufficient cells was confirmed by measuring Csf3r mRNA expression in bone marrow cells 6–8 weeks after transplantation (Fig. 1F). Of note, the number of osteoclasts (as measured by bone marrow cathepsin K and TRAP expression) was comparable for Csf3r−/− recipients reconstituted with wild-type bone marrow and those reconstituted with Rank−/− bone marrow (Fig. 1G, H). As expected, these mice are not osteopetrotic, as evidenced by bone marrow histology (Supplementary Figure 1, online only, available at www.exphem.org) and normal bone marrow and spleen cellularity (Supplementary Figure 2, online only, available at www.exphem.org). At baseline, the numbers of bone marrow, peripheral blood, and spleen Kit+ Sca+ lineage− (KLS) cells and colony-forming units in cells (CFU-C) were similar in Rank−/− Csf3r−/− and Rank+/+ Csf3r−/− chimeras (Fig. 1I, J). Moreover, a similar robust mobilization response to G-CSF with both types of chimeras was observed. The Rank−/− Csf3r−/− chimera data indicate that G-CSF signaling in osteoclasts is not required for HSPC mobilization by G-CSF. However, it is possible that osteoclasts may regulate HSPC trafficking independent of the G-CSF receptor. To address this possibility, we acutely depleted osteoclasts by treating wild-type mice with OPG-Fc. OPG is a decoy receptor for RANKL [15Simonet W.S. Lacey D.L. Dunstan C.R. et al.Osteoprotegerin: A novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4299) Google Scholar]. Recombinant OPG-Fc is a chimeric molecule fusing the RANKL binding domain of OPG with the Fc portion of human immunoglobulin. OPG-Fc has a long circulating half-life, and treatment with OPG-Fc reversibly depletes osteoclasts in rats [16Ominsky M.S. Li X. Asuncion F.J. et al.RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.J Bone Miner Res. 2008; 23: 672-682Crossref PubMed Scopus (114) Google Scholar]. As expected, we observed a marked suppression of osteoclasts in wild-type mice after a single injection of OPG-Fc that persisted for at least 9 days. Cathepsin K and TRAP mRNA expression in the bone marrow decreased 50- and 8.4-fold, respectively (Fig. 2A, B ), and TRAP + cells were nearly absent (Fig. 2C, D and Supplementary Figure 3, online only, available at www.exphem.org) (number of osteoclasts per millimeter of bone surface: 3.5 ± 0.11 [saline treated] vs. 0.19 ± 0.02 [day 7 after OPG-Fc treatment], p < 0.001). Of note, OPG-Fc had no effect on the numbers of monocytes, macrophages, and myeloid dendritic cells (Supplementary Figure 4, online only, available at www.exphem.org). Osteoclast depletion by itself did not result in mobilization of KLS cells to blood or spleen (Fig. 2E). We next treated mice with OPG-Fc followed by G-CSF (Fig. 2F). Again, severe osteoclast suppression was observed in OPG-Fc-treated mice, whether or not G-CSF also was given (Fig. 2G). Importantly, G-CSF-induced mobilization of KLS cells and CFU-C to blood and spleen was similar in OPG-Fc treated and control mice (Fig. 2H, I). Collectively, our data indicate that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF. Consequently, the increase in basal and G-CSF-induced HSPC mobilization observed in constitutive models of osteoclast deficiency is likely secondary to osteopetrosis. Of note, treatment with bisphosphonates augments HSPC mobilization by G-CSF in mice [7Winkler I.G. Sims N.A. Pettit A.R. et al.Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.Blood. 2010; 116: 4815-4828Crossref PubMed Scopus (582) Google Scholar, 10Miyamoto K. Yoshida S. Kawasumi M. et al.Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.J Exp Med. 2011; 208: 2175-2181Crossref PubMed Scopus (75) Google Scholar], suggesting that off-target effects of these agents, rather than osteoclast suppression, are responsible for the enhanced mobilization response. Collectively, these data suggest that pharmacologic strategies to augment HSPC mobilization by disrupting osteoclast function are unlikely to be effective. We thank Amgen for their generous gift of OPG-Fc. This work was supported by a grant from the National Institutes of Health : RO1 HL60772 (DCL). Conflict of interest disclosureThe authors declare no competing financial interests. The authors declare no competing financial interests. Supplementary dataSupplementary Figure 2Bone marrow and spleen cellularity in Rank−/− fetal liver chimeras. Wild-type or Rank−/− fetal liver cells were transplanted into 6-week-old male recipients. Illustrated are total mononuclear cell counts for the bone marrow and spleen. Data represent the mean ± SEM of n = 5 or 6 mice. BM = bone marrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 3TRAP staining of OPG-Fc treated mice bone marrow. Wild-type mice were treated with a single dose of OPG-Fc and analyzed 7 days later. Shown are representative bone sections stained for tartrate-resistant acid phosphatase from untreated (left) or OPG-Fc treated (right) mice. OPG = osteoprotegerin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 4OPG-Fc ablates osteoclasts, but not macrophages, myeloid dendritic cells, or monocytes. (A) Mice were treated with a single dose of OPG-Fc, and 3 days later, bone marrow cathepsin K mRNA expression relative to β-actin mRNA was measured. At the same time, the numbers of (B) macrophages, (C) myeloid dendritic cells, and (D) monocytes in the bone marrow were measured by flow cytometry. Macrophages were defined as F4/80+ CX3CR1− cells; myeloid dendritic cells as CX3CR1+MHC IIhiCD11chi cells, and monocytes as CX3CR1+MHC IIlo/neg CD11clo/neg Gr-1intCD115+ cells. Data represent the mean ± SEM of n = 3 mice per cohort. *p < 0.05. OPG = osteoprotegerin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)" @default.
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