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Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells... A r t i c l e Bone marrow CD169 macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche 1,2,3 2,3 2,4 Andrew Chow, Daniel Lucas, Andrés Hidalgo, 2,5 1,2 2,3 Simón Méndez-Ferrer, Daigo Hashimoto, Christoph Scheiermann, 2 1,2 2,3 Michela Battista, Marylene Leboeuf, Colette Prophete, 6 7 1,2 Nico van Rooijen, Masato Tanaka, Miriam Merad, 2,3 and Paul S. Frenette 1 2 Department of Gene and Cell Medicine and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461 4 5 Department of Epidemiology, Atherothrombosis, and Imaging and Cardiovascular Developmental Biology Department, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain Department of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, Netherlands Laboratory for Innate Cellular Immunity, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, 230-0045, Japan Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear hi lo phagocytes that include Gr-1 monocytes (MOs), Gr-1 MOs, and macrophages (M) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and M conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, spe- cific depletion of CD169 M, which spares BM MOs, was sufficient to induce HSC/ progenitor egress. M depletion also enhanced mobilization induced by a CXCR4 antago- nist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which M cross talk with the Nestin niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM M hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly. The BM is the preferred site for adult hemato- to mobilize HSCs/progenitors out of the BM CORRESPONDENCE Paul S. Frenette: poiesis. Transplantation of BM cells containing into the peripheral blood has allowed for effi - [email protected] hematopoietic stem cells (HSCs) and progeni - cient, less invasive HSC procurement in clinical OR tors has been a remarkable medical advance - stem cell transplantation. However, up to 30% Miriam Merad: [email protected] ment that allows for the replacement of the of patients previously treated with cytotoxic hematopoietic compartment after preparative anticancer therapies do not mobilize sufficient Abbreviations used: BMDM, regimens. HSCs are retained in perivascular numbers of stem cells using current protocols BM-derived M; BMEF, BM extracellular u fl id; DT, diphtheria niches that are distributed near osteoblasts and (Bensinger et al., 2009). toxin; G-CSF, granulocyte within the nonendosteal parenchyma (Kiel et al., colony-stimulating factor; HSC, 2005; Sugiyama et al., 2006; Lo Celso et al., hematopoietic stem cell; M, © 2010 Chow et al. This article is distributed under the terms of an Attribution– 2009; Méndez-Ferrer et al., 2010b). The ability macrophage; MO, monocyte; Noncommercial–Share Alike–No Mirror Sites license for the first six months after Q-PCR, quantitative RT-PCR; the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share SNS, sympathetic nervous system. A. Chow and D. Lucas and M. Merad and P. Frenette con- Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ tributed equally to this paper. by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 208 No. 2 261-271 www.jem.org/cgi/doi/10.1084/jem.20101688 The Journal of Experimental Medicine Sympathetic neural tone is crucial for both steady state population were positive for F4/80 (Fig. S1 A), CD11c, and (Méndez-Ferrer et al., 2008) and granulocyte colony - MHC class II (Fig. 1 C). – int + stimulating factor (G-CSF)–enforced (Katayama et al., 2006) The Gr-1 CD115 F4/80 fraction consisted of two release of HSCs/progenitors from the BM. Recent studies populations: eosinophils (Fig. 1 A, gate V) and a population of indicate that mesenchymal stem cells (MSCs), identified by mononuclear cells (gate IV), which could be discriminated by – int the expression of the intermediate filament protein Nestin, forward and side scatter characteristics. The Gr-1 CD115 + hi comprise a critical cellular constituent of the stem cell niche F4/80 subset, after exclusion of SSC eosinophils, comprised that is under the control of the sympathetic nervous system 2.6 ± 0.2% of BM cells and was the only population of BM (SNS; Méndez-Ferrer et al., 2010b). Because previous studies mononuclear phagocytes that showed expression of CD169 using G-CSF receptor–deficient mice showed that expression (Fig. 1 C). CD169, also known as sialoadhesin or SIGLEC-1, of the receptor on transplantable hematopoietic cells was r -e is a sialic acid binding molecule that was initially described quired for G-CSF–induced mobilization (Liu et al., 2000), we over two decades ago to have high activity on BM stromal have previously speculated that at least two distinct pathways, and lymph node M, but not on blood MO (Crocker and neural and hematopoietic, acted in concert to promote HSC/ Gordon, 1986). CD169 is recognized by the M antibody progenitor egress (Katayama et al., 2006). MOMA-1, which has long been used to stain M in the Hypothesizing that mononuclear phagocytes are crucial spleen, lymph nodes, lamina propria, Peyer’s patches, and CNS for stromal function of the BM, we sought to eliminate these (Oetke et al., 2006). Based on this marker, we will sb usequently – int populations to evaluate their contributions to HSC trac ffi king. call this cell population BM M. These Gr-1 CD115 + + Unexpectedly, we have found that BM macrophages (M) F4/80 CD169 M also expressed intermediate levels of did not promote the egress of HSCs/progenitors, but rather MHC class II, CD11c, and CD68; low expression of CD11b; contributed to the retention of HSCs in the BM by acting on and negligible expression of CX3CR1 (Fig. 1 C). Nestin MSCs. These data uncover a new role for the innate immune system in regulating stem cell niche functions. Depletion of BM mononuclear phagocytes correlates with HSC/progenitor egress and reduction in marrow CXCL12 RESULTS We first depleted BM MO/M  using clodronate liposome Phenotypic markers of BM mononuclear phagocytes injection. 14 h after clodronate liposome administration, BM hi Depletion of monocytes (MO) and/or M from the BM has M were reduced by 84% (Fig. 2, A and B), whereas Gr-1 lo been accomplished with injection of clodronate liposomes MO, Gr-1 MO, and total BM cellularity were reduced by 79, (Giuliani et al., 2001) and injection of the FK-binding pro - 88, and 24%, respectively, compared with PBS-treated animals tein dimerizer AP20187 in transgenic Maa fi mice (Burnett et al., (Fig. 2, A and C–E). Depletion of the noneosinophil Gr-1 + + 2004; Chang et al., 2008). Mafia mice have a Fas suicide/ CD115 F4/80 population with clodronate further supports apoptotic system driven by the CD115 (M-CSF receptor) pro- the conclusion that these CD169 cells are indeed M. moter. Previous phenotypic descriptions of BM M have We assessed the effect of BM MO/M  depletion on cir- exclusively relied on F4/80 expression (Hume et al., 1983; culating hematopoietic progenitors and found a marked in- Giuliani et al., 2001; Chang et al., 2008). However, this marker crease in colony-forming unit activity (4.5-fold; Fig. 2 F) and +  hi – + + is also expressed on BM neutrophils (Gr-1 CD115 ), Gr-1 Lineage Sca-1 c-kit (LSK) cells (6.2-fold; Fig. 2 G) in blood + + lo  + MO (Gr-1 CD115 ), Gr-1 MO (Gr-1 CD115 ; Gordon after mononuclear phagocyte depletion. Notably, circulating hi + – and Taylor, 2005), and eosinophils (SSC Siglec-F ; Zhang LSK Flk2 cells, enriched in long-term repopulating HSCs et al., 2004; Fig. S1). To distinguish among BM mononuclear (Christensen and Weissman, 2001), increased by 12.9-fold phagocytes and to elucidate their differential surface pheno - after clodronate treatment (Fig. 2 H). types, we purified different BM populations via cell sorting We next determined the effect of mononuclear phago - based on three markers: Gr-1 (Ly6C/G), CD115, and F4/80. cyte depletion in modulating the levels of CXCL12, a che - As expected, neutrophil granulocytes were homogenously mokine produced by stromal niche cells that is crucial in +  represented in the Gr-1 CD115 gate (Fig. 1 A, gate I) and retention and maintenance of HSCs/progenitors in the BM represented 49.6 ± 1.1% of the total BM nucleated cells. (Méndez-Ferrer and Frenette, 2007). Clodronate-induced de- In mice, there are two subsets of CD115 MO that differen - pletion of BM MO/M and HSC/progenitor mobilization tially express Gr-1 (Gordon and Taylor, 2005). In concordance, was associated with a 44% reduction in CXCL12 mRNA + + the Gr-1 CD115 portion (Fig. 1 A, gate II) represented a levels in total BM (Fig. 2 I) and a 40% reduction in CXCL12 homogenous population of MO (Fig. 1 B) that constituted protein in the BM extracellular u fl id (BMEF; Fig. 2 J). Because hi 9.8 ± 0.3% of the BM and is characterized as F4/80 BM MO/M do not produce CXCL12 (Fig. S2), these data hi int int – – – CD11b CD68 CX3CR1 MHCII CD11c CD169 suggest that MO/M depletion causes a reduction in CXCL12 hi (Fig. 1 C and Fig. S1 A), and will herein be termed Gr-1 expression by BM stromal cells. – + MO. The Gr-1 CD115 population (Fig. 1 A, gate III) rep- These results were further confirmed using other con - resenting 1.4 ± 0.1% of BM consisted of a population of ditional depletion models of mononuclear phagocytes, i-n hi int int MO (Fig. 1 B) characterized as CX3CR1 CD11b CD68 cluding transgenic mice expressing the diphtheria toxin – lo CD169 and will be termed Gr-1 MO. Subsets of this (DT) receptor under the CD11b promoter (CD11b-DTR; 262 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e Figure 1. Mononuclear phagocytes can be distinguished in the BM with Gr-1, CD115, and F4/80. (A) Gating strategy of BM mononuclear phagocytes. The Gr-1 pop- ulations were divided into a CD115 fraction comprised of neutrophils (I; left) and CD115 hi lo fraction of Gr-1 MOs (II; left). The Gr-1 fraction was further subdivided into two pop- + lo ulations (middle): CD115 Gr-1 MOs (III) and + int a F4/80 CD115 population, which can be hi subdivided into SSC eosinophils (V) and int/lo SSC M (IV). (B) Morphology of cell popula- tions I–IV (63× magnification; bars, 10 µm). (C) Overlay histograms show the differential expression of CD11b, CD11c, MHC class II, CX3CR1, CD68, and CD169 (blue line) among mononuclear phagocyte populations. Gray histograms represent isotype control. All results are representative of two indepen- dent experiments. 47% reduction in BMEF CXCL12 (Fig. S3 S). These data are consistent with a recent study also using liposo - mal clodronate and Mafia mice that demonstrated the association of BM mononuclear phagocyte depletion with HSC/progenitor egress (Winkler et al., 2010). Progenitor release cannot be explained by nonspecic fi cell death be- cause in vivo depletion of neutrophils hi and Gr-1 MO using anti–Gr-1 (Ly6G/ C) antibody or depletion of dendritic cells using DT administration in CD11c-DTR mice (Jung et al., 2002) did not lead to any progenitor mobiliza - Cailhier et al., 2005) and the Mafia mice (Burnett et al., 2004). tion (unpublished data). Collectively, these results sugge st that mononuclear phagocytes play a critical role in the rete-n In these studies, we generated BM chimeric mice in which wild-type recipients were reconstituted with BM cells iso - tion of HSCs/progenitors in the BM. lated from these transgenic animals to minimize toxicity of the depleting agents to nonhematopoietic organs. Treatment Mononuclear phagocytes regulate Nestin niche cells that of CD11b-DTR chimeric animals with DT reduced BM maintain HSCs in the BM M counts by >40% (Fig. S3 A) and MO subsets by >50% Recent studies have revealed that GFP expression, when (Fig. S3, B and C). Consistent with the expression of CD11b, driven by Nestin regulatory elements, identifies rare MSCs also on neutrophils, total BM cellularity was reduced by that form HSC niches (Méndez-Ferrer et al., 2010b). We ob- + + 40% in this model (Fig. S3 D). Concomitantly, progenitors served CD68 and CD169 cells throughout the BM and in circulating in blood were significantly increased by 1.6-fold the vicinity of rare Nestin MSC niche cells F ( ig. 3 A). To evalu- (Fig. S3 E) and BMEF CXCL12 was significantly reduced ate whether mononuclear phagocytes regulate Nestin niche (Fig. S3 F). AP20187-treated Mafia BM chimeras depleted cells, we used clodronate liposomes to deplete BM MO/M. BM M, MO, and total BM cells by 40, >80, and 30%, r- e Because the number of Nestin cells was unchanged by this spectively (Fig. S3, G–J). This was associated with an 6-fold treatment (Fig. S4 D), we assessed alterations in gene expres-   + increase in circulating progenitors (Fig. S3 K), and 46% re- sion in these cells. We sorted CD45 Ter119 Nestin and      duction in BM CXCL12 levels (Fig. S3 L). In nontransplanted CD45 Ter119 Nestin from the BM and CD45 Ter119   + Mafia mice, mobilization was even more robust; AP20187- CD31 Sca-1 CD51 osteoblasts (Semerad et al., 2005; treated Mafia animals exhibited a >80% reduction in MO/ Winkler et al., 2010) from the bone 14 h or 7 d after depletion     M (Fig. S3, M–P), 31% reduction in BM cellularity (Fig. S3 Q), (Fig. S4, A and B). Bone CD45 Ter119 CD31 Sca-1 >20-fold increase in circulating progenitors (Fig. S3R), and CD51 cells were conr fi med to be enriched in osteoblasts by JEM VOL. 208, February 14, 2011 263 Figure 2. Depletion of mononuclear phagocytes is associated with HSC/progenitor mobilization and CXCL12 reduction. (A–H) C57BL/6 mice were treated with PBS (blue) or clodronate-encapsulated liposomes (red). (A) Representative dot plots show the percentages hi lo of neutrophils, Gr-1 MOs, Gr-1 MOs, and M, as de- scribed in Fig. 1 A. (B–E) Absolute numbers of mono- nuclear phagocytes and total nucleated cells in the BM (n = 11). (F) Absolute numbers of colony-forming units in culture in the peripheral blood (CFU-C; n = 12–15).  + + (G–H) Enumeration of Lineage Sca-1 c-kit (LSK; G) and LSKFlk2 (H) cells in the peripheral blood (n = 9–10). B–H represents pooled data from at least three independent experiments. (I) RT-PCR analysis of Cxcl12 mRNA levels in total BM cells (n = 5 mice per group). Data representative of two independent experiments are shown. (J) CXCL12 levels in the BMEF. Data are pooled from three indepen- dent experiments. controls (Fig. S4, D–G). These results, together with the gene expression analyses (Fig. 3, B–E; and Fig. S5), suggest that BM mononuclear phagocytes play a role in HSC/progenito r retention by regulating maintenance of reten- tion gene expression specifically in Nestin niche cells, but not osteoblasts. Microarray expression analyses have shown that Nestin cells express high levels of Csf1 (Méndez-Ferrer et al., 2010b), a critical cytokine for M development and survival (Hamilton, 2008). To determine whether Nestin cells in the BM regulate mononuclear phagocyte num - bers, we depleted Nestin cells by administering ERT2 DT into tamoxifen-treated Nes-Cre /iDTR   high Osteocalcin expression compared with CD45 Ter119 animals (Méndez-Ferrer et al., 2010b). We found no di- f CD31 endothelial cells (Fig. S4 C). From these populations, we ference in BM M (Fig. S6 A) or MO (Fig. S6, B and C) in ERT2 performed quantitative real-time PCR (Q-PCR) of genes en- Nes-Cre /iDTR mice treated with tamoxifen and DT, coding molecules previously implicated in HSC maintenance compared with control iDTR animals. and retention (Cxcl12, Angpt1, Kitl, and Vcam1). These genes are highly expressed by Nestin cells and down-regulated BM M produce a protein factor that raises CXCL12 during G-CSF–induced mobilization or upon 3 adrenergic production by stromal cells in vitro signaling (Méndez-Ferrer et al., 2010b). As previously reported, The aforementioned results demonstrate a robust in vivo cor- Nestin cells from the BM had markedly higher expression relation between BM MO/M depletion, CXCL12 reduc- of the four retention genes, compared with the Nestin frac- tion, and HSC/progenitor mobilization. To further dissect the tion (Méndez-Ferrer et al., 2010b). Interestingly, we found effect of mononuclear phagocytes on stromal cell function, that the expression of Cxcl12, Angpt1, Kitl, and Vcam1 was we established long-term murine Dexter BM cultures con - 48-, 18-, 65-, and 35-fold higher, respectively, in Nestin cells sisting of an adherent stromal layer and attached hematopoi - in the BM compared with bone osteoblasts in the steady state etic cells (Dexter et al., 1977). Consistent with the in vivo (Fig. 3, B–E). Strikingly, we observed a 65–80% reduction in data, we found that clodronate liposome treatment of Dexter the expression of these four genes 14 h after treatment with cultures dramatically reduced the cellularity in the we lls, clodronate, compared with PBS liposome-treated animals especially among the adherent M, compared with PBS (Fig. 3, B–E). The reduced expression persisted at least until liposome-treated wells Fig. ( 4 A). The number of CD115 day 7 after treatment with clodronate (Fig. S5). In contrast, mononuclear phagocytes was indeed reduced by >50% the expression levels of these retention genes in sorted osteo - after liposomal clodronate treatment (Fig. 4 B). These changes blasts were unchanged 14 h and 7 d after clodronate treatment. in mononuclear phagocytes were associated with decreased In addition, the numbers of Nestin cells and osteoblasts 14 h stromal production of CXCL12 at 24 h (30%↓) and 72 h and 7 d after administration of clodronate were similar to (40%↓; Fig. 4 C). 264 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e Figure 3. Anatomical and functional relationships between mononuclear phagocytes and Nestin niche cells. (A) Distribution of BM CD68 (white) and CD169 (red) cells in 5-µm femoral sections of Nes-GFP mice. Dashed line demarcates separation of bone and BM. Images were acquired at 40× magnification (bar, 20 µm). Isotype control is shown in inset. (B–E) Relative expression of Cxcl12 (B), Angpt1 (C), Kitl (D), and Vcam1   + +  (E) in CD45 Ter119 Nestin (Nestin ) and CD45   Ter119 Nestin (Nestin ) fractions sorted from the BM and osteoblasts sorted from the bone 14 h after treat- ment with PBS- (blue bars) or clodronate-encapsulated (red bars) liposomes. Data are presented with the mean expression of the PBS liposome-treated Nestin fraction set at 100% and are representative of three indepen- dent experiments (n = 4–5). Data analyzed by one-way ANOVA/Newman-Keuls test. ***, P < 0.001. Depletion of BM CD169 M mobilizes HSCs/progenitors The MS-5/M co-culture experiments sug- gest that differentiated BM M , rather than MO, are the mononuclear phagocytes that promote HSC/progenitor retention. To test di- rectly whether BM M are promoting HSC/ progenitor retention in the BM, we took ad- vantage of the differential expression of CD169 between MO and M and mice expressing Because adherent M appear to interdigitate the stroma DTR under the endogenous CD169 promoter (Miyake et al., in Dexter cultures, we sought to determine the relative con - 2007). Treatment of heterozygous CD169-DTR mice with tribution of M and MO in stromal cell–mediated pro - DT-depleted M (Fig. 5, A and B), but not MO (Fig. 5, A, genitor retention. Thus, we used the murine stromal cell C, and D), in the BM. DT treatment was associated with a 3.5- line MS-5 to determine whether the addition of M or fold increase in circulating hematopoietic progenitors, a s M-synthesized products could affect the stromal niche. assessed by LSK cell enumeration (Fig. 5 E), and a 5.4-fold MS-5 stromal cells have been used as an appropriate in vitro increase in the stem cell-enriched LSKFlk2 fraction (Fig. 5 F). model in which to replicate in vivo modulation of CXCL12 Moreover, depletion of CD169 M from long-term Dexter production by noradrenergic signals (Méndez-Ferrer et al., 2008). culture resulted in a 42% reduction in the ability of BM When grown in medium conditioned by the M cell line stromal cells to produce CXCL12 (Fig. 5 G). Thus, CD169 RAW264.7 (Raschke et al., 1978), CXCL12 production by M in the BM promote stromal production of CXCL12, MS-5 was significantly increased ( Fig. S7 A), whereas no sig- and their specific depletion in vivo is sufficient to mobilize nificant difference was observed in medium conditioned by HSCs/progenitors. the myeloid myeloblast cell line M1 (Ralph et al., 19 83; Fig. S7B). Co-culture of BM-derived M (BMDM) or media Parallel and antagonistic roles of M and SNS in regulating conditioned by BMDM increased MS-5 production of HSC/progenitor release CXCL12, indicating that a secreted soluble factor induces The SNS is crucial in HSC/progenitor trafficking (Katayama CXCL12 up-regulation by MS-5 cells (Fig. 4, D and E). This et al., 2006; Méndez-Ferrer et al., 2008) where 3-adrenergic secreted factor was a protein, as digestion with Proteinase K receptor (3R) signaling plays a key role in circadian oscilla - abrogated the ability of M-conditioned medium to raise tions of HSC release, and both 2-adrenergic receptor (2R) CXCL12 production by M (Fig. 4 F). We have evaluated and 3R signaling cooperate in G-CSF–enforced egress putative candidate factors using antibodies or knockout ani- (Méndez-Ferrer et al., 2010a). M could act independently, mals for IGF-1, IL-1, TNF, or IL-10. However, the loss-of- or alternatively, through alterations in the sympathetic tone. function of any one of these factors did not alter the ability of Thus, to assess whether M operate through a distinct path- BMDM-conditioned medium to induce CXCL12 synthesis way, we examined whether clodronate treatment was capable (Fig. S7, C–F). These data suggest that BM M, through the of inducing mobilization in sympathectomized animals . secretion of a yet undefined protein factors, directly promote We found that mice chemically sympathectomized with the retention of HSCs/progenitors by raising CXCL12 pro- 6-hydroxydopamine (6OHDA) still exhibited signic fi ant pro - duction in BM niche cells. genitor mobilization (>7-fold) in response to clodronate JEM VOL. 208, February 14, 2011 265 Figure 4. M in culture promote CXCL12 production. (A) Morphology of ad- herent layer of Dexter culture 24 h after addi- tion of PBS or clodronate liposomes (10×; bars, 100 µm). (B) Adherent cells were ana- lyzed by flow cytometry for CD115 cells. (A and B) Representative data from two inde- pendent experiments are shown. (C) CXCL12 levels were assessed by ELISA at 24 h (left bars) and 72 h (right bars) after liposomal incubation. (D) CXCL12 levels were assessed 3 d after MS-5 cells were co-cultured with (M) or without (Ctrl) BMDM. (E) Levels of CXCL12 secreted from MS-5 cells after culture with medium conditioned by BMDM (M CM) or with control medium (Ctrl). (F) CXCL12 levels were measured after culture of MS-5 cells with control (Ctrl, blue) or M-conditioned (M CM, red) medium that was untreated began their recovery treatment; however, the absolute number of mobilized pro - (left two bars) or treated with Proteinase genitors did not reach the level of SNS-intact Clodronate- by day 7 after clodro- K (right two bars). (C–F) Representative data from at least two independent experiments treated mice (Fig. 6 A, mid-left bars). To evaluate this issue nate administration / are shown. using another model, we used mice dec fi ient in 2R (Adrb2 ) (unpublished data). treated with an antagonist to the 3R. Clodronate treatment Interestingly, levels was still able to cause HSC/progenitor mobilization (>3-fold) of LSK (Fig. 7 B) and LSKFlk2 (Fig. 7 C) inversely matched in these mice, but again HSCs/progenitors did not mobilize BM M counts. HSCs/progenitors were still elevated in the to the same level as wild type animals (Fig. 6 A, mid-right circulation 10 d after clodronate administration (4.2- and 3.2- bars). To dissect further the relative contribution of the 2R fold, respectively), and mobilization persisted at least until day and 3R in SNS promotion of HSC/progenitor release, we 16 (Fig. 7 C). These data further support the specific role of BM M, but not MO, in promoting the retention of HSCs/ evaluated the effect of M  depletion in mice singly lacking 2R (Fig. 6 A, right bars) or 3R (Fig. 6 B). Whereas clodro- progenitors in the BM. nate treatment led to a robust increase in circulating progeni - Because BM M promote HSC/progenitor retention, / tors in Adrb2 animals compared with wild-type animals we examined whether elimination of this population would / (Fig. 6 A, right bars), the response was blunted in Adrb3 enhance mobilization using the CXCR4 antagonist AMD3100 mice (Fig. 6 B). These data are consistent with previous stud- or G-CSF, both of which are clinically approved mobilizing ies demonstrating that 3R, but not 2R, signaling is critical agents. We found that clodronate treatment 14 h before harvest for physiological HSC/progenitor release (Méndez-Ferrer et al., doubled HSC/progenitor mobilization in AMD3100-treated 2008). The fact that M depletion can still mobilize HSCs— animals and resulted in a significant increase in the number of albeit at lower amplitude—when SNS signaling is disrupted, HSCs/progenitors mobilized by G-CSF (Fig. 7, D-E). To further assess the role of M in a situation where progenitor mobi- argues that the SNS and the BM M act through distinct parallel pathways. Thus, these data suggest antagonistic func - lization is suboptimal, we treated mice with G-CSF (Hidalg o tions of the autonomic nervous system and innate immunity et al., 2004) for 2 d, instead of 4 d. We found that CFU-C, LSK, in regulating the niche (Fig. 6 C), where the SNS promotes and LSKFlk2 cells in the peripheral blood mobilized by 2 d of egress by reducing the expression of key retention factors by G-CSF were increased 3.8-, 5.9-, and 9.3-fold (Fig. 7, F-H), the niche cell (Méndez-Ferrer et al., 2010b), and in contrast, respectively, in mice that were treated with clodronate lipo - BM M promotes the expression of these genes and HSC/ somes 14 h before harvest. Furthermore, we tested whether progenitor retention in the BM. depleting M 10 d before blood collection, rather than 14 h before collection, could synergize with 4 d G-CSF treatment. BM M depletion synergizes with enforced Indeed, when mice were preinfused with clodronate liposomes 10 d before harvest, they had 5.2-, 3.4-, 2.7- fold higher HSC/progenitor mobilization Because the expression of retention genes is still reduced in CFU-C, LSK, and LSKFlk2 in the peripheral blood, respec - the Nestin niche cells 7 d after clodronate treatment (Fig. S5), tively (Fig. 7, I, J, K). Thus, targeting BM M may be a novel we sought to determine the duration of M reduction and modality by which to enhance mobilization yields in patients. the kinetics of recovery. We found that BM M remain markedly (>90%) reduced 10 d after clodronate treatment DISCUSSION (Fig. 7 A). Recovery started by day 16 (58% reduction) and In this study, we sought to identify the role of BM mono- nuclear phagocytes in HSC/progenitor mobilization. Unex - clodronate-treated mice demonstrated no reduction in M counts by day 28. Alternatively, MO populations in the BM pectedly, we found that depletion of mononuclear phagocytes 266 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e Figure 5. Depletion of BM CD169 M, but not CD169 MOs, mobilizes HSCs/progenitors. (A–D) Wild-type (WT) or heterozygous CD169-DTR DTR/+ (CD169 ) mice were treated with DT. (A) Representa- tive dot plots show the percentages of BM mononuclear DTR/+ phagocytes in wild-type (top) or CD169 mice treated with DT (bottom). (B–D) Bar graphs depict the absolute numbers of mononuclear phagocytes (n = 6) in wild type DTR/+ mice and wild type or CD169 mice injected with DT.  + + (E–F) Bar graphs enumerate Lineage Sca-1 c-kit (LSK; E) and LSKFlk2 cells (F) per milliliter of blood in the same mice analyzed in B–D (n = 6). Data are pooled from two independent experiments. (G) CXCL12 levels were mea- sured 72 h after administration of PBS or 1 µg/ml DT into Dexter cultures plated from the BM of CD169-DTR animals (n = 3–4 wells). Representative data from two independent experiments are shown. of niche cells that express Nestin (Méndez-Ferrer et al., 2010b). Although osteoblasts have been proposed to represent a HSC niche, selective modulation of osteoblast numbers do not necessarily alter HSC numbers (Wilson and Trumpp, 2006; Kiel et al., 2007; Zhu et al., 2007), and the lack of osteoblasts in sites of extramedullary hematopoiesis suggest that they are dispensable to support HSCs. Recent studies have suggested that a more primitive pre- cursor of osteoblasts compose the stem cell niche (Méndez-Ferrer et al., 2010b; Omatsu et al., 2010). Steady-state Nestin MSCs express sub- stantially higher levels of genes required in HSC/ progenitor maintenance and retention, including Cxcl12, compared with in vivo sorted (Fig. 3 and using four in vivo models was sufficient to mobilize HSCs/ Fig. S5) or cultured osteoblasts (Méndez-Ferrer et al., 2010b). progenitors. Before this study, mononuclear phagocytes in the The relatively low expression of Cxcl12 detected in sorted BM had been poorly characterized and relied on F4/80 , osteoblasts is consistent with a recent study showing that a marker with a promiscuous expression profile in the BM DT administration into Cxcl12-DTR-GFP mice did not hi (Fig. S1). Herein, we rigorously discriminated among Gr-1 result in loss of spindle-shaped N-cadherin–expressing osteo - lo MOs, Gr1 MOs, and M in the BM by die ff rential expression blasts (Omatsu et al., of Gr-1, CD115, F4/80, CD11b, CD11c, MHC class II, 2010). It has been Figure 6. Opposite influences of the M  DTR/+ CX3CR1, CD68, and CD169. Using CD169 animals, proposed that M and the SNS on HSC/progenitor retention. we were able to implicate M, constituting 2.6% of total depletion mobilizes (A) CFU-C after treatment with PBS (blue BM cells, in the promotion of HSC/progenitor retention HSCs/progenitors bars) or clodronate liposomes (red bars) in through interaction with the recently characterized population wild-type C57BL/6 (n = 11–13), 6OHDA- by disruption of the treated mice (n = 12–13), 2-adrenergic re- / ceptor–deficient ( Adrb2 ) mice treated with an antagonist to the 3-adrenergic receptor / (3R; n = 10), and Adrb2 mice (n =7). Data are pooled from three independent experiments and analyzed with one-way ANOVA/Newman-Keuls test. (B) CFU-C after treatment with PBS (blue bars) or clodronate (red bars) in wild-type FVB mice (n = 5) and / 3-adrenergic receptor-deficient ( Adrb3 ) mice (n = 8). Data are pooled from two inde- pendent experiments. (C) Schematic of an- tagonistic regulation of HSC/progenitor retention by M and the SNS. JEM VOL. 208, February 14, 2011 267 osteoblasts either 14 h or 7 d after the depletion of M, de- spite the fact that HSCs/progenitors were clearly elevated at both time points. In contrast, significant reductions in the ex - pression of HSC retention genes were observed in Nestin cells, and correlated with persistent HSC/progenitor mobili - zation even 7 d after clodronate treatment. These results thus indicate that CD169 BM M promote HSC retention by acting specifically on the Nestin HSC niche in the BM. Previous studies have revealed that a transplantable cell expressing the G-CSF receptor (G-CSFR) is essential for G-CSF–induced mobilization (Liu et al., 2000). An accom - panying study, using an elegant mouse model in which the G-CSFR is expressed exclusively in CD68-expressing cells, also implicates mononuclear phagocytes in G-CSF mobilization (see Christopher et al. in this issue). Thus, G-CSF signaling exclusively in M is sufficient to reduce niche retention and promote HSC/progenitor mobilization. However, because G-CSF–induced HSC/progenitor mobilization is at least three times more potent than M depletion with clodronate liposomes (this study; Winkler et al., 2010), it must also be acting on cells other than M. The SNS is required for progenitor egress (Katayama et al., 2006; Méndez-Ferrer et al., 2010a), suggesting that G-CSF– mediated increase of sympathetic tone in the BM may repre- sent a putative M-independent target. Our results using models with impaired sympathetic activity suggest that BM M exert antagonistic, independent regulatory functions in the HSC niche compared with the SNS. Although G-CSF likely has several targets in the BM microenvironment, the present data Figure 7. M depletion synergizes with AMD3100 and G-CSF uncover two distinct opposing activities that lead to major mobilization. (A–C) Kinetics of M reduction (A) and LSK (B) and LSKFlk2 changes in HSC retention by Nestin niche cells. We thus (C) mobilization, at the indicated time points, after administration of PBS- (blue) or clodronate-encapsulated (red) liposomes (n = 3–4). Data are propose that G-CSF induces HSC mobilization by inhibiting pooled from two independent experiments. (D–E) CFU-C from peripheral M-mediated retention signals and simultaneously enhanc - blood of mice that were treated with PBS (blue) or clodronate liposomes ing sympathetic-mediated progenitor release (Fig. 6 C). (red; 14 h before harvest) and mobilized with AMD3100 (D; 1 h before har- These results expand our understanding of HSC niche vest) or G-CSF (E; 4 d). Data are pooled from two independent experiments. components by implicating a cellular constituent of the innate (F–H) CFU-C (F), LSK (G), and LSKFlk2 (H) cells from the peripheral blood immune system, the CD169 M, as a niche regulator. Because of mice that were mobilized for 2 d with G-CSF and treated with PBS- or targeted reduction of BM M can enhance HSC/progenitor clodronate-encapsulated liposomes 14 h before harvest. Experiment was mobilization, the use of antibodies against CSF-1 (M-CSF), performed once (n = 4). (I–K) CFU-C (I), LSK (J), and LSKFlk2 (K) from its receptor, or small molecule inhibitors of M-CSF signaling 16-wk-old female mice that were pretreated with PBS- or clodronate- encapsulated liposomes 10 d before harvest and mobilized with G-CSF may provide a novel strategy to increase the ec ffi iency of HSC/ for 4 d. Data are representative of two independent experiments (n = 4). progenitor mobilization for autologous transplantation. osteoblastic niche (Winkler et al., 2010). Winkler et al. ob - MATERIALS AND METHODS served a reduction in osteoblast numbers, as determined by Mice. All experiments, unless otherwise noted, were performed on 8–10- histomorphometry, on day 4 after two treatments with clodro- wk-old C57BL/6 male mice from Charles River Laboratories (Frederick nate liposomes on days 0 and 2. Although we have not ob - Cancer Research Center, Frederick, Maryland). 2-adrenergic receptor- served any significant reduction in osteoblast counts 14 h tm1Bkk deficient ( Adrb2 /J; Chruscinski et al., 1999; gift from G. Karsenty , or 7 d after clodronate treatment, it remains possible that osteo - Columbia University, New York, NY), 3-adrenergic receptor-deficient tm1low blast numbers may have transiently decreased and recovered (FVB/N-Adrb3 l/J; Susulic et al., 1995; The Jackson Laboratory), FVB/ N-CD11b-DTR (Cailhier et al., 2005; gift from C. Aloman, Mount Sinai after M ablation. In addition to CXCL12, the regulation Medical Center, New York, NY), Mafia (C57BL/6-Tg[Csf1r-EGFP-NGFR/ of other key retention genes appears to correlate with HSC/ FKBP1A/TNFRSF6]2Bck/J; Burnett et al., 2004; The Jackson Laboratory), progenitor egress. For example, treatment with G-CSF or 3R CX3CR1/GFP (Jung et al., 2000; gift from D. Littman, New York University, agonists down-regulates Cxcl12, Angiopoietin-1, Kitl, and New York, NY), CD169-DTR (Miyake et al., 2007), Nes-Gfp (Mignone Vcam-1 (Méndez-Ferrer et al., 2010b). We have not detected ERT2 et al., 2004), Nes-Cre (Balordi and Fishell, 2007), iDTR (C57BL/6-Gt[R / any reductions in the expression of HSC retention genes in OSA]26Sortm1[HBEGF]Awai/J; Buch et al., 2005), IL-10 (Berg et al., 1996; 268 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e gift from H. Xiong, Mount Sinai Medical Center, New York, NY), and CFU-C assays and mobilization. Colony-forming assays were performed / TNF (Marino et al., 1997; gift from M. van den Brink, Memorial Sloan as previously described (Frenette et al., 1998). Mobilization experiments with Kettering Cancer Center, New York, NY) mice were also used in these stud- AMD3100 and 4-d G-CSF were performed as previously described (Katayama ies. FVB/N-CD11b-DTR and Mafia (CD45.2) BM chimeras were gener - et al., 2006; Lucas et al., 2008). Some experiments were performed with subopti - 6 6 ated by transplanting 2.0 × 10 and 1.9 × 10 BM-nucleated cells from male mal G-CSF treatment (2 d), as previously described (Hidalgo et al., 2004). donors into lethally irradiated 8-wk-old FVB/N (The Jackson Laboratory) and C57BL/6 Ly5.2 (CD45.1) male mice, respectively. BM and blood of RNA isolation, reverse transcription, and Q-PCR. For measurement Mafia BM chimeras showed >95% donor chimerism as assessed by flow cyto- of BM Cxcl12 gene expression, femurs were flushed and mixed with 0.5 ml metry 1 mo after transplantation. Mice were maintained on a 12 h light/ Trizol (Invitrogen) and stored at 80°C. Conventional reverse transcrip- 12 h darkness lighting schedule. All in vivo experiments were harvested be - tion, using the Sprint PowerScript reverse transcription (Takara Bio Inc.) tween 12:00 and 1:00 p.m. (Zeitgeber time 5:00 and 6:00) to limit circadian was performed in accordance with the manufacturer’s instructions. Q-PCR variations in HSC/progenitor release (Méndez-Ferrer et al., 2008) and mo - was performed with SYBR GREEN on an ABI PRISM 7900HT Sequence bilization (Lucas et al., 2008). All mice were housed in specific pathogen–free Detection System (Applied Biosystems). The PCR protocol consisted of one facilities at the Mount Sinai School of Medicine or Albert Einstein College cycle at 95°C (10 min) followed by 40 cycles of 95°C (15 s) and 60°C of Medicine animal facility. Experimental procedures performed on the mice (1 min). Expression of Gapdh was used as a standard. The mean threshold cycle were approved by the Animal Care and Use Committee of the Mount Sinai number (C ) for each tested mRNA was used to quantify the relative ex- School of Medicine and Albert Einstein College of Medicine. pression of each gene: 2^(C [Gapdh]  C [gene]). Primers used are listed t t below: Cxcl12_fwd, 5-CGCCAAGGTCGTCGCCG-3; Cxcl12_rev, 5-TTGGCTCTGGCGATGTGGC-3; Angpt1_fwd, 5-CTCGTCAG- In vivo cell depletion. Cl2MDP (or clodronate) was a gift from Roche ACATTCATCATCCAG-3; Angpt1_rev, 5-CACCTTCTTTAGTG- (Van Rooijen and Sanders, 1994). Clodronate liposomes (250 µl) were in - CAAAGGCT-3; Kitl_fwd, 5-CCCTGAAGACTCGGGCCTA-3; Kitl_ fused i.v. at 1 d (14 h), 7 d, 10 d, 16 d, or 28 d before harvest. CD11b-DTR rev, 5-CAATTACAAGCGAAATGAGAGCC-3; Vcam1_fwd, 5-GACC- mice were treated with DT i.p. 25 ng/g on days 1 and 3 before harvest. DT TGTTCCAGCGAGGGTCTA-3; Vcam1_rev, 5-CTTCCATCCT- was purchased from Sigma-Aldrich. AP20187 was a gift from Ariad Pharma- CATAGCAATTAAGGTG-3; Osteocalcin_fwd, 5-GGGCAATAAGG- ceuticals. Lyophilized AP20187 was dissolved in 100% ethanol at a concentration TAGTGAACAG-3; Osteocalcin_rev, 5-GCAGCACAGGTCCTAAA- of 62.5 mg/ml stock solution and was stored at –20°C. As recommended TAGT-3; Gapdh_fwd, 5-TGTGTCCGTCGTGGATCTGA-3; Gapdh_ by Ariad Pharmaceuticals, injection solutions were prepared with a diluent rev, 5-CCTGCTTCACCACCTTCTTGA-3. composed of 4% ethanol, 10% PEG-400, and 2% Tween-20 in water. All in - jections were administered i.v. within 30 min after preparation. The volume CXCL12 ELISA. 96-well ELISA plates were coated overnight at 4°C wit h of injection solution was adjusted according to the average mouse body 50 µl of 2 µg/ml anti-CXCL12 coating antibody (MAB350; R&D Systems). weight to deliver a dose of 10 mg/kg AP20187 per mouse in an mean vol - Next, the wells were washed three times with wash bue ff r (0.05% Tween 20 in ume of 100 µl. Mice were injected daily for 5 d before harvest. Heterozygous DTR/+ PBS) and incubated for 1 h at room temperature with 200 µl of blocking bue ff r CD169-DTR (CD169 ) or control C57BL/6 were injected i.p. with (1% BSA, 5% D-Sucrose, and 0.05% NaN in PBS; all from Thermo Fisher Sc- i 10 µg/kg DT 48 h before harvest. Depletion of Nestin cells was accom - entic fi ). After 3 washes, 100 µl of samples diluted 1:2 in PBS were added and plished as previously described (Méndez-Ferrer et al., 2010b). incubated for 2 h at room temperature. After 3 washes, 100 µl of 0.250 µg/ml polyclonal biotinylated anti–human/mouse SDF-1 (BAF310; R&D Systems) Flow cytometry and cell sorting. Fluorochrome-conjugated or biotinylated was added and incubated for 2 h at room temperature. After 3 washes, 100 µl of mAbs specific to mouse Gr-1 (Ly6G/C; clone RB6-8C5), CD115 (clone 0.1 µg/ml Neutravidin-HRP (Thermo Fisher Scientic fi ) was added and incu - AFS98), Siglec-F (clone E50-2440), CD11b (clone M1/70), CD11c (clone bated for 30 min. After 3 additional washes, the reaction was developed by incu - N418), I-A/I-E clone (clone M5/114.15.2), CD45 (clone 30-F11), Sca- 1 bation for 20–30 min with 50 µl of TMB substrate solution (Sigma-Aldrich) and (clone D7), Flk2 (clone A2F10), CD117 (clone 2B8), CD3 (clone 145-2C11), stopped by adding 50 µl of 1M HCl solution (Thermo Fisher Scientic fi ). Optical B220 (clone RA3-6B2), Ter119 (clone TER-119), CD51 (clone RMV-7), and density was determined with a microplate reader set at 450 nm. Optical density CD31 (clone MEC13.3), corresponding isotype controls, and secondary of PBS control wells was subtracted from optical density of samples. Recom-bi reagents (eu fl or450-, APC-eu fl or780–, and PE-Cy7–conjugated streptavidin) nant mSDF-1 (PeproTech) was used to generate a linear standard curve. were purchased from eBioscience. Anti-F4/80 (clone CI:A3.1), CD68 (clone FA-11), and CD169 (clone 3D6.112) were purchased from AbD Serotec. CD68 Immunou fl orescence. Anesthetized Nes-GFP transgenic animals (Mignone was stained extracellularly and subsequently intracellularly with the Cyt/ ox fi et al., 2004) were perfused, and femurs and tibia were sectioned and stored as Cytoperm kit (BD) according to the manufacturer’s protocol. Multiparameter previously described (Méndez-Ferrer et al., 2008). Slides were washed three analyses of stained cell suspensions were performed on an LSRII (BD) and an-a  times in Coplin jars (Sigma-Aldrich) to remove OCT solution residue, and lyzed with FlowJo software (Tree Star, Inc.). DAPI single cells were evaluated for then incubated for 1 h at room temperature in 20% goat serum (Sgma- all analyses except for intracellular stains. To purify mononuclear phagocyte p-op Aldrich) diluted in PBS + 0.1% Tween solution (PBSTw). After 3 washes in ulations, BM was sorted with an InFlux cell sorter (BD) to achieve >97% purity. PBSTw, slides were incubated in PBSTw + 2% goat serum + 0.5% Triton Sorted mononuclear phagocytes were cytospun with Cytospin 3 (Thermo Fisher X-100 for 1 h at room temperature. After 3 washes in PBSTw, slides were Scientic fi ) and stained with Hema 3 manual staining system (Thermo Fisher +  incubated in primary antibody (anti-CD68-Alexa Fluor 647 [clone FA-11; Scientic fi ). To isolate Nestin and Nestin cells from the BM for Q-PCR, RBC- AbD Serotec] and anti–CD169-biotin [clone MOMA-1; AbCam]) at a 1:100 lysed BM cells were digested with collagenase, trypsin, and DNase, as previously concentration in PBSTw + 2% goat serum overnight in the dark at room described (Méndez-Ferrer et al., 2010b). Endothelial cells and osteoblasts were temperature. After three washes in PBSTw, CD169 staining was continued isolated similar to previous studies (Semerad et al., 2005; Winkler et al., 2010 ). with streptavidin-PE staining for 5 min. Slides were mounted with Vector- In brief, tibias, femurs, and humeri of mice were u fl shed thoroughly of BM shield + DAPI, covered, and sealed with nail polish. Images were acquired cells, chopped with a scalpel, and washed three times through a 5-ml polystyrene on an Examiner microscope (Carl Zeiss, Inc.) and all images were processed tube with blue-top cell strainer (BD) to further remove residual BM cells. The using Slidebook software (Intelligent Imaging Innovations, Inc). bone fragments were then digested at 37°C with Type IA collagenase (Sigma- Aldrich) for 40 min while spinning. RBC-lysed pellet was then stained for so -rt ing. Cells were sorted by Moflo Cell sorter (Dako) at the Flow Cytometry Long-term BM Dexter cultures. 3.7 × 10 BM-nucleated cell s Core Facility at Mount Sinai School of Medicine or Aria Cell sorter (BD) at the were plated in 1 ml Dexter medium (Myelocult M5300 media [Stem Cell Flow Cytometry Core Facility at Albert Einstein College of Medicine. Technologies] supplemented with 1% penicillin–streptomycin [Cellgr o], JEM VOL. 208, February 14, 2011 269 + 6 CXCL12 reduction. Fig. S4 shows the sorting strategy for Nestin and Nes- 1% amphotericin [Cellgro], and 10 M freshly thawed Hydrocortisone [Sigma- tin fractions and bone endothelial cell and osteoblast fractions. Fig. S5 shows Aldrich]) in 12-well plates. Cultures were maintained in a water-jacketed - in that retention gene expression is reduced in Nestin cells seven days after cubator at 33°C and 5% CO . Half the media was changed weekly for 6 wk. In the sixth week, the culture media was removed and replaced with 1 ml of mononuclear phagocyte treatment. Fig. S6 shows that mononuclear phago - Dexter medium containing 40% PBS- or clodronate-encapsulated liposomes cytes are not reduced 7 d after depletion of Nestin cells. Fig. S7 shows that by volume in some experiments. After 24-h incubation, the culture media soluble factor from a M, but not myeloblast, cell line enhances stromal was removed and 1 ml fresh media was added. 24 h and 72 h later, the media CXCL12 production. Online supplemental material is available at http:// was collected and frozen to assess CXCL12 levels by ELISA and the adherent www.jem.org/cgi/content/full/jem.20101688/DC1. layer was Hema 3-stained to assess cell morphology or detached by cell We would like to acknowledge experimental help and mice provided by scraper (BD) for o fl w cytometric analysis. In experiments with Dexter cultures DTR/+ O.M. Smith, M. van den Brink, and all the investigators that provided mice derived from CD169 BM, the media was removed and replaced with 1 ml for these experiments. fresh Dexter medium containing PBS or 1 µg/ml DT. After 72 h of incuba- This work was supported by the National Institutes of Health grants tion, the media was frozen and later assessed for CXCL12 levels by ELISA. R01DK056638 and R01HL097819 to P.S Frenette, R01CA112100 to M. Merad, and P30CA013330 to the AECOM Flow Cytometry Core Facility. A. Chow, D. Lucas, MS-5 cell culture. MS-5 cells were grown in monolayers in complete me - A. Hidalgo, S. Mendez-Ferrer, C. Scheiermann, and M. Battista were supported by dium (-MEM medium supplemented with 10% FBS [Stem Cell Technolo - fellowships from NHLBI (1F30HL099028-01; A. Chow), Fundación Ramón Areces gies], penicillin–streptomycin [Invitrogen], 5% glutamine [Invitrogen], and (D. Lucas), American Heart Association Scientist Development Grant (0735165N; 5% sodium pyruvate [Invitrogen]). Cultures were maintained at 37°C and A. Hidalgo), Ramón y Cajal Fellowship from the Spanish Ministry of Science and 1:10 split with 0.05% trypsin-EDTA (Invitrogen) every 3 or 4 d, when cells Innovation (A. Hidalgo and S. Méndez-Ferrer), Scholar Award from the American reached 80% confluence. 5,000 MS-5 cells were plated in 300 µl complete Society for Hematology (S. Méndez-Ferrer), German Academic Exchange Service medium in 48-well plates for 24 h before addition of BMDM or medium (DAAD; C. Scheiermann) and Cooley’s Anemia Foundation (M. Battista). P.S. Frenette conditioned by BMDM (M CM), RAW264.7, or M1 cells (see below). is an Established Investigator of the American Heart Association. The authors have no financial conflict with these studies. BM MO/M cell culture. The M1 myeloblast cell line was cultured in Submitted: 13 August 2010 DME medium (Cellgro) supplemented with 10% FBS, 2 mM l-glutamine Accepted: 7 January 2011 (Sigma-Aldrich), 1% sodium pyruvate (Invitrogen), and 1% penicillin - streptomycin. RAW264.7 M cell line (a gift from B. Tenoever, Mount Sinai Medical Center, New York, NY) was cultured identically to the M1 myelo - REFERENCES blast cell line, except for addition of 100 mM Hepes (Sigma-Aldrich). 50,000 Balordi, F., and G. Fishell. 2007. 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B-lymphocyte commitment and differentiation from hematopoietic 94:8093–8098. doi:10.1073/pnas.94.15.8093 stem cells.Blood . 109:3706–3712. doi:10.1182/blood-2006-08-041384 JEM VOL. 208, February 14, 2011 271 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

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Abstract

A r t i c l e Bone marrow CD169 macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche 1,2,3 2,3 2,4 Andrew Chow, Daniel Lucas, Andrés Hidalgo, 2,5 1,2 2,3 Simón Méndez-Ferrer, Daigo Hashimoto, Christoph Scheiermann, 2 1,2 2,3 Michela Battista, Marylene Leboeuf, Colette Prophete, 6 7 1,2 Nico van Rooijen, Masato Tanaka, Miriam Merad, 2,3 and Paul S. Frenette 1 2 Department of Gene and Cell Medicine and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461 4 5 Department of Epidemiology, Atherothrombosis, and Imaging and Cardiovascular Developmental Biology Department, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain Department of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, Netherlands Laboratory for Innate Cellular Immunity, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, 230-0045, Japan Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear hi lo phagocytes that include Gr-1 monocytes (MOs), Gr-1 MOs, and macrophages (M) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and M conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, spe- cific depletion of CD169 M, which spares BM MOs, was sufficient to induce HSC/ progenitor egress. M depletion also enhanced mobilization induced by a CXCR4 antago- nist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which M cross talk with the Nestin niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM M hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly. The BM is the preferred site for adult hemato- to mobilize HSCs/progenitors out of the BM CORRESPONDENCE Paul S. Frenette: poiesis. Transplantation of BM cells containing into the peripheral blood has allowed for effi - [email protected] hematopoietic stem cells (HSCs) and progeni - cient, less invasive HSC procurement in clinical OR tors has been a remarkable medical advance - stem cell transplantation. However, up to 30% Miriam Merad: [email protected] ment that allows for the replacement of the of patients previously treated with cytotoxic hematopoietic compartment after preparative anticancer therapies do not mobilize sufficient Abbreviations used: BMDM, regimens. HSCs are retained in perivascular numbers of stem cells using current protocols BM-derived M; BMEF, BM extracellular u fl id; DT, diphtheria niches that are distributed near osteoblasts and (Bensinger et al., 2009). toxin; G-CSF, granulocyte within the nonendosteal parenchyma (Kiel et al., colony-stimulating factor; HSC, 2005; Sugiyama et al., 2006; Lo Celso et al., hematopoietic stem cell; M, © 2010 Chow et al. This article is distributed under the terms of an Attribution– 2009; Méndez-Ferrer et al., 2010b). The ability macrophage; MO, monocyte; Noncommercial–Share Alike–No Mirror Sites license for the first six months after Q-PCR, quantitative RT-PCR; the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share SNS, sympathetic nervous system. A. Chow and D. Lucas and M. Merad and P. Frenette con- Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ tributed equally to this paper. by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 208 No. 2 261-271 www.jem.org/cgi/doi/10.1084/jem.20101688 The Journal of Experimental Medicine Sympathetic neural tone is crucial for both steady state population were positive for F4/80 (Fig. S1 A), CD11c, and (Méndez-Ferrer et al., 2008) and granulocyte colony - MHC class II (Fig. 1 C). – int + stimulating factor (G-CSF)–enforced (Katayama et al., 2006) The Gr-1 CD115 F4/80 fraction consisted of two release of HSCs/progenitors from the BM. Recent studies populations: eosinophils (Fig. 1 A, gate V) and a population of indicate that mesenchymal stem cells (MSCs), identified by mononuclear cells (gate IV), which could be discriminated by – int the expression of the intermediate filament protein Nestin, forward and side scatter characteristics. The Gr-1 CD115 + hi comprise a critical cellular constituent of the stem cell niche F4/80 subset, after exclusion of SSC eosinophils, comprised that is under the control of the sympathetic nervous system 2.6 ± 0.2% of BM cells and was the only population of BM (SNS; Méndez-Ferrer et al., 2010b). Because previous studies mononuclear phagocytes that showed expression of CD169 using G-CSF receptor–deficient mice showed that expression (Fig. 1 C). CD169, also known as sialoadhesin or SIGLEC-1, of the receptor on transplantable hematopoietic cells was r -e is a sialic acid binding molecule that was initially described quired for G-CSF–induced mobilization (Liu et al., 2000), we over two decades ago to have high activity on BM stromal have previously speculated that at least two distinct pathways, and lymph node M, but not on blood MO (Crocker and neural and hematopoietic, acted in concert to promote HSC/ Gordon, 1986). CD169 is recognized by the M antibody progenitor egress (Katayama et al., 2006). MOMA-1, which has long been used to stain M in the Hypothesizing that mononuclear phagocytes are crucial spleen, lymph nodes, lamina propria, Peyer’s patches, and CNS for stromal function of the BM, we sought to eliminate these (Oetke et al., 2006). Based on this marker, we will sb usequently – int populations to evaluate their contributions to HSC trac ffi king. call this cell population BM M. These Gr-1 CD115 + + Unexpectedly, we have found that BM macrophages (M) F4/80 CD169 M also expressed intermediate levels of did not promote the egress of HSCs/progenitors, but rather MHC class II, CD11c, and CD68; low expression of CD11b; contributed to the retention of HSCs in the BM by acting on and negligible expression of CX3CR1 (Fig. 1 C). Nestin MSCs. These data uncover a new role for the innate immune system in regulating stem cell niche functions. Depletion of BM mononuclear phagocytes correlates with HSC/progenitor egress and reduction in marrow CXCL12 RESULTS We first depleted BM MO/M  using clodronate liposome Phenotypic markers of BM mononuclear phagocytes injection. 14 h after clodronate liposome administration, BM hi Depletion of monocytes (MO) and/or M from the BM has M were reduced by 84% (Fig. 2, A and B), whereas Gr-1 lo been accomplished with injection of clodronate liposomes MO, Gr-1 MO, and total BM cellularity were reduced by 79, (Giuliani et al., 2001) and injection of the FK-binding pro - 88, and 24%, respectively, compared with PBS-treated animals tein dimerizer AP20187 in transgenic Maa fi mice (Burnett et al., (Fig. 2, A and C–E). Depletion of the noneosinophil Gr-1 + + 2004; Chang et al., 2008). Mafia mice have a Fas suicide/ CD115 F4/80 population with clodronate further supports apoptotic system driven by the CD115 (M-CSF receptor) pro- the conclusion that these CD169 cells are indeed M. moter. Previous phenotypic descriptions of BM M have We assessed the effect of BM MO/M  depletion on cir- exclusively relied on F4/80 expression (Hume et al., 1983; culating hematopoietic progenitors and found a marked in- Giuliani et al., 2001; Chang et al., 2008). However, this marker crease in colony-forming unit activity (4.5-fold; Fig. 2 F) and +  hi – + + is also expressed on BM neutrophils (Gr-1 CD115 ), Gr-1 Lineage Sca-1 c-kit (LSK) cells (6.2-fold; Fig. 2 G) in blood + + lo  + MO (Gr-1 CD115 ), Gr-1 MO (Gr-1 CD115 ; Gordon after mononuclear phagocyte depletion. Notably, circulating hi + – and Taylor, 2005), and eosinophils (SSC Siglec-F ; Zhang LSK Flk2 cells, enriched in long-term repopulating HSCs et al., 2004; Fig. S1). To distinguish among BM mononuclear (Christensen and Weissman, 2001), increased by 12.9-fold phagocytes and to elucidate their differential surface pheno - after clodronate treatment (Fig. 2 H). types, we purified different BM populations via cell sorting We next determined the effect of mononuclear phago - based on three markers: Gr-1 (Ly6C/G), CD115, and F4/80. cyte depletion in modulating the levels of CXCL12, a che - As expected, neutrophil granulocytes were homogenously mokine produced by stromal niche cells that is crucial in +  represented in the Gr-1 CD115 gate (Fig. 1 A, gate I) and retention and maintenance of HSCs/progenitors in the BM represented 49.6 ± 1.1% of the total BM nucleated cells. (Méndez-Ferrer and Frenette, 2007). Clodronate-induced de- In mice, there are two subsets of CD115 MO that differen - pletion of BM MO/M and HSC/progenitor mobilization tially express Gr-1 (Gordon and Taylor, 2005). In concordance, was associated with a 44% reduction in CXCL12 mRNA + + the Gr-1 CD115 portion (Fig. 1 A, gate II) represented a levels in total BM (Fig. 2 I) and a 40% reduction in CXCL12 homogenous population of MO (Fig. 1 B) that constituted protein in the BM extracellular u fl id (BMEF; Fig. 2 J). Because hi 9.8 ± 0.3% of the BM and is characterized as F4/80 BM MO/M do not produce CXCL12 (Fig. S2), these data hi int int – – – CD11b CD68 CX3CR1 MHCII CD11c CD169 suggest that MO/M depletion causes a reduction in CXCL12 hi (Fig. 1 C and Fig. S1 A), and will herein be termed Gr-1 expression by BM stromal cells. – + MO. The Gr-1 CD115 population (Fig. 1 A, gate III) rep- These results were further confirmed using other con - resenting 1.4 ± 0.1% of BM consisted of a population of ditional depletion models of mononuclear phagocytes, i-n hi int int MO (Fig. 1 B) characterized as CX3CR1 CD11b CD68 cluding transgenic mice expressing the diphtheria toxin – lo CD169 and will be termed Gr-1 MO. Subsets of this (DT) receptor under the CD11b promoter (CD11b-DTR; 262 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e Figure 1. Mononuclear phagocytes can be distinguished in the BM with Gr-1, CD115, and F4/80. (A) Gating strategy of BM mononuclear phagocytes. The Gr-1 pop- ulations were divided into a CD115 fraction comprised of neutrophils (I; left) and CD115 hi lo fraction of Gr-1 MOs (II; left). The Gr-1 fraction was further subdivided into two pop- + lo ulations (middle): CD115 Gr-1 MOs (III) and + int a F4/80 CD115 population, which can be hi subdivided into SSC eosinophils (V) and int/lo SSC M (IV). (B) Morphology of cell popula- tions I–IV (63× magnification; bars, 10 µm). (C) Overlay histograms show the differential expression of CD11b, CD11c, MHC class II, CX3CR1, CD68, and CD169 (blue line) among mononuclear phagocyte populations. Gray histograms represent isotype control. All results are representative of two indepen- dent experiments. 47% reduction in BMEF CXCL12 (Fig. S3 S). These data are consistent with a recent study also using liposo - mal clodronate and Mafia mice that demonstrated the association of BM mononuclear phagocyte depletion with HSC/progenitor egress (Winkler et al., 2010). Progenitor release cannot be explained by nonspecic fi cell death be- cause in vivo depletion of neutrophils hi and Gr-1 MO using anti–Gr-1 (Ly6G/ C) antibody or depletion of dendritic cells using DT administration in CD11c-DTR mice (Jung et al., 2002) did not lead to any progenitor mobiliza - Cailhier et al., 2005) and the Mafia mice (Burnett et al., 2004). tion (unpublished data). Collectively, these results sugge st that mononuclear phagocytes play a critical role in the rete-n In these studies, we generated BM chimeric mice in which wild-type recipients were reconstituted with BM cells iso - tion of HSCs/progenitors in the BM. lated from these transgenic animals to minimize toxicity of the depleting agents to nonhematopoietic organs. Treatment Mononuclear phagocytes regulate Nestin niche cells that of CD11b-DTR chimeric animals with DT reduced BM maintain HSCs in the BM M counts by >40% (Fig. S3 A) and MO subsets by >50% Recent studies have revealed that GFP expression, when (Fig. S3, B and C). Consistent with the expression of CD11b, driven by Nestin regulatory elements, identifies rare MSCs also on neutrophils, total BM cellularity was reduced by that form HSC niches (Méndez-Ferrer et al., 2010b). We ob- + + 40% in this model (Fig. S3 D). Concomitantly, progenitors served CD68 and CD169 cells throughout the BM and in circulating in blood were significantly increased by 1.6-fold the vicinity of rare Nestin MSC niche cells F ( ig. 3 A). To evalu- (Fig. S3 E) and BMEF CXCL12 was significantly reduced ate whether mononuclear phagocytes regulate Nestin niche (Fig. S3 F). AP20187-treated Mafia BM chimeras depleted cells, we used clodronate liposomes to deplete BM MO/M. BM M, MO, and total BM cells by 40, >80, and 30%, r- e Because the number of Nestin cells was unchanged by this spectively (Fig. S3, G–J). This was associated with an 6-fold treatment (Fig. S4 D), we assessed alterations in gene expres-   + increase in circulating progenitors (Fig. S3 K), and 46% re- sion in these cells. We sorted CD45 Ter119 Nestin and      duction in BM CXCL12 levels (Fig. S3 L). In nontransplanted CD45 Ter119 Nestin from the BM and CD45 Ter119   + Mafia mice, mobilization was even more robust; AP20187- CD31 Sca-1 CD51 osteoblasts (Semerad et al., 2005; treated Mafia animals exhibited a >80% reduction in MO/ Winkler et al., 2010) from the bone 14 h or 7 d after depletion     M (Fig. S3, M–P), 31% reduction in BM cellularity (Fig. S3 Q), (Fig. S4, A and B). Bone CD45 Ter119 CD31 Sca-1 >20-fold increase in circulating progenitors (Fig. S3R), and CD51 cells were conr fi med to be enriched in osteoblasts by JEM VOL. 208, February 14, 2011 263 Figure 2. Depletion of mononuclear phagocytes is associated with HSC/progenitor mobilization and CXCL12 reduction. (A–H) C57BL/6 mice were treated with PBS (blue) or clodronate-encapsulated liposomes (red). (A) Representative dot plots show the percentages hi lo of neutrophils, Gr-1 MOs, Gr-1 MOs, and M, as de- scribed in Fig. 1 A. (B–E) Absolute numbers of mono- nuclear phagocytes and total nucleated cells in the BM (n = 11). (F) Absolute numbers of colony-forming units in culture in the peripheral blood (CFU-C; n = 12–15).  + + (G–H) Enumeration of Lineage Sca-1 c-kit (LSK; G) and LSKFlk2 (H) cells in the peripheral blood (n = 9–10). B–H represents pooled data from at least three independent experiments. (I) RT-PCR analysis of Cxcl12 mRNA levels in total BM cells (n = 5 mice per group). Data representative of two independent experiments are shown. (J) CXCL12 levels in the BMEF. Data are pooled from three indepen- dent experiments. controls (Fig. S4, D–G). These results, together with the gene expression analyses (Fig. 3, B–E; and Fig. S5), suggest that BM mononuclear phagocytes play a role in HSC/progenito r retention by regulating maintenance of reten- tion gene expression specifically in Nestin niche cells, but not osteoblasts. Microarray expression analyses have shown that Nestin cells express high levels of Csf1 (Méndez-Ferrer et al., 2010b), a critical cytokine for M development and survival (Hamilton, 2008). To determine whether Nestin cells in the BM regulate mononuclear phagocyte num - bers, we depleted Nestin cells by administering ERT2 DT into tamoxifen-treated Nes-Cre /iDTR   high Osteocalcin expression compared with CD45 Ter119 animals (Méndez-Ferrer et al., 2010b). We found no di- f CD31 endothelial cells (Fig. S4 C). From these populations, we ference in BM M (Fig. S6 A) or MO (Fig. S6, B and C) in ERT2 performed quantitative real-time PCR (Q-PCR) of genes en- Nes-Cre /iDTR mice treated with tamoxifen and DT, coding molecules previously implicated in HSC maintenance compared with control iDTR animals. and retention (Cxcl12, Angpt1, Kitl, and Vcam1). These genes are highly expressed by Nestin cells and down-regulated BM M produce a protein factor that raises CXCL12 during G-CSF–induced mobilization or upon 3 adrenergic production by stromal cells in vitro signaling (Méndez-Ferrer et al., 2010b). As previously reported, The aforementioned results demonstrate a robust in vivo cor- Nestin cells from the BM had markedly higher expression relation between BM MO/M depletion, CXCL12 reduc- of the four retention genes, compared with the Nestin frac- tion, and HSC/progenitor mobilization. To further dissect the tion (Méndez-Ferrer et al., 2010b). Interestingly, we found effect of mononuclear phagocytes on stromal cell function, that the expression of Cxcl12, Angpt1, Kitl, and Vcam1 was we established long-term murine Dexter BM cultures con - 48-, 18-, 65-, and 35-fold higher, respectively, in Nestin cells sisting of an adherent stromal layer and attached hematopoi - in the BM compared with bone osteoblasts in the steady state etic cells (Dexter et al., 1977). Consistent with the in vivo (Fig. 3, B–E). Strikingly, we observed a 65–80% reduction in data, we found that clodronate liposome treatment of Dexter the expression of these four genes 14 h after treatment with cultures dramatically reduced the cellularity in the we lls, clodronate, compared with PBS liposome-treated animals especially among the adherent M, compared with PBS (Fig. 3, B–E). The reduced expression persisted at least until liposome-treated wells Fig. ( 4 A). The number of CD115 day 7 after treatment with clodronate (Fig. S5). In contrast, mononuclear phagocytes was indeed reduced by >50% the expression levels of these retention genes in sorted osteo - after liposomal clodronate treatment (Fig. 4 B). These changes blasts were unchanged 14 h and 7 d after clodronate treatment. in mononuclear phagocytes were associated with decreased In addition, the numbers of Nestin cells and osteoblasts 14 h stromal production of CXCL12 at 24 h (30%↓) and 72 h and 7 d after administration of clodronate were similar to (40%↓; Fig. 4 C). 264 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e Figure 3. Anatomical and functional relationships between mononuclear phagocytes and Nestin niche cells. (A) Distribution of BM CD68 (white) and CD169 (red) cells in 5-µm femoral sections of Nes-GFP mice. Dashed line demarcates separation of bone and BM. Images were acquired at 40× magnification (bar, 20 µm). Isotype control is shown in inset. (B–E) Relative expression of Cxcl12 (B), Angpt1 (C), Kitl (D), and Vcam1   + +  (E) in CD45 Ter119 Nestin (Nestin ) and CD45   Ter119 Nestin (Nestin ) fractions sorted from the BM and osteoblasts sorted from the bone 14 h after treat- ment with PBS- (blue bars) or clodronate-encapsulated (red bars) liposomes. Data are presented with the mean expression of the PBS liposome-treated Nestin fraction set at 100% and are representative of three indepen- dent experiments (n = 4–5). Data analyzed by one-way ANOVA/Newman-Keuls test. ***, P < 0.001. Depletion of BM CD169 M mobilizes HSCs/progenitors The MS-5/M co-culture experiments sug- gest that differentiated BM M , rather than MO, are the mononuclear phagocytes that promote HSC/progenitor retention. To test di- rectly whether BM M are promoting HSC/ progenitor retention in the BM, we took ad- vantage of the differential expression of CD169 between MO and M and mice expressing Because adherent M appear to interdigitate the stroma DTR under the endogenous CD169 promoter (Miyake et al., in Dexter cultures, we sought to determine the relative con - 2007). Treatment of heterozygous CD169-DTR mice with tribution of M and MO in stromal cell–mediated pro - DT-depleted M (Fig. 5, A and B), but not MO (Fig. 5, A, genitor retention. Thus, we used the murine stromal cell C, and D), in the BM. DT treatment was associated with a 3.5- line MS-5 to determine whether the addition of M or fold increase in circulating hematopoietic progenitors, a s M-synthesized products could affect the stromal niche. assessed by LSK cell enumeration (Fig. 5 E), and a 5.4-fold MS-5 stromal cells have been used as an appropriate in vitro increase in the stem cell-enriched LSKFlk2 fraction (Fig. 5 F). model in which to replicate in vivo modulation of CXCL12 Moreover, depletion of CD169 M from long-term Dexter production by noradrenergic signals (Méndez-Ferrer et al., 2008). culture resulted in a 42% reduction in the ability of BM When grown in medium conditioned by the M cell line stromal cells to produce CXCL12 (Fig. 5 G). Thus, CD169 RAW264.7 (Raschke et al., 1978), CXCL12 production by M in the BM promote stromal production of CXCL12, MS-5 was significantly increased ( Fig. S7 A), whereas no sig- and their specific depletion in vivo is sufficient to mobilize nificant difference was observed in medium conditioned by HSCs/progenitors. the myeloid myeloblast cell line M1 (Ralph et al., 19 83; Fig. S7B). Co-culture of BM-derived M (BMDM) or media Parallel and antagonistic roles of M and SNS in regulating conditioned by BMDM increased MS-5 production of HSC/progenitor release CXCL12, indicating that a secreted soluble factor induces The SNS is crucial in HSC/progenitor trafficking (Katayama CXCL12 up-regulation by MS-5 cells (Fig. 4, D and E). This et al., 2006; Méndez-Ferrer et al., 2008) where 3-adrenergic secreted factor was a protein, as digestion with Proteinase K receptor (3R) signaling plays a key role in circadian oscilla - abrogated the ability of M-conditioned medium to raise tions of HSC release, and both 2-adrenergic receptor (2R) CXCL12 production by M (Fig. 4 F). We have evaluated and 3R signaling cooperate in G-CSF–enforced egress putative candidate factors using antibodies or knockout ani- (Méndez-Ferrer et al., 2010a). M could act independently, mals for IGF-1, IL-1, TNF, or IL-10. However, the loss-of- or alternatively, through alterations in the sympathetic tone. function of any one of these factors did not alter the ability of Thus, to assess whether M operate through a distinct path- BMDM-conditioned medium to induce CXCL12 synthesis way, we examined whether clodronate treatment was capable (Fig. S7, C–F). These data suggest that BM M, through the of inducing mobilization in sympathectomized animals . secretion of a yet undefined protein factors, directly promote We found that mice chemically sympathectomized with the retention of HSCs/progenitors by raising CXCL12 pro- 6-hydroxydopamine (6OHDA) still exhibited signic fi ant pro - duction in BM niche cells. genitor mobilization (>7-fold) in response to clodronate JEM VOL. 208, February 14, 2011 265 Figure 4. M in culture promote CXCL12 production. (A) Morphology of ad- herent layer of Dexter culture 24 h after addi- tion of PBS or clodronate liposomes (10×; bars, 100 µm). (B) Adherent cells were ana- lyzed by flow cytometry for CD115 cells. (A and B) Representative data from two inde- pendent experiments are shown. (C) CXCL12 levels were assessed by ELISA at 24 h (left bars) and 72 h (right bars) after liposomal incubation. (D) CXCL12 levels were assessed 3 d after MS-5 cells were co-cultured with (M) or without (Ctrl) BMDM. (E) Levels of CXCL12 secreted from MS-5 cells after culture with medium conditioned by BMDM (M CM) or with control medium (Ctrl). (F) CXCL12 levels were measured after culture of MS-5 cells with control (Ctrl, blue) or M-conditioned (M CM, red) medium that was untreated began their recovery treatment; however, the absolute number of mobilized pro - (left two bars) or treated with Proteinase genitors did not reach the level of SNS-intact Clodronate- by day 7 after clodro- K (right two bars). (C–F) Representative data from at least two independent experiments treated mice (Fig. 6 A, mid-left bars). To evaluate this issue nate administration / are shown. using another model, we used mice dec fi ient in 2R (Adrb2 ) (unpublished data). treated with an antagonist to the 3R. Clodronate treatment Interestingly, levels was still able to cause HSC/progenitor mobilization (>3-fold) of LSK (Fig. 7 B) and LSKFlk2 (Fig. 7 C) inversely matched in these mice, but again HSCs/progenitors did not mobilize BM M counts. HSCs/progenitors were still elevated in the to the same level as wild type animals (Fig. 6 A, mid-right circulation 10 d after clodronate administration (4.2- and 3.2- bars). To dissect further the relative contribution of the 2R fold, respectively), and mobilization persisted at least until day and 3R in SNS promotion of HSC/progenitor release, we 16 (Fig. 7 C). These data further support the specific role of BM M, but not MO, in promoting the retention of HSCs/ evaluated the effect of M  depletion in mice singly lacking 2R (Fig. 6 A, right bars) or 3R (Fig. 6 B). Whereas clodro- progenitors in the BM. nate treatment led to a robust increase in circulating progeni - Because BM M promote HSC/progenitor retention, / tors in Adrb2 animals compared with wild-type animals we examined whether elimination of this population would / (Fig. 6 A, right bars), the response was blunted in Adrb3 enhance mobilization using the CXCR4 antagonist AMD3100 mice (Fig. 6 B). These data are consistent with previous stud- or G-CSF, both of which are clinically approved mobilizing ies demonstrating that 3R, but not 2R, signaling is critical agents. We found that clodronate treatment 14 h before harvest for physiological HSC/progenitor release (Méndez-Ferrer et al., doubled HSC/progenitor mobilization in AMD3100-treated 2008). The fact that M depletion can still mobilize HSCs— animals and resulted in a significant increase in the number of albeit at lower amplitude—when SNS signaling is disrupted, HSCs/progenitors mobilized by G-CSF (Fig. 7, D-E). To further assess the role of M in a situation where progenitor mobi- argues that the SNS and the BM M act through distinct parallel pathways. Thus, these data suggest antagonistic func - lization is suboptimal, we treated mice with G-CSF (Hidalg o tions of the autonomic nervous system and innate immunity et al., 2004) for 2 d, instead of 4 d. We found that CFU-C, LSK, in regulating the niche (Fig. 6 C), where the SNS promotes and LSKFlk2 cells in the peripheral blood mobilized by 2 d of egress by reducing the expression of key retention factors by G-CSF were increased 3.8-, 5.9-, and 9.3-fold (Fig. 7, F-H), the niche cell (Méndez-Ferrer et al., 2010b), and in contrast, respectively, in mice that were treated with clodronate lipo - BM M promotes the expression of these genes and HSC/ somes 14 h before harvest. Furthermore, we tested whether progenitor retention in the BM. depleting M 10 d before blood collection, rather than 14 h before collection, could synergize with 4 d G-CSF treatment. BM M depletion synergizes with enforced Indeed, when mice were preinfused with clodronate liposomes 10 d before harvest, they had 5.2-, 3.4-, 2.7- fold higher HSC/progenitor mobilization Because the expression of retention genes is still reduced in CFU-C, LSK, and LSKFlk2 in the peripheral blood, respec - the Nestin niche cells 7 d after clodronate treatment (Fig. S5), tively (Fig. 7, I, J, K). Thus, targeting BM M may be a novel we sought to determine the duration of M reduction and modality by which to enhance mobilization yields in patients. the kinetics of recovery. We found that BM M remain markedly (>90%) reduced 10 d after clodronate treatment DISCUSSION (Fig. 7 A). Recovery started by day 16 (58% reduction) and In this study, we sought to identify the role of BM mono- nuclear phagocytes in HSC/progenitor mobilization. Unex - clodronate-treated mice demonstrated no reduction in M counts by day 28. Alternatively, MO populations in the BM pectedly, we found that depletion of mononuclear phagocytes 266 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e Figure 5. Depletion of BM CD169 M, but not CD169 MOs, mobilizes HSCs/progenitors. (A–D) Wild-type (WT) or heterozygous CD169-DTR DTR/+ (CD169 ) mice were treated with DT. (A) Representa- tive dot plots show the percentages of BM mononuclear DTR/+ phagocytes in wild-type (top) or CD169 mice treated with DT (bottom). (B–D) Bar graphs depict the absolute numbers of mononuclear phagocytes (n = 6) in wild type DTR/+ mice and wild type or CD169 mice injected with DT.  + + (E–F) Bar graphs enumerate Lineage Sca-1 c-kit (LSK; E) and LSKFlk2 cells (F) per milliliter of blood in the same mice analyzed in B–D (n = 6). Data are pooled from two independent experiments. (G) CXCL12 levels were mea- sured 72 h after administration of PBS or 1 µg/ml DT into Dexter cultures plated from the BM of CD169-DTR animals (n = 3–4 wells). Representative data from two independent experiments are shown. of niche cells that express Nestin (Méndez-Ferrer et al., 2010b). Although osteoblasts have been proposed to represent a HSC niche, selective modulation of osteoblast numbers do not necessarily alter HSC numbers (Wilson and Trumpp, 2006; Kiel et al., 2007; Zhu et al., 2007), and the lack of osteoblasts in sites of extramedullary hematopoiesis suggest that they are dispensable to support HSCs. Recent studies have suggested that a more primitive pre- cursor of osteoblasts compose the stem cell niche (Méndez-Ferrer et al., 2010b; Omatsu et al., 2010). Steady-state Nestin MSCs express sub- stantially higher levels of genes required in HSC/ progenitor maintenance and retention, including Cxcl12, compared with in vivo sorted (Fig. 3 and using four in vivo models was sufficient to mobilize HSCs/ Fig. S5) or cultured osteoblasts (Méndez-Ferrer et al., 2010b). progenitors. Before this study, mononuclear phagocytes in the The relatively low expression of Cxcl12 detected in sorted BM had been poorly characterized and relied on F4/80 , osteoblasts is consistent with a recent study showing that a marker with a promiscuous expression profile in the BM DT administration into Cxcl12-DTR-GFP mice did not hi (Fig. S1). Herein, we rigorously discriminated among Gr-1 result in loss of spindle-shaped N-cadherin–expressing osteo - lo MOs, Gr1 MOs, and M in the BM by die ff rential expression blasts (Omatsu et al., of Gr-1, CD115, F4/80, CD11b, CD11c, MHC class II, 2010). It has been Figure 6. Opposite influences of the M  DTR/+ CX3CR1, CD68, and CD169. Using CD169 animals, proposed that M and the SNS on HSC/progenitor retention. we were able to implicate M, constituting 2.6% of total depletion mobilizes (A) CFU-C after treatment with PBS (blue BM cells, in the promotion of HSC/progenitor retention HSCs/progenitors bars) or clodronate liposomes (red bars) in through interaction with the recently characterized population wild-type C57BL/6 (n = 11–13), 6OHDA- by disruption of the treated mice (n = 12–13), 2-adrenergic re- / ceptor–deficient ( Adrb2 ) mice treated with an antagonist to the 3-adrenergic receptor / (3R; n = 10), and Adrb2 mice (n =7). Data are pooled from three independent experiments and analyzed with one-way ANOVA/Newman-Keuls test. (B) CFU-C after treatment with PBS (blue bars) or clodronate (red bars) in wild-type FVB mice (n = 5) and / 3-adrenergic receptor-deficient ( Adrb3 ) mice (n = 8). Data are pooled from two inde- pendent experiments. (C) Schematic of an- tagonistic regulation of HSC/progenitor retention by M and the SNS. JEM VOL. 208, February 14, 2011 267 osteoblasts either 14 h or 7 d after the depletion of M, de- spite the fact that HSCs/progenitors were clearly elevated at both time points. In contrast, significant reductions in the ex - pression of HSC retention genes were observed in Nestin cells, and correlated with persistent HSC/progenitor mobili - zation even 7 d after clodronate treatment. These results thus indicate that CD169 BM M promote HSC retention by acting specifically on the Nestin HSC niche in the BM. Previous studies have revealed that a transplantable cell expressing the G-CSF receptor (G-CSFR) is essential for G-CSF–induced mobilization (Liu et al., 2000). An accom - panying study, using an elegant mouse model in which the G-CSFR is expressed exclusively in CD68-expressing cells, also implicates mononuclear phagocytes in G-CSF mobilization (see Christopher et al. in this issue). Thus, G-CSF signaling exclusively in M is sufficient to reduce niche retention and promote HSC/progenitor mobilization. However, because G-CSF–induced HSC/progenitor mobilization is at least three times more potent than M depletion with clodronate liposomes (this study; Winkler et al., 2010), it must also be acting on cells other than M. The SNS is required for progenitor egress (Katayama et al., 2006; Méndez-Ferrer et al., 2010a), suggesting that G-CSF– mediated increase of sympathetic tone in the BM may repre- sent a putative M-independent target. Our results using models with impaired sympathetic activity suggest that BM M exert antagonistic, independent regulatory functions in the HSC niche compared with the SNS. Although G-CSF likely has several targets in the BM microenvironment, the present data Figure 7. M depletion synergizes with AMD3100 and G-CSF uncover two distinct opposing activities that lead to major mobilization. (A–C) Kinetics of M reduction (A) and LSK (B) and LSKFlk2 changes in HSC retention by Nestin niche cells. We thus (C) mobilization, at the indicated time points, after administration of PBS- (blue) or clodronate-encapsulated (red) liposomes (n = 3–4). Data are propose that G-CSF induces HSC mobilization by inhibiting pooled from two independent experiments. (D–E) CFU-C from peripheral M-mediated retention signals and simultaneously enhanc - blood of mice that were treated with PBS (blue) or clodronate liposomes ing sympathetic-mediated progenitor release (Fig. 6 C). (red; 14 h before harvest) and mobilized with AMD3100 (D; 1 h before har- These results expand our understanding of HSC niche vest) or G-CSF (E; 4 d). Data are pooled from two independent experiments. components by implicating a cellular constituent of the innate (F–H) CFU-C (F), LSK (G), and LSKFlk2 (H) cells from the peripheral blood immune system, the CD169 M, as a niche regulator. Because of mice that were mobilized for 2 d with G-CSF and treated with PBS- or targeted reduction of BM M can enhance HSC/progenitor clodronate-encapsulated liposomes 14 h before harvest. Experiment was mobilization, the use of antibodies against CSF-1 (M-CSF), performed once (n = 4). (I–K) CFU-C (I), LSK (J), and LSKFlk2 (K) from its receptor, or small molecule inhibitors of M-CSF signaling 16-wk-old female mice that were pretreated with PBS- or clodronate- encapsulated liposomes 10 d before harvest and mobilized with G-CSF may provide a novel strategy to increase the ec ffi iency of HSC/ for 4 d. Data are representative of two independent experiments (n = 4). progenitor mobilization for autologous transplantation. osteoblastic niche (Winkler et al., 2010). Winkler et al. ob - MATERIALS AND METHODS served a reduction in osteoblast numbers, as determined by Mice. All experiments, unless otherwise noted, were performed on 8–10- histomorphometry, on day 4 after two treatments with clodro- wk-old C57BL/6 male mice from Charles River Laboratories (Frederick nate liposomes on days 0 and 2. Although we have not ob - Cancer Research Center, Frederick, Maryland). 2-adrenergic receptor- served any significant reduction in osteoblast counts 14 h tm1Bkk deficient ( Adrb2 /J; Chruscinski et al., 1999; gift from G. Karsenty , or 7 d after clodronate treatment, it remains possible that osteo - Columbia University, New York, NY), 3-adrenergic receptor-deficient tm1low blast numbers may have transiently decreased and recovered (FVB/N-Adrb3 l/J; Susulic et al., 1995; The Jackson Laboratory), FVB/ N-CD11b-DTR (Cailhier et al., 2005; gift from C. Aloman, Mount Sinai after M ablation. In addition to CXCL12, the regulation Medical Center, New York, NY), Mafia (C57BL/6-Tg[Csf1r-EGFP-NGFR/ of other key retention genes appears to correlate with HSC/ FKBP1A/TNFRSF6]2Bck/J; Burnett et al., 2004; The Jackson Laboratory), progenitor egress. For example, treatment with G-CSF or 3R CX3CR1/GFP (Jung et al., 2000; gift from D. Littman, New York University, agonists down-regulates Cxcl12, Angiopoietin-1, Kitl, and New York, NY), CD169-DTR (Miyake et al., 2007), Nes-Gfp (Mignone Vcam-1 (Méndez-Ferrer et al., 2010b). We have not detected ERT2 et al., 2004), Nes-Cre (Balordi and Fishell, 2007), iDTR (C57BL/6-Gt[R / any reductions in the expression of HSC retention genes in OSA]26Sortm1[HBEGF]Awai/J; Buch et al., 2005), IL-10 (Berg et al., 1996; 268 Bone marrow macrophages promote HSC retention | Chow et al. A r t i c l e gift from H. Xiong, Mount Sinai Medical Center, New York, NY), and CFU-C assays and mobilization. Colony-forming assays were performed / TNF (Marino et al., 1997; gift from M. van den Brink, Memorial Sloan as previously described (Frenette et al., 1998). Mobilization experiments with Kettering Cancer Center, New York, NY) mice were also used in these stud- AMD3100 and 4-d G-CSF were performed as previously described (Katayama ies. FVB/N-CD11b-DTR and Mafia (CD45.2) BM chimeras were gener - et al., 2006; Lucas et al., 2008). Some experiments were performed with subopti - 6 6 ated by transplanting 2.0 × 10 and 1.9 × 10 BM-nucleated cells from male mal G-CSF treatment (2 d), as previously described (Hidalgo et al., 2004). donors into lethally irradiated 8-wk-old FVB/N (The Jackson Laboratory) and C57BL/6 Ly5.2 (CD45.1) male mice, respectively. BM and blood of RNA isolation, reverse transcription, and Q-PCR. For measurement Mafia BM chimeras showed >95% donor chimerism as assessed by flow cyto- of BM Cxcl12 gene expression, femurs were flushed and mixed with 0.5 ml metry 1 mo after transplantation. Mice were maintained on a 12 h light/ Trizol (Invitrogen) and stored at 80°C. Conventional reverse transcrip- 12 h darkness lighting schedule. All in vivo experiments were harvested be - tion, using the Sprint PowerScript reverse transcription (Takara Bio Inc.) tween 12:00 and 1:00 p.m. (Zeitgeber time 5:00 and 6:00) to limit circadian was performed in accordance with the manufacturer’s instructions. Q-PCR variations in HSC/progenitor release (Méndez-Ferrer et al., 2008) and mo - was performed with SYBR GREEN on an ABI PRISM 7900HT Sequence bilization (Lucas et al., 2008). All mice were housed in specific pathogen–free Detection System (Applied Biosystems). The PCR protocol consisted of one facilities at the Mount Sinai School of Medicine or Albert Einstein College cycle at 95°C (10 min) followed by 40 cycles of 95°C (15 s) and 60°C of Medicine animal facility. Experimental procedures performed on the mice (1 min). Expression of Gapdh was used as a standard. The mean threshold cycle were approved by the Animal Care and Use Committee of the Mount Sinai number (C ) for each tested mRNA was used to quantify the relative ex- School of Medicine and Albert Einstein College of Medicine. pression of each gene: 2^(C [Gapdh]  C [gene]). Primers used are listed t t below: Cxcl12_fwd, 5-CGCCAAGGTCGTCGCCG-3; Cxcl12_rev, 5-TTGGCTCTGGCGATGTGGC-3; Angpt1_fwd, 5-CTCGTCAG- In vivo cell depletion. Cl2MDP (or clodronate) was a gift from Roche ACATTCATCATCCAG-3; Angpt1_rev, 5-CACCTTCTTTAGTG- (Van Rooijen and Sanders, 1994). Clodronate liposomes (250 µl) were in - CAAAGGCT-3; Kitl_fwd, 5-CCCTGAAGACTCGGGCCTA-3; Kitl_ fused i.v. at 1 d (14 h), 7 d, 10 d, 16 d, or 28 d before harvest. CD11b-DTR rev, 5-CAATTACAAGCGAAATGAGAGCC-3; Vcam1_fwd, 5-GACC- mice were treated with DT i.p. 25 ng/g on days 1 and 3 before harvest. DT TGTTCCAGCGAGGGTCTA-3; Vcam1_rev, 5-CTTCCATCCT- was purchased from Sigma-Aldrich. AP20187 was a gift from Ariad Pharma- CATAGCAATTAAGGTG-3; Osteocalcin_fwd, 5-GGGCAATAAGG- ceuticals. Lyophilized AP20187 was dissolved in 100% ethanol at a concentration TAGTGAACAG-3; Osteocalcin_rev, 5-GCAGCACAGGTCCTAAA- of 62.5 mg/ml stock solution and was stored at –20°C. As recommended TAGT-3; Gapdh_fwd, 5-TGTGTCCGTCGTGGATCTGA-3; Gapdh_ by Ariad Pharmaceuticals, injection solutions were prepared with a diluent rev, 5-CCTGCTTCACCACCTTCTTGA-3. composed of 4% ethanol, 10% PEG-400, and 2% Tween-20 in water. All in - jections were administered i.v. within 30 min after preparation. The volume CXCL12 ELISA. 96-well ELISA plates were coated overnight at 4°C wit h of injection solution was adjusted according to the average mouse body 50 µl of 2 µg/ml anti-CXCL12 coating antibody (MAB350; R&D Systems). weight to deliver a dose of 10 mg/kg AP20187 per mouse in an mean vol - Next, the wells were washed three times with wash bue ff r (0.05% Tween 20 in ume of 100 µl. Mice were injected daily for 5 d before harvest. Heterozygous DTR/+ PBS) and incubated for 1 h at room temperature with 200 µl of blocking bue ff r CD169-DTR (CD169 ) or control C57BL/6 were injected i.p. with (1% BSA, 5% D-Sucrose, and 0.05% NaN in PBS; all from Thermo Fisher Sc- i 10 µg/kg DT 48 h before harvest. Depletion of Nestin cells was accom - entic fi ). After 3 washes, 100 µl of samples diluted 1:2 in PBS were added and plished as previously described (Méndez-Ferrer et al., 2010b). incubated for 2 h at room temperature. After 3 washes, 100 µl of 0.250 µg/ml polyclonal biotinylated anti–human/mouse SDF-1 (BAF310; R&D Systems) Flow cytometry and cell sorting. Fluorochrome-conjugated or biotinylated was added and incubated for 2 h at room temperature. After 3 washes, 100 µl of mAbs specific to mouse Gr-1 (Ly6G/C; clone RB6-8C5), CD115 (clone 0.1 µg/ml Neutravidin-HRP (Thermo Fisher Scientic fi ) was added and incu - AFS98), Siglec-F (clone E50-2440), CD11b (clone M1/70), CD11c (clone bated for 30 min. After 3 additional washes, the reaction was developed by incu - N418), I-A/I-E clone (clone M5/114.15.2), CD45 (clone 30-F11), Sca- 1 bation for 20–30 min with 50 µl of TMB substrate solution (Sigma-Aldrich) and (clone D7), Flk2 (clone A2F10), CD117 (clone 2B8), CD3 (clone 145-2C11), stopped by adding 50 µl of 1M HCl solution (Thermo Fisher Scientic fi ). Optical B220 (clone RA3-6B2), Ter119 (clone TER-119), CD51 (clone RMV-7), and density was determined with a microplate reader set at 450 nm. Optical density CD31 (clone MEC13.3), corresponding isotype controls, and secondary of PBS control wells was subtracted from optical density of samples. Recom-bi reagents (eu fl or450-, APC-eu fl or780–, and PE-Cy7–conjugated streptavidin) nant mSDF-1 (PeproTech) was used to generate a linear standard curve. were purchased from eBioscience. Anti-F4/80 (clone CI:A3.1), CD68 (clone FA-11), and CD169 (clone 3D6.112) were purchased from AbD Serotec. CD68 Immunou fl orescence. Anesthetized Nes-GFP transgenic animals (Mignone was stained extracellularly and subsequently intracellularly with the Cyt/ ox fi et al., 2004) were perfused, and femurs and tibia were sectioned and stored as Cytoperm kit (BD) according to the manufacturer’s protocol. Multiparameter previously described (Méndez-Ferrer et al., 2008). Slides were washed three analyses of stained cell suspensions were performed on an LSRII (BD) and an-a  times in Coplin jars (Sigma-Aldrich) to remove OCT solution residue, and lyzed with FlowJo software (Tree Star, Inc.). DAPI single cells were evaluated for then incubated for 1 h at room temperature in 20% goat serum (Sgma- all analyses except for intracellular stains. To purify mononuclear phagocyte p-op Aldrich) diluted in PBS + 0.1% Tween solution (PBSTw). After 3 washes in ulations, BM was sorted with an InFlux cell sorter (BD) to achieve >97% purity. PBSTw, slides were incubated in PBSTw + 2% goat serum + 0.5% Triton Sorted mononuclear phagocytes were cytospun with Cytospin 3 (Thermo Fisher X-100 for 1 h at room temperature. After 3 washes in PBSTw, slides were Scientic fi ) and stained with Hema 3 manual staining system (Thermo Fisher +  incubated in primary antibody (anti-CD68-Alexa Fluor 647 [clone FA-11; Scientic fi ). To isolate Nestin and Nestin cells from the BM for Q-PCR, RBC- AbD Serotec] and anti–CD169-biotin [clone MOMA-1; AbCam]) at a 1:100 lysed BM cells were digested with collagenase, trypsin, and DNase, as previously concentration in PBSTw + 2% goat serum overnight in the dark at room described (Méndez-Ferrer et al., 2010b). Endothelial cells and osteoblasts were temperature. After three washes in PBSTw, CD169 staining was continued isolated similar to previous studies (Semerad et al., 2005; Winkler et al., 2010 ). with streptavidin-PE staining for 5 min. Slides were mounted with Vector- In brief, tibias, femurs, and humeri of mice were u fl shed thoroughly of BM shield + DAPI, covered, and sealed with nail polish. Images were acquired cells, chopped with a scalpel, and washed three times through a 5-ml polystyrene on an Examiner microscope (Carl Zeiss, Inc.) and all images were processed tube with blue-top cell strainer (BD) to further remove residual BM cells. The using Slidebook software (Intelligent Imaging Innovations, Inc). bone fragments were then digested at 37°C with Type IA collagenase (Sigma- Aldrich) for 40 min while spinning. RBC-lysed pellet was then stained for so -rt ing. Cells were sorted by Moflo Cell sorter (Dako) at the Flow Cytometry Long-term BM Dexter cultures. 3.7 × 10 BM-nucleated cell s Core Facility at Mount Sinai School of Medicine or Aria Cell sorter (BD) at the were plated in 1 ml Dexter medium (Myelocult M5300 media [Stem Cell Flow Cytometry Core Facility at Albert Einstein College of Medicine. Technologies] supplemented with 1% penicillin–streptomycin [Cellgr o], JEM VOL. 208, February 14, 2011 269 + 6 CXCL12 reduction. Fig. S4 shows the sorting strategy for Nestin and Nes- 1% amphotericin [Cellgro], and 10 M freshly thawed Hydrocortisone [Sigma- tin fractions and bone endothelial cell and osteoblast fractions. Fig. S5 shows Aldrich]) in 12-well plates. Cultures were maintained in a water-jacketed - in that retention gene expression is reduced in Nestin cells seven days after cubator at 33°C and 5% CO . Half the media was changed weekly for 6 wk. In the sixth week, the culture media was removed and replaced with 1 ml of mononuclear phagocyte treatment. Fig. S6 shows that mononuclear phago - Dexter medium containing 40% PBS- or clodronate-encapsulated liposomes cytes are not reduced 7 d after depletion of Nestin cells. Fig. S7 shows that by volume in some experiments. After 24-h incubation, the culture media soluble factor from a M, but not myeloblast, cell line enhances stromal was removed and 1 ml fresh media was added. 24 h and 72 h later, the media CXCL12 production. Online supplemental material is available at http:// was collected and frozen to assess CXCL12 levels by ELISA and the adherent www.jem.org/cgi/content/full/jem.20101688/DC1. layer was Hema 3-stained to assess cell morphology or detached by cell We would like to acknowledge experimental help and mice provided by scraper (BD) for o fl w cytometric analysis. In experiments with Dexter cultures DTR/+ O.M. Smith, M. van den Brink, and all the investigators that provided mice derived from CD169 BM, the media was removed and replaced with 1 ml for these experiments. fresh Dexter medium containing PBS or 1 µg/ml DT. After 72 h of incuba- This work was supported by the National Institutes of Health grants tion, the media was frozen and later assessed for CXCL12 levels by ELISA. R01DK056638 and R01HL097819 to P.S Frenette, R01CA112100 to M. Merad, and P30CA013330 to the AECOM Flow Cytometry Core Facility. A. Chow, D. Lucas, MS-5 cell culture. MS-5 cells were grown in monolayers in complete me - A. Hidalgo, S. Mendez-Ferrer, C. Scheiermann, and M. Battista were supported by dium (-MEM medium supplemented with 10% FBS [Stem Cell Technolo - fellowships from NHLBI (1F30HL099028-01; A. Chow), Fundación Ramón Areces gies], penicillin–streptomycin [Invitrogen], 5% glutamine [Invitrogen], and (D. Lucas), American Heart Association Scientist Development Grant (0735165N; 5% sodium pyruvate [Invitrogen]). Cultures were maintained at 37°C and A. Hidalgo), Ramón y Cajal Fellowship from the Spanish Ministry of Science and 1:10 split with 0.05% trypsin-EDTA (Invitrogen) every 3 or 4 d, when cells Innovation (A. Hidalgo and S. Méndez-Ferrer), Scholar Award from the American reached 80% confluence. 5,000 MS-5 cells were plated in 300 µl complete Society for Hematology (S. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Feb 14, 2011

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