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Distinct Biological Effects of Macrophage Inflammatory Protein-1 and Stroma-Derived Factor-1 on CD34⁺ Hemopoietic Cells

^a Department of Haematology, University Hospital Essen, Essen, Germany;
^b CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom

Key Words. Chemokines • MIP-1 • SDF-1 • Hemopoietic cells • Calcium flux • Growth inhibition • Adhesion

Dr. Clare Heyworth, CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemokines are important regulators of both hemopoietic progenitor cell (HPC) proliferation and adhesion to extracellular matrix molecules. Here, we compared the biological effects of the CC chemokine macrophage inflammatory protein-1 (MIP-1) with those of the CXC chemokine stroma-derived factor-1 (SDF-1) on immunomagnetically purified CD34⁺ cells from leukapheresis products (LP CD34⁺). In particular, studies on chemokine-induced alterations of LP CD34⁺ cell attachment to fibronectin-coated plastic surfaces, proliferation of these cells in colony-forming cell (CFC) assays and intracellular calcium mobilization were performed. MIP-1 but not SDF-1 was found to increase the adhesion of LP CD34⁺ cells to fibronectin in a dose-dependent manner. Both chemokines elicited growth-suppressive effects on LP CD34⁺ cells in CFC assays. While MIP-1 reduced the number of granulomonocytic (CFC-GM) and erythroid (BFU-E) colonies to the same extent, SDF-1 showed a significantly greater inhibitory effect on CFC-GM than BFU-E. Finally, we demonstrated that SDF-1 but not MIP-1 triggers increases in intracellular calcium in LP CD34⁺ cells. The SDF-1-induced calcium response was rapid and concentration-dependent, with a maximal stimulation observed at 15 ng/ml. In conclusion, our data suggest distinct biological properties of SDF-1 and MIP-1 in terms of modulation of LP CD34⁺ cell adhesion to fibronectin and intracellular calcium levels. However, comparable growth-suppressive effects on HPC proliferation were observed, indicating that this feature may be independent of chemokine-induced calcium responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemokines—chemotactic cytokines—are important regulators of hemopoietic cell proliferation, adhesion to extracellular matrix molecules, and migration. A number of recent articles have reviewed their structural and functional properties [1-3]. They can be classified into at least four different subfamilies on the basis of the relative position of their cysteine residues. The largest families are the and ß chemokines. The chemokines are characterized by a CXC motif and include PF-4, interleukin 8 (IL-8), and stroma-derived factor-1 (SDF-1). The ß chemokines are defined by two adjacent cysteine residues (CC motif) and include macrophage inflammatory protein-1 (MIP-1) MIP-1, MCP-1 and RANTES. Chemokines are produced by an array of stromal cell types, including endothelial cells [4], macrophages [5], and fibroblasts [6] in response to pro-inflammatory stimuli [7].

Chemokines elicit their effects on primitive hemopoietic cells via G-protein-coupled receptors, several of which have recently been cloned [8-12]. To date, 10 human CC chemokine receptors (CCR-1 through CCR-10) and five CXC chemokine receptors (CXCR-1 through CXCR-5) have been identified. MIP-1 has been shown to bind to at least three of these CCR: 1, 5, and 9 [9, 11-13], and possibly CCR-3 and CCR-4 [8, 10, 13], whereas SDF-1 has been shown to interact with CXCR-4 [14, 15]. Most of these receptors, i.e., CCR-1, CCR-4, CCR-5, and CXCR-4, have been shown to be expressed by CD34⁺ cells [16-20]. Furthermore, recent studies revealed that chemokine receptors from both subfamilies, in particular CCR-3, CCR-5, and CXCR-4, can serve as coreceptors in association with CD4 for HIV binding to hemopoietic cells, thereby offering an explanation for the myelosuppression observed in the course of the HIV infection [14, 15, 21-24].

While the pattern of chemokine receptor expression in immature hemopoietic progenitor cells begins to be elucidated, comparably little is known about the functional changes and signal transduction events elicited as a consequence of chemokine binding to its respective receptor(s) in CD34⁺ cells. We have recently investigated the growth-inhibitory effects of MIP-1 on CD34⁺ cells isolated from human umbilical cord blood (CB) and normal bone marrow. Our own results and those of others show that MIP-1 affects the proliferative state of hemopoietic progenitor cells in a complex way depending on the origin [16] and the maturational stage of the target cells [25, 26]. In the present study, we compare the biological effects of MIP-1 with those of the CXC chemokine SDF-1 on immunomagnetically purified CD34⁺ cells from leukapheresis products (LP CD34⁺).

	Materials and Methods

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Cells
Human umbilical CB samples were collected from full-term normal deliveries. Leukapheresis products (LP) were obtained from 31 patients with hematological malignancies or solid tumors (11 multiple myeloma, four acute lymphoblastic leukemia, three acute myeloid leukemia, three teratoma, three non-Hodgkin's lymphoma, six breast cancer, and one chronic lymphocytic leukemia). In order to mobilize hemopoietic progenitor cells into the peripheral blood, all patients received appropriate cytotoxic chemotherapy followed by recombinant G-CSF (lenograstim, 263 µg/day s.c.). Leukapheresis was performed on these patients when the rising blood white cell count exceeded 3 x 10⁹/l. At the time of the leukapheresis, all patients were in remission. All samples were obtained with informed consent. Samples were collected in sterile tubes containing preservative-free heparin, and the mononuclear cells (MNC) were isolated by centrifugation on Ficoll-Hypaque (Lymphoprep, 1.077 g/ml, Nycomed; Birmingham, UK) at 400 g for 25 min. The MNC at the interface were collected and washed in phosphate buffered saline (PBS) containing 0.5% (w/v) bovine serum albumin (BSA).

Isolation of CD34⁺ Cells
CD34⁺ cells from LP were isolated using the Mini-Macs^® immunomagnetic separation system (Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer's instructions. Briefly, 10⁸ cells were suspended in 300 µl of sorting buffer (PBS supplemented with 5 mM EDTA and 0.5 % (w/v) BSA) and incubated with 100 µl human IgG FcR blocking antibody and 100 µl monoclonal microbead-conjugated CD34 antibody (clone QBEND/10; Miltenyi Biotec) for 30 min at 4°C. Thereafter, the cells were washed and passed through a 30-µm nylon mesh and separated in a column exposed to the magnetic field of the Macs device. The column was washed four times with sorting buffer (500-µl aliquots) and removed from the separator. The retained cells were eluted in 1 ml sorting buffer and counted using a hemocytometer. The purity of CB and LP CD34⁺ cell preparations was not routinely assessed but usually exceeded 80% as determined by flow cytometry, and this was in agreement with our previous data [18].

Clonogenic Assays
Two to three thousand CD34⁺ cells were plated, as previously described [27]. Briefly, cells were added to a 1 ml mixture containing 30% (v/v) fetal calf serum (FCS), 10% (w/v) deionized BSA, 10% (v/v) 5637-conditioned medium, two units erythropoietin (EPO), and 1.35% (w/v) methylcellulose in Iscove's modified Dulbecco's medium ([IMDM]; GIBCO Laboratories; Grand Island, NY). After thorough mixing, cells were plated in triplicate and incubated for 14 days at 37°C in 5% CO₂ and 5% O₂ in nitrogen. Colonies of granulocyte-macrophage cells (CFC-GM) or erythroid cells (BFU-E) were assessed.

Cell Adhesion Assay
Adhesion assays were performed in 96-well flat-bottom microtiter plates (Costar; Cambridge, MA), as previously described [28]. Briefly, wells were coated for 60 min at room temperature with 50 µl aliquots of human fibronectin (Sigma; Poole, Dorset, UK) at 50 µg/ml in PBS, and nonspecific binding sites blocked with 50 µl of 10 mg/ml heat-denatured BSA. CD34⁺ cells recovered from the mini-Macs were resuspended at 1.5 x 10⁶/ml in IMDM (serum-free medium). One hundred-µl aliquots of cell suspensions were then added to the wells in duplicate and incubated for 60 min at 37°C in a CO₂ incubator. MIP-1 (MIP-1, BB10010, with improved solution properties and of clinical grade was supplied by British Biotech; Oxford, UK, [29]) or SDF-1 (supplied by R&D Systems; Abingdon, UK) was immediately added. Nonadherent cells were removed by shaking the plates horizontally on an electronic shaker set at 100 excursions/min for 30 seconds and washing twice with 100 µl PBS. Bound cells were removed by aspiration with PBS. Both fractions were counted with a hemocytometer and adhesion expressed as the percentage of bound cells/(unbound + bound cells). Aliquots of each fraction (containing 2,000-3,000 cells) were also plated in colony-forming cell assays, and the percentage of adhering colonies of granulocyte-macrophage cells (CFC-GM) and erythroid cells (BFU-E) was assessed using standard criteria after 14 days' incubation.-

Ca²⁺ Mobilization Assay
This assay was performed as previously described with minor modifications [30]. MNC and CD34⁺ cells recovered from suspension cultures (overnight incubation of LP CD34⁺ cells in IMDM/15% [v/v] FCS in the presence or absence of tumor necrosis factor- (TNF-) [2 ng/ml, R&D Systems]) were loaded for 30 min at 37°C with 3 µM Indo-1/acetoxymethylester per 5 x 10⁶ cells/ml in RPMI 1640 with 1% (v/v) FCS. At the time of the analysis, 100 µl of the cell suspension were added to 400 µl of prewarmed (37°C) RPMI 1640/1% (v/v) FCS containing CaCl₂ (1 mM). Analyses were performed on a Becton Dickinson FACS Vantage (Becton Dickinson; San Jose, CA). The fluorescent signal induced by the changes in intracellular Ca²⁺ was measured by monitoring violet and blue fluorescent emissions at 395 and 530 nm, respectively. The ratio of indo-1 violet to blue fluorescence was digitally calculated by the PC Lysis II software package (Becton Dickinson) and displayed against the elapsed time. Cells were analyzed at 37°C at a flow rate of approximately 200 cells/sec. Forward and side scatter were used to gate live cells from dead cells and debris. For each experiment, a baseline unstimulated measurement was obtained which was followed by addition of the indicated stimulus. Results were assessed as chemokine induced increase of the indo-1 violet to blue fluorescence ratio over baseline values.

Statistical Analysis
Data are expressed as mean ± standard error (SE). The Student's t-test for paired samples was used to compare results between MIP-1- and SDF-1-treated LP CD34⁺ cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MIP-1 but not SDF-1 Increases the Adhesion of LP CD34⁺ Cells to Fibronectin
Figure 1 shows a time course of baseline and MIP-1-stimulated adhesion of LP CD34⁺ cells to immobilized fibronectin. Unstimulated adhesion reached a plateau after 30 min at 37°C, and adhesion remained at this level for the entire observation period of 120 min. In contrast, MIP-1-stimulated adhesion curve profile peaked at 60 min post initiation of the assay. At that time, MIP-1 was found to increase LP CD34⁺ cell adhesion to a maximum of 59 ± 11% as compared with 31 ± 9% in the control group (mean ± SE; n = 3, p < 0.05). Based on these findings, 60-min incubation periods were used for dose-response studies of MIP-1 and SDF-1.

View larger version (15K): [in this window] [in a new window]   Figure 1. Kinetics of LP CD34+ cell adhesion to fibronectin; effect of MIP-1. LP CD34+ cells were cultured in fibronectin-coated tissue culture plates for various time periods in the presence or absence of MIP-1 as described in Materials and Methods. MIP-1 (15 ng/ml) was directly added to the culture medium at the beginning of each assay. Results are mean± SE of three independent experiments. * Denotes significant difference between MIP-1-treated and -untreated cells.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of MIP-1 (A and B) and SDF-1 (C and D) on the adhesion of LP CD34+ cells to fibronectin. All adhesion experiments were performed in serum-free medium for 60 min at 37°C. Adhesion was quantified by counting the numbers of fibronectin adherent and nonadherent cells (A and C) and also by determining the total numbers of colonies (CFC-GM and BFU-E) generated from these cells in CFC assays (B and D). Chemokines were directly added at the indicated concentration to the adhesion medium at the beginning of each assay. Results are mean ± SE of five independent experiments. 0/BSA indicates attachment of CD34+ cells to BSA in the absence of chemokine stimulants and served as a control for nonspecific cell binding. * Denotes significant difference between MIP-1-treated cells and the respective controls (FN, no MIP-1)

The Effects of MIP-1 and SDF-1 on Colony Formation from CD34⁺ Cells
To minimize contributory effects of accessory cell types—in particular, macrophages and T lymphocytes that have been reported to be functionally activated by MIP-1 and SDF-1—we used immunomagnetically purified CD34⁺ cells. Also, this approach allowed direct comparisons of dose-effect curves between clonogenic and adhesion assays. In Figures 3A and 3B, the effects of varying concentrations of MIP-1 and SDF-1 on colony formation from LP CD34⁺cells are compared. The addition of MIP-1 and SDF-1 to LP CD34⁺ cell suspensions in clonogenic assays resulted in significant concentration-dependent reductions of the number of CFC-GM formed, at a chemokine concentration of 150 ng/ml to 53 ± 9% (n = 10) and 36 ± 10% (n = 8; mean ± SE, p < 0.05) of the respective controls ( Fig. 3A and 3B). It is noteworthy that MIP-1 exerted comparable growth-suppressive effects on CFC-GM and BFU-E (p >0.05), whereas SDF-1 inhibition of CFC-GM growth was significantly greater than that of BFU-E (p < 0.05). Similar results were obtained for SDF-1-promoted colony growth inhibition of CD34⁺ cells isolated from umbilical CB samples (Fig. 3C), although here the SDF-1-induced suppression of BFU-E reached statistically significant levels.

View larger version (14K): [in this window] [in a new window]   Figure 3. Effects of MIP-1 (A) and SDF-1 (B and C) on the proliferation of colony-forming cells in clonogenic methylcellulose cultures. 2,000 to 3,000 LP CD34+ cells were cultured in the continuous presence of MIP-1 (A) and SDF-1 (B) or 2,000 CB CD34+ (C) at the indicated concentrations. Results of n (shown) experiments are expressed as the total numbers of colonies (CFC-GM and BFU-E) generated from these cells in the percentage of untreated controls. Error bars indicate SE. Control cultures contained 37 ± 6 CFC-GM and 216 ± 39 BFU-E. * Denotes significant difference (p < 0.05) between chemokine-treated cultures and untreated controls. # Denotes significant difference (p < 0.05) between CFC-GM and BFU-E in SDF-1-treated cultures.

View this table: [in this window] [in a new window]   Table 1. Effects of different growth factor combinations on MIP-1- and SDF-1-mediated inhibition of hemopoietic colony formation

The Effects of MIP-1 and SDF-1 on Cytosolic Calcium Levels in LP CD34⁺ and Mononuclear Cells

View larger version (19K): [in this window] [in a new window]   Figure 4. A) Calcium mobilization responses of LP CD34+ cells to MIP-1 and SDF-1 (both at 150 ng/ml). LP CD34+ cells were harvested from 10-h suspension cultures and loaded with indo-1 before stimulation with the indicated chemokine. Details of the indo-1 staining protocol and flow cytometric analysis are given in Materials and Methods. One representative experiment of three performed is shown. B) Dose-response effects of SDF-1 on calcium mobilization in LP CD34+ cells. The assay was performed as described in the legend to Figure 4A andMaterials and Methods.

View larger version (20K): [in this window] [in a new window]   Figure 5. A) Comparison of SDF-1 and MIP-1 (both at 150 ng/ml) induced intracellular calcium responses in LP MNC. B) Dose-response effects of MIP-1 on calcium mobilization in LP MNC cells. One representative experiment of three performed is shown. C) MIP-1 induced calcium responses in LP MNC are restricted to the monocytic cell population. Intracellular calcium elevations in response to MIP-1 (at 150 ng/ml) were analyzed separately for the total MNC population and cells comprised in the lymphocyte and monocyte gates using standard side and forward scatter criteria.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In a second series of experiments, we investigated the effects of MIP-1 and SDF-1 on colony formation from LP CD34⁺ cells. Immunomagnetically isolated CD34⁺ cells were incubated in the presence of various concentrations of the respective chemokine and various cytokines for 14 days. We used CD34⁺ cells, since this allowed us to eliminate contributory effects of accessory cells such as lymphocytes and monocytes that are known to be activated by these chemokines [32]. Both the chemokines used induced a dose-dependent inhibition of colony formation. MIP-1 and SDF-1 exerted a greater effect on hemopoietic progenitor cells of the granulomonocytic (CFC-GM) than of the erythroid (BFU-E) lineage, but these differences reached statistical significance only in SDF-1-treated cultures.

A possible problem in the interpretation of experiments using primary CD34⁺ cells is potential differences in the biological properties of CD34⁺ cells from different cell sources. Importantly, Aiuti et al. [30] have shown that CD34⁺ cells isolated from peripheral blood exhibit a reduced chemotactic response to SDF-1 when compared with CD34⁺ isolated from bone marrow. Similarly, we have shown that MIP-1 has a differential response depending on the source of CD34⁺ cells. If cells are isolated from normal bone marrow, MIP-1 inhibits colony formation; however, if CD34⁺ cells are isolated from umbilical CB, MIP-1 stimulates the formation of CFC-GM [16]. In this context, it is noteworthy that our experiments revealed comparable SDF-1-induced growth-suppressive effects in LP CD34⁺ cells and CD34⁺ cells isolated from CB and bone marrow. While the growth-suppressive action of MIP-1 has been previously established by our group [16, 33] and others [26, 31], SDF-1 inhibition of human hemopoietic colony-forming cell growth has not yet been reported in the literature. However, recently it was shown that SDF-1 binding to its receptor, CXCR-4, can confer myelosuppressive effects in the murine 32D cell line model [34].

MIP-1 binding to its receptors [8-13] is associated with transient elevations of intracellular calcium levels [Ca²⁺]_i, which has been used as an indicator of receptor activation [9, 33]. Other signal transduction pathways further downstream of the receptors have only been partially elucidated. Aronica et al. [35] showed that treatment of MO7e cells with MIP-1 inhibits upregulation of Raf-1 kinase activity in response to GM-CSF and SCF. The same authors recently reported that the chemokine interferon-inducible protein 10 and MIP-1 block the stimulatory effects of GM-CSF and SCF on MAP kinase activity in MO7e cells [36]. These biochemical findings support earlier data suggesting that MIP-1 only exhibits maximal inhibition of colony growth when combinations of growth factors are used, not single cytokines [31]. To test whether similar conditions are required for the inhibitory action of SDF-1, we modified the assay in that we omitted the 5637 conditioned medium (which is known to contain G-CSF, GM-CSF, IL-1ß, M-CSF, and SCF [27, 37]) and EPO and substituted recombinant human G-CSF (2,500 U/ml) for them. As expected [31], the inhibitory action of MIP-1 was completely abrogated, whereas SDF-1 still exerted some, if considerably reduced, growth-suppressive effects.

We next tried to further define and compare the signal transduction events associated with the binding of the two chemokines to their respective receptors. The ability of the two chemokines to rapidly and transiently increase intracellular calcium levels [Ca²⁺]_i in LP CD34⁺ cells was measured. Interestingly, SDF-1 but not MIP-1 was found to be a potent mobilizer of intracellular free calcium in LP CD34⁺ cells. Dose-response studies revealed a concentration-dependent SDF-1 effect with a limit of detection at 1.5 to 15 ng/ml and a maximum response observed at 150 ng/ml. MIP-1, by contrast, did not affect intracellular calcium levels over a concentration range of 1.5 to 300 ng/ml. The unresponsiveness of LP CD34⁺ cells to MIP-1 could not be explained by the absence of MIP-1 receptors, since flow cytometric analysis revealed that the majority of LP CD34⁺ cells bound biotinylated MIP-1 molecules [18].

When similar experiments were performed on LP MNC, a dose-dependent calcium response to MIP-1 became evident. However, gating revealed that this response was entirely due to calcium elevations in the monocytic subpopulation, while the cell population in the lymphocyte gate (which includes the CD34⁺ cells) remained unaffected. Flow cytometric analysis of MIP-1 receptor expression on monocytes (identified by the presence of the CD14 antigen and their characteristic forward and side scatter profile) yielded comparable levels to those observed for the LP CD34⁺ cells. Differential expression of MIP-1 receptor subtypes may account for the observed differences of the MIP-1-induced calcium responses in the monocytic and the lymphocytic cell populations. It is also possible that the variety of responses to MIP-1 is a reflection of both the receptor ligand interaction and the intracellular signaling cascades elicited as a consequence and may be specific for different cell types.

Taken together, these findings raise several points: First, both MIP-1 and SDF-1 exhibit comparable suppressive effects on colony growth from CD34⁺ hemopoietic cells. This effect was more pronounced in the presence of several rather than a single stimulating growth factor, suggesting similarities in the working mechanism or even a shared intracellular signal transduction pathway for chemokine-induced growth-suppressive signals.

Second, the differential response of CD34⁺ cells and monocytes to MIP-1 in terms of calcium mobilization supports the concept of cell-type-dependent signal sorting for chemokines proposed by Murphy [38]. MIP-1 triggered marked elevations of [Ca²⁺]_i in mature monocytic cell populations, and this correlates well with the chemotactic response of these cells in transwell assays reported in the literature [39]. However, we did not observe a reproducible calcium response in LP CD34⁺ cells, and this finding was associated with a weak chemotactic response to MIP-1 in transwell assays (our own unpublished findings, and Aiuti et al. [30]). In contrast, we showed that SDF-1 is a potent inducer of intracellular calcium elevations in CD34⁺ and monocytic cells, and this is paralleled by marked chemotactic responses in both cell types ([40] and Dürig et al., unpublished results). Moreover, it has recently been demonstrated that actin polymerization in SDF-1-treated MO7e cells closely follows the kinetics of intracellular calcium alterations in the same cells. Thus, it appears that chemokine-induced signal transduction events involving intracellular calcium elevations may be associated with migration responses in primitive hemopoietic cells.

Third, the finding that SDF-1 but not MIP-1 was capable of eliciting a calcium response in LP CD34⁺ cells suggests that the stimulation of adhesion and growth inhibition observed with MIP-1 are independent of chemokine-induced calcium responses. This is further supported by the apparent disparities in the dose-response curves for SDF-1-induced growth suppression and calcium responses in LP CD34⁺ cells; while increases of intracellular calcium levels could be detected at SDF-1 concentrations of 1.5 to 15 ng/ml, significant growth-suppressive effects only became evident at 50 and 150 ng/ml.

Fourth and finally, the difference in the dose-response curves for MIP-1-induced adhesion and growth-suppressive effects suggests a concentration-dependent signal sorting in CD34⁺ cells. While a maximum adhesion effect was achieved between 1.5 to 15 ng/ml, MIP-1-induced inhibition of colony formation plateaued between 50 and 150 ng/ml. Analogous to this finding, two distinct concentration-dependent T-cell signaling pathways were found for RANTES [41], one linked to activation of pertussis-toxin-sensitive G proteins and the other linked to protein-tyrosine kinase activation.

In general, recent experience in chemokine research has demonstrated that in vitro findings have to be interpreted with caution, as these systems often do not take into account the redundancy between chemokines observed in vivo [42]. For example, although the stem cell inhibitory activity of MIP-1 has been validated in vitro [31, 33, 43-45] and also in vivo [46-48], its homeostatic functions in hemopoiesis remain unclear since MIP-1 knockout mice exhibited a normal hemopoietic phenotype [49]. In contrast, mice lacking the SDF-1 or the CXCR-4 gene have severely impaired lymphopoiesis and abnormally low numbers of bone-marrow precursors [50, 51]. In addition, unlike most chemokines, SDF-1 is expressed constitutively in several tissues, including bone marrow fibroblasts and is thought to be involved in the homing of hemopoietic progenitor cells to the bone marrow microenvironment [40]. Based on the results described, we are currently employing neutralizing antibodies to the SDF-1 receptor CXCR-4 to further define the physiological function of SDF-1 in the bone marrow microenvironment.

	Acknowledgments

 
We thank T.M. Dexter for support and British Biotech for supplying MIP-1. We are grateful to M. Hughes and J. Barry for excellent technical assistance with flow cytometry. J.D. was supported by a grant from ESMO. We thank members of the Medical Oncology Department at Christie Hospital for the supply of clinical samples.

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