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(Journal of Leukocyte Biology. 2001;70:455-460.)
© 2001 by Society for Leukocyte Biology

CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors

Erika A. de Wynter^*, Clare M. Heyworth^*, Naofumi Mukaida, Ewa Jaworska^*, Almeriane Weffort-Santos^*, Kouji Matushima and Nydia G. Testa^*

^* Paterson Institute for Cancer Research, Manchester, United Kingdom; and
Kanazawa University, Ishikawa, and
Molecular Preventive Medicine, University of Tokyo, Japan

Correspondence: E. A. de Wynter, Molecular Medicine Unit, Clinical Sciences Building, St James’s University Hospital, Leeds LS9 7TF, United Kingdom. E-mail: medeadw{at}leeds.ac.uk

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

 
Simple methods that separate progenitor cells of different hemopoietic lineages would facilitate studies on lineage commitment and differentiation. We used an antibody specific for the chemokine receptor CCR1 to examine mononuclear cells isolated from cord blood samples. When CD34⁺ cells were separated into CD34⁺CCR1⁺ and CD34⁺CCR1^- cells and plated in colony-forming assays, the granulocyte/macrophage progenitors were found almost exclusively in the CD34⁺CCR1⁺ cells. In contrast, the CD34⁺CCR1^- cells contained the majority of the erythroid progenitors. There was a highly significant difference (P<0.002) in the total percentage distribution of both granulocyte-macrophage colony-forming cells and erythroid burst-forming units between the two populations. This is the first report of separation of erythroid progenitors from granulocyte/macrophage progenitors using a chemokine receptor antibody in cord blood samples. These results suggest that at the clonogenic progenitor cell stage the expression of CCR1 might be lineage-specific. This method should prove useful for studies on erythroid progenitor and granulocyte/macrophage differentiation.

Key Words: myeloid progenitors • CCR1 antibody • CD34⁺ cells • MIP-1

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

 
Mature blood cells arise from a primitive hemopoietic stem cell population in the bone marrow. The stem cells proliferate and differentiate and give rise to more developmentally restricted progeny of different lineages. The committed progenitor cells cannot be identified by morphological criteria, although they are detected by their capacity to originate discrete colonies in clonogenic assays. Progress in understanding the processes of commitment and differentiation has been hampered by the lack of markers that can distinguish specific progenitor cell populations. Although the CD34 antigen is used widely as a marker of primitive hemopoietic cells, the selected CD34⁺ populations are heterogeneous and contain stem cells, progenitor cells, and more mature cells. A number of methods have been reported that allow purification and concentration of normal myeloid progenitors. These include differential density centrifugation, immune selection using rosetting, negative or positive selection by immunomagnetic separation, and fluorescein-activated cell sorting. [^{1 2 3 4 5 6 7 8 9} ]. To obtain high purities, multistep procedures are necessary, although this often compromises the yield. To overcome this, other reports describe conditions that favor selective amplification of the CD34⁺ cells in liquid culture to generate the appropriate myeloid progenitors [¹⁰ , ¹¹ ].

Macrophage inflammatory protein-1 (MIP-1), a cysteine cysteine (CC) chemokine, has diverse actions that inhibit or enhance proliferation of subpopulations of myeloid progenitor cells [^{12 13 14 15} ], inhibit stem cell proliferation [^{16 17 18 19 20} ], and mobilize progenitor cells into the blood [¹⁶ , ²¹ ]. The enhancing effect of MIP-1 on colony formation by granulocyte-macrophage colony-forming cells (GM-CFCs) was more apparent on progenitor cells isolated from cord blood [¹⁴ ]. Under similar assay conditions, growth of GM-CFCs from bone marrow cells was suppressed. The effects of MIP-1 on hemopoietic progenitors are direct and appear to be mediated by specific receptors [¹⁵ ]. At least three chemokine receptors, CCR1, CCR5, and D6, bind to MIP-1, although D6 does not signal in response to interaction with the molecule [^{22 23 24 25} ]. It is not clear which of these receptors mediate the different MIP-1 responses.

mRNA for CCR1 is expressed abundantly in CD34⁺ cells isolated from bone marrow and cord blood [¹⁴ ]. Recently, a specific antibody to the CCR1 receptor has been described, and the receptor has been shown to be expressed on some lymphocytes and monocytes but not on granulocytes [²⁶ ]. Here we show that the majority of CD34⁺ cells expressed high levels of CCR1 and that this expression may be used to separate erythroid and granulocyte/macrophage progenitors. Therefore, expression of the CCR1 receptor on CD34⁺ cells allows rapid discrimination between erythroid and granulocyte/macrophage progenitors.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

 
Reagents
Recombinant human cytokines and growth factors were obtained from the following sources: stem cell factor (SCF), Amgen (Thousand Oaks, CA); thrombopoietin (TPO) and Flt-3 ligand (FL3), R&D Systems Europe (Abingdon, England); granulocyte-macrophage colony-stimulating factor (GM-CSF), Glaxo (Greenford, England); interleukin 3 (IL-3), Sandoz (Basel, Switzerland); human erythropoietin (EPO), Boehringer Mannheim UK Ltd. (Lewes, England); and human recombinant MIP-1 BB10010 (British Biotech, Oxford, U.K.) BB10010 is an active, nonaggregating variant of human MIP-1 that exhibits similar biological effects to native MIP-1 [²⁷ , ²⁸ ].

Isolation and enrichment of CD34⁺ cord blood cells
Human cord blood was obtained from normal, full-term deliveries with the local ethical committee’s approval. Samples were diluted 1:1 with phosphate buffered saline (PBS) and centrifuged over Ficoll-Hypaque gradients (1.077 g/mL) (Lymphoprep; Life Technologies) for 25 min at 400 g. Mononuclear cells (MNCs) were recovered from the interface, washed in PBS, and resuspended in ice-cold PBS containing 5 mM EDTA and 0.5% (w/v) bovine serum albumin (PBE).

CD34⁺ cells were isolated as previously described [¹⁴ ] using the Miltenyi Isolation System (Miltenyi Biotech, Bergisch Gladbach, Germany). Briefly, MNCs were labeled with CD34 antibody conjugated to superparamagnetic beads by incubating them for 30 min at 4°C. The cells were washed, and the CD34⁺ cells were separated using MiniMACS columns according to the manufacturer’s instructions. The CD34-enriched fractions were collected by flushing the column with cold PBE buffer.

Labeling of MNCs and CD34⁺ cells with CCR1 antibody
To analyze CCR1 expression, 5–10 x 10⁵ MNCs or 2–5 x 10⁵ CD34⁺ cells were first incubated in antibody diluent (DAKO Ltd., Ely, U.K.) containing 1% (v/v) human AB serum and previously titrated anti-CCR1 antibody at an optimal concentration of 25 µg/mL [²⁶ ]. After the cells were washed, primary antibody was detected with a secondary swine anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody (DAKO Ltd.). For two-color analysis, the washed CD34⁺ cells were labeled first with anti-CCR1 and then with swine anti-rabbit-FITC. They were subsequently labeled with anti-CD34 phycoerythrin (PE)-conjugated antibody (HPCA-2) (Becton Dickinson, Cowley, Oxford, U.K.). Portions of the cells were stained with the appropriate control antibodies. All labeled cells were analyzed and sorted by flow cytometry.

Fluorescein-activated cell sorter analysis and cell sorting
Analysis of the labeled cells was performed with a FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 488-nm argon laser. Data acquisition was performed with LYSYS II software, and at least 30,000 events were collected.

Cell sorting of MNCs and double-labeled CD34⁺ cells was also performed with the FACS Vantage flow cytometer. The labeled cells were suspended at a concentration of 10⁶/mL in PBE buffer, and any residual erythrocytes and dead cells were gated out using forward- and side-scatter channels. A gate was set around the lymphocyte population, and CD34 expression in this gate was assessed. Positively labeled CD34⁺ cells were then gated and sorted according to CCR1 expression.

The fractions obtained after fluorescein-activated cell sorting were cultured in appropriate assays or stained for morphological evaluation as described in Results.

Colony assays
Sorted CD34⁺CCR1⁺ or CD34⁺CCR1^- cells (1,000–2,000) were plated in a mixture consisting of 1.35% (v/v) methylcellulose (Sigma, Dorsetshire, England), 30% (v/v) fetal calf serum (Autogen Bioclear, Calne, England), 1% (v/v) bovine serum albumin (Sigma), 10% (v/v) 5637 conditioned medium from the EJ bladder carcinoma cell line, and 2 U/mL of EPO to detect GM-CFC and erythroid-forming unit, bursts (BFU-E) progenitors. The cultures were plated in triplicate and incubated for 14 days at 37°C in a humidified atmosphere containing 5% CO₂ and 5% O₂. GM-CFCs and BFU-Es were counted according to standard criteria [²⁹ ]. In some experiments, colonies were removed after 14 days, cytospins were prepared, and the cells were evaluated for morphology after being stained with May-Grünwald/Giemsa.

Serum-free cultures
Cells (n=5,000) from the selected cell populations described in Results were cultured in X-VIVO 10 serum-free medium (Bio-Whittaker, Workingham, England) containing 2 mM L-glutamine and cytokines, which promoted either growth of erythroid cells or granulocytes and macrophages. Growth of erythroid cells was stimulated with SCF (100 ng/mL), EPO (5 U/mL), and TPO (50 ng/mL), and cultures to promote granulocytes and macrophages were supplemented with SCF (100 ng/mL), GM-CSF (10 ng/mL), and IL-3 (10 ng/mL). The cells were incubated for 7–14 days at 37°C in a humidified atmosphere containing 5% CO₂ and 5% O₂. At days 7 and 14, the cultures were assessed for progenitor cell content.

Statistical analysis
The Wilcoxon rank sum test and the Wilcoxon signed rank test were used for paired samples.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

 
Expression of CCR1 on MNCs
We investigated the expression of CCR1 on MNCs from cord blood samples. In four samples, an average of 5.6 ± 0.6% of the MNCs expressed CCR1. The MNCs were sorted into CCR1⁺ and CCR1^- populations, and then cytospins were prepared and stained with May-Grünwald/Giemsa. The majority of the early and late erythroblasts were present in the CCR1^- population (Table 1 ), suggesting that erythroblasts in cord blood lack the CCR1 receptor or express very low levels of CCR1. Most of the undifferentiated blasts were confined to the CCR1⁺ population.

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Table 1. Morphological Analysis of CCR1+ and CCR1- Mononuclear Cells from Cord Blood

Expression of CCR1 on CD34⁺ cells
We previously observed by Northern blot analysis that CCR1 is abundantly expressed in CD34⁺ cells [¹⁴ ]. Using the anti-CCR1 antibody and flow cytometry analysis, Figure 1 shows that the majority of CD34⁺ cord blood cells expressed CCR1. A mean of 80.9 ± 4.7% CD34⁺ cord blood cells was labeled, although this number was reduced to 66% in the CD34⁺ bone marrow cells (data not shown).

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Figure 1. Fluorescein-activated cell sorting of CD34+ cells expressing CCR1. CD34+ cells were sequentially stained with anti-CCR1 and anti-rabbit-FITC. Cord blood CD34+ cells were double stained with CD34-PE antibody, and all cells in the lymphocyte gate were analyzed for CD34 expression. After gating on CD34+ cells, CCR1 staining was determined. (A) Lymphocyte gate set on light scatter of CD34 selected cells, (B) CD34-PE expression determined on cells double labeled with secondary FITC-conjugated antibody to set the negative CCR1 population, (C) CCR1 negative and positive expression on CD34-PE labeled cells, and (D) CD34+ cells labeled with FITC and PE control antibodies.

Progenitor cell content in sorted CD34⁺CCR1⁺ and CD34⁺CCR1^- cells
To determine whether the absence of CCR1 on cord blood erythroblasts was preserved on progenitor cells, we examined the colony-forming ability in cultures that allow formation of both erythroid and granulocyte/macrophage colonies. There was a clear distinction in the types of colonies generated by the two cell fractions. The CD34⁺CCR1⁺ cells formed mainly GM-CFC colonies, whereas the CD34⁺CCR1^- cells generated almost exclusively erythroid colonies (Table 2 ). When the percentage distribution of the total progenitors was determined, >96% of the GM-CFCs was restricted to the CD34⁺CCR1⁺ population, with 80% of the erythroid progenitors located in the CD34⁺CCR1^- cell fraction, as shown in Table 2 . In each case, the percentage distributions in the two populations were highly significant (P<0.002).

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Table 2. Colonies Generated from CD34+ Cord Blood Cell Subpopulations Stained with Anti-CCR1 Antibody

When colonies were picked and cytospins were prepared from these cells, it was clear that the GM-CFC colonies contained largely macrophages and neutrophils, whereas the BFU-E colonies consisted of erythroid cells (Fig. 2 ).

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Figure 2. Colonies and morphology of cells generated from CD34+CCR1+ and CD34+CCR1- cells. Colonies observed when sorted cell populations were plated in clonogenic assays. (A) Whole plate and (D) colonies in A at higher magnification; colonies generated by CD34+CCR1- cells (B) whole plate and (F) erythroid colonies in B at higher magnification. Colonies from each plate were pelleted, and methylcellulose was removed (C and E). Cytospins were prepared from the cell pellets and stained with May-Grünwald/Giemsa. Macrophages and granulocytes (G) from CD34+CCR1+ cultures and erythroid cells (H) from the CD34+CCR1- cultures._art>

Effect of MIP-1 on colony formation by CD34⁺CCR1⁺ and CD34⁺CCR1^- cells
Our previous observations indicated that MIP-1 enhanced GM-CFC colony formation on CD34⁺ cells from cord blood in a dose-dependent manner. Next, we examined the effect of MIP-1 on the selected CD34⁺CCR1⁺ and CD34⁺CCR1^-cells. As shown in Figure 3 A, MIP-1 significantly enhanced colony formation by CD34⁺CCR1⁺ cells, with colony numbers increasing to between 135 and 191% of untreated control values. The colonies that were formed in the assay by these cells with or without MIP-1 were composed mainly of granulocytes and macrophages. In the CD34⁺CCR1^- cultures, the erythroid colony numbers were reduced to between 68.5 and 98.3% of the untreated controls in the presence of MIP-1, as illustrated in Figure 3B , showing that the inhibitory response on the erythroid colony-forming cells was maintained in the absence of the CCR1 receptor.

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Figure 3. Effect of human MIP-1 on colony formation by GM-CFCs in standard clonogenic assays from CCR1+ and CCR1- cells. Results shown are from individual experiments with CCR1+ cells (n=6) and CCR1- cells (n=5) and are expressed as a percentage of the untreated controls. Control colony numbers ranged from 26–53 for GM-CFCs and 87–220 for BFU-Es. There was significant enhancement in colony formation of GM-CFCs in the presence of MIP-1 (P<0.03).

Suspension cultures in serum-free media
To examine the effects of growth factors on proliferation and differentiation of the sorted cells, 5,000 cells were sorted into 1 mL of serum-free medium containing various combinations of cytokines. The highest proliferation, 207-fold, was found when CD34⁺CCR1^- cells were cultured in the presence of SCF, EPO, and TPO. After 14 days, these cells had a predominantly erythroid phenotype, with 81% being CD71⁺ and 49% being glycophorin A⁺ (Fig. 4 ). In the CD34⁺CCR1⁺ fraction, cell numbers increased 94-fold and contained <9% and 4% CD71 and glycophorin A⁺ labeled cells, respectively. In these cultures, the majority of the cells were macrophages and immature neutrophils.

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Figure 4. Phenotypic analysis of cultured CCR1+ and CCR1- cells. Cells were sorted as indicated in Figure 1C . The sorted CCR1+ cells were cultured in serum-free medium in the presence of SCF, GM-CSF, and IL-3, and CCR1- cells were cultured in serum-free medium in the presence of SCF, EPO, and TPO (see Materials and Methods). After 14 days of culture, the cells were stained with PE-labeled CD71 or PE-labeled glycophorin A and analyzed by flow cytometry. CCR1+ cultured cells are shown in A and C, and CCR1- cells are shown in B and D. Control antibodies (red) and specific staining (black) are illustrated._art>

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

 
In this study, we analyzed CCR1 expression in human cord blood MNCs and CD34⁺ selected cells and showed that it is present on mature neutrophils and granulocyte/macrophage progenitors but largely absent on erythroblasts and erythroid progenitors. In liquid suspension cultures, CD34⁺CCR1^- cells produced erythroblasts, whereas CD34⁺CCR1⁺ cells produced macrophages and immature neutrophils. To our knowledge, this is the first report of separation of distinct myeloid lineages using a chemokine receptor as a marker.

The clonal assay we used allowed the simultaneous development of GM-CFC-originating cells from the granulocyte and macrophage lineages and of BFU-Es, which generated colonies of erythroid cells. Both CD34⁺CCR1⁺ and CD34⁺CCR1^- cell fractions were examined in this assay, and the findings that CD34⁺CCR1^- cells predominantly gave rise to erythroid colonies by 14 days in culture suggested that the BFU-Es are mainly contained in the CD34⁺CCR1^- cells. In line with this finding, CD34⁺CCR1^- cells produced erythroblasts in liquid suspension cultures, and these cells expressed high levels of CD71, the human transferrin receptor, and glycophorin A, another marker of the erythroid lineage. Moreover, >96% of the GM-CFCs segregated to the CD34⁺CCR1⁺ cell fraction and generated granulocyte/macrophage colonies (Table 2) . Proliferation of these cells in liquid culture was also extensive, and macrophages and immature neutrophils were apparent at 14 days. The enrichment of colonies in the two subfractions indicated that these isolated populations are biologically distinct in lineage commitment programs. The distinction between the erythroid and monomyelocytic lineages was also seen in freshly isolated cord blood cells, when expression of CCR1 was examined on MNCs (Table 1) . Thus, CCR1 is mainly expressed on immature and mature cells of the granulocyte and macrophage lineages, and cells destined to develop (BFU-Es) and those already developing along the erythroid lineage exhibit at best low levels of the receptor.

Some GM-CFCs were also found in the CD34⁺CCR1^- population, which might reflect insufficient separation of this subset and the necessity for a more stringent gating strategy. In the gating strategy depicted in Figure 1 , we designated the cells as CD34⁺CCR1^- rather than CD34⁺CCR1^low because, although there might be a few negative cells in the positive cell population, there are no positive cells in the negative cell population. Indeed, in three separate experiments, when the cell populations exhibiting the lowest or the highest levels of immunofluorescence were isolated, there was better discrimination between erythroid and granulocyte/macrophage progenitors. In fact, 99% of erythroid colonies were obtained in the CD34⁺CCR1^- fraction, with 96% granulocyte/macrophage colonies in the CD34⁺CCR1⁺ subset. Therefore, we suggest that CCR1^- designates a population of cells that do not express the receptor.

Primary human erythroid progenitors are difficult to obtain as homogeneous cell populations, and various strategies for isolation or enrichment have been reported. These include differential density centrifugation and negative and positive selection by immunomagnetic separation with purities ranging from 18–56% [^{1 2 3} , ⁵ ]. Alternative methods include ex vivo culture of MNCs or CD34⁺ cells in defined growth factor combinations to generate large numbers of specific progenitors, although contamination by mature cells that also develop in the cultures is often a problem [⁵ , ³⁰ ]. Two recent studies distinguished GM-CFCs and BFU-Es on the basis of their ability to bind biotinylated c-kit ligand or multiple cell surface antigens [³¹ , ³² ]. The results of these studies compare favorably with results in the present study, in which 97% of the GM-CFCs were present among CD34⁺CCR1⁺ cells, with 81% of BFU-Es in the CD34⁺CCR1^- fraction. Monoclonal antibodies against a number of other cell surface antigens including c-kit, CD71, HTK, and the EPO receptor have been generated [⁴ , ^{33 34 35} ]. All can distinguish erythroid precursors and have similar patterns of expression, in that their expression reaches maximum at the BFU-E stage and declines during terminal differentiation. CCR1 is distinct in that expression is absent from the progenitor stage onwards. CD34⁺ cells expressing high levels of the receptor are restricted to the granulocyte/macrophage lineages.

The exact expression pattern of CCR1 during hemopoietic cell differentiation is not known, and some conflicting results have been reported. Although mRNA for CCR1 has been detected in CD34⁺ cells in cord blood and also in bone marrow using reverse transcriptase-PCR and Northern blotting [¹⁴ , ²⁶ , ³⁶ ], Majka was unable to demonstrate the presence of CCR1 on CD34⁺ cells in human bone marrow MNCs [³⁷ ]. Previously published data indicated that CCR1 is present on mature erythroblasts and erythroid progenitors in bone marrow [³⁸ ], but the data presented here show that this is not the case with cord blood. It is possible that expression of CCR1 varies depending on the sample source, because we noted a differential response to MIP-1 depending on the source of the hemopoietic cells [¹⁴ ].

MIP-1 enhanced colony formation of granulocyte-macrophage progenitors by the CD34⁺CCR1⁺ cells (Figure 3) , suggesting that the proliferation response is mediated by the CCR1 receptor. Erythroid progenitors, which are CCR1^-, maintained an inhibitory response, as shown by a decrease in formation of erythroid colonies, indicating that this inhibitory effect is mediated by a MIP-1 receptor other than CCR1. This agrees with compelling evidence that the CCR1 receptor might mediate the proliferative response to MIP-1. In bone marrow cells from mice with a targeted disruption of CCR1, MIP-1 failed to enhance colony formation in clonogenic assays stimulated with GM-CSF or macrophage colony-stimulating factor. In contrast, marrow cells from wild-type animals responded normally with enhanced proliferation in the presence of MIP-1 [³⁹ ]. The antibody used did not seriously interfere with the function of the cells in the biological assays, given the fact that the positively labeled cells (CCR1⁺) were able to respond effectively when MIP-1 was added.

Simplified methods for isolation of specific lineage-committed progenitors would be advantageous for a variety of applications. It would allow for definitive studies of selective expression of specific genes at distinct stages of differentiation and commitment as well as examination of genes whose targeted disruptions lead to defects in myeloid lineages. The procedure outlined here using the anti-CCR1 antibody should prove useful in distinguishing the myeloid lineages.

 
The work was supported by the Cancer Research Campaign. The authors would like to thank Jeff Barry and Mike Hughes for flow cytometry and Clare Hart for excellent technical assistance.

Received January 2, 2001; accepted January 17, 2001.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

 

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