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and the Expression of MIP-1 Receptors on These Immature Cells
CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, United Kingdom
Key Words. MIP-1
receptors • Chemokines • Hemopoietic cells • Cell cycle
Dr. E.A. de Wynter, CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Withington, Manchester, M20 4BX, United Kingdom.
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Abstract |
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) has been shown to have a role in the control of myeloid stem and progenitor cell proliferation. Recent evidence suggests that MIP-1 also has a stimulatory effect on proliferation of mature progenitors as well as an inhibitory effect on immature progenitors in vitro. We have compared the effect of MIP-1 on myeloid and erythroid colony formation of CD34+ cells isolated from bone marrow and cord blood. In the presence of MIP-1, bone marrow granulocyte-macrophage-colony forming cells (GM-CFC) were inhibited over a dose range of 15 ng/ml to 500 ng/ml, and GM-CFC from cord blood CD34+ cells were stimulated over the same dose range. MIP-1 suppressed BFU-E colonies in both bone marrow and cord blood. Using thymidine suicide assays, the influence of MIP-1 on the cycling status of the cells was assessed. A good correlation between the effect of MIP-1 on colony formation and cell cycle progression was observed. These results suggest that there is a differential response to MIP-1 when bone marrow and cord blood CD34+ cells are compared.
Using flow cytometry and a biotinylated human MIP-1/avidin fluorescein conjugate, the expression of MIP-1 receptors on CD34+ cells was assessed. The data indicated that there was little quantitative difference in overall expression of receptors (82.9% versus 93%) from bone marrow or cord blood, respectively. However, when Northern blot analysis was used, mRNA for two different MIP-1 receptors CCR1 and CCR5 could be detected in bone marrow, but only CCR1 mRNA was seen in cord blood CD34+ samples. Therefore, the expression of different receptor subtypes on CD34+ cells may be responsible for the difference in MIP-1 responsiveness observed.
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Introduction |
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), a member of the ß chemokine family of proinflammatory molecules. Although MIP-1 has a wide range of biological activities, much interest has focused on its effects on primitive hemopoietic cells.
Potential hemopoietic inhibitors are generally assayed on the basis of suppression of colony formation or inhibition of DNA synthesis. Using the thymidine suicide assay, Clements et al. [3] demonstrated that MIP-1 prevented murine stem cells from entering S phase, thus inhibiting DNA synthesis and further proliferation. The ability of MIP-1 to induce proliferative quiescence in vivo has been exploited to protect primitive hemopoietic cells against the cytotoxic effects of cell-cycle-specific drugs [4-6]. Although MIP-1 does show cell-cycle-related effects this may not be confined simply to a block on proliferation; in mice treated with a combination of cytotoxic drugs and MIP-1, stem cell numbers recovered at a faster rate and to a higher level than in untreated mice [4].
The in vitro effects are less clear, and a number of conflicting results have been reported. Some studies showed that MIP-1 suppressed the growth of immature progenitors in CFU-GEMM (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte), CFU-A (colony-forming unit-agar), and HPP-CFC (high proliferative potential-colony-forming cell) assays [7-10]. In contrast, other workers found no such inhibitory effects with MIP-1 on HPP-CFC and only weak inhibition on isolated populations of primitive bone marrow cells [11-13]. Nevertheless, MIP-1 may have a role in maintaining the long-term culture initiating cells (LTC-IC) as LTC-IC were maintained for up to eight weeks in vitro, in the presence of MIP-1 [14].
Recent work indicates that the effect of this chemokine on stem and progenitor cells is the result of direct interactions between MIP-1 and its target cells, implying that the responsive cells must bear the appropriate receptors [12, 15]. To date, at least three human receptors which bind MIP-1 have been identified and cloned: CCR1, CCR4, and CCR5 [16-19]. All three are members of the seven-transmembrane domain superfamily of receptors which transduce their signal by coupling G-proteins. CCR1 appears to be closely involved in MIP-1-induced monocyte chemotaxis and is predominantly expressed in human and murine monocytes [20]. Recently, CCR5 was identified as the major coreceptor for the macrophage-tropic HIV. The receptor is expressed in lymphoid organs and peripheral blood leukocytes and is so far the only human chemokine receptor which signals in response to MIP-1ß [16, 21]. However, the expression of these three receptors on hemopoietic stem and progenitor cells and their role in hemopoiesis is not well documented.
In the present study, we have examined the effects of MIP-1 on CD34+ cells isolated from cord blood and bone marrow. Using flow cytometry, we demonstrate that a population of these cells expresses receptors for MIP-1 and identifies the pattern of expression of mRNA for the known receptors by Northern blot analysis.
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Materials and Methods |
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CD34+ Isolation
Mononuclear cells were prepared by centrifugation of diluted samples on Ficoll-Hypaque (Lymphoprep, 1.077 g/ml) at 400 x g for 25 min. CD34+ cells were then isolated as previously described [22]. Briefly, mononuclear cells were suspended at a concentration of 108 cells in 300 µl of PBE (phosphate buffered saline [PBS]/0.5% [w/v] bovine serum albumin [BSA]/5 mM EDTA). The cell suspension was incubated with blocking reagent and a CD34 antibody from the CD34 Isolation Kit (MACS, Miltenyi Biotec; Bisley, Surrey, UK) for 15 min at 4-8°C. After incubation, the cells were washed in PBE and resuspended in 400 µl of the same buffer. Added to the cells were 100 µl of immunomagnetic bead suspension conjugated to an antimouse antibody, and incubation continued for a further 15 min in the cold. After washing, the CD34+ cells were isolated by passing the cell suspension through a column placed in a magnetic field which allowed retention of the target cells. The magnetic field was then removed, and the CD34+ cells were flushed from the column with PBE buffer. Purity of the cell population was determined by flow cytometry.
MIP-1 Receptor Labeling
We have previously reported isolation of human CD34+ cells by immunomagnetic beads, resulting in a greater than 80% pure population [23]. We were interested in examining the distribution of MIP-1 receptors on CD34+ cells to determine if there was a significant difference between bone marrow and cord blood. Enriched CD34+ populations were labeled with biotinylated MIP-1, followed by a second incubation with streptavidin-conjugated FITC.
CD34+ cells isolated by MiniMACS were washed twice in PBS, and the cells resuspended at a concentration of 4 x 106 cells/ml in PBS. Cells were then labeled with a Fluorokine Kit for Human MIP-1 (Cytokine Flow Cytometry Reagent Biotin conjugate, R & D Systems; Abingdon, Oxford, UK) according to the manufacturer's instructions. Briefly, 10 µl of biotinylated cytokine reagent were added to 25 µl aliquots of washed cells (105) and incubated for 60 min at 2-8°C. At the end of the incubation period, 10 µl of avidin-fluorescein isothiocyante (FITC) reagent were added, and the cells were incubated for a further 30 min at 2-8°C in the dark. The cells were then washed twice, using the buffer provided to remove unreacted avidin-fluorescein, and analyzed by flow cytometry. As a negative control, an identical sample of washed cells was incubated with 10 µl of biotinylated negative control reagent (supplied with the kit). Specificity of the MIP-1 biotin reaction was tested by mixing 20 µl of anti-human MIP-1 blocking antibody with 10 µl of MIP-1 biotin and incubating for 15 min at room temperature. Then 105 cells were added to the mixture, and labeling continued as described above.
3H-Thymidine Incorporation
BB10010, a genetically engineered variant of human MIP-1 with improved solution properties was used throughout these experiments [24] and was a gift from British Biotech (Oxford, UK).
To assay the cell cycle inhibitory effects of MIP-1 (BB10010), the tritiated thymidine suicide (3H-TdR) technique was used. Isolated cord blood CD34+ cells were resuspended in Iscove's modified Dulbecco's medium (IMDM). Aliquots of 2 ml containing 2 x 105 cells in each aliquot were incubated with or without MIP-1 at 15 ng/ml for four h at 37°C. 100 µCi 3H-TdR (specific activity 20 Ci/mmol) was then added to one tube and an equal volume of unlabeled thymidine added to a second tube as a control. Both tubes were incubated for a further 30 min at 37°C. At the end of the incubation period, MIP-1 and radioactive label were removed by washing the cells twice with cold IMDM containing 100 µg/ml unlabeled thymidine. Cells were plated in the CFC-Mix assay. The reduction in the number of colonies produced as a result of 3H-TdR incorporation was taken as a measure of the proportion of cells in DNA synthesis versus colony numbers obtained in the absence of labeled thymidine.
CFC Assays
CFC-Mix assays were performed as previously described [25]. CD34+ cells 2 x 103 were plated in a 1 ml mixture containing final concentrations of 30% (v/v) fetal calf serum, 1% (w/v) deionized bovine serum albumin, 10% (v/v) conditioned medium from the 5637 EJ bladder carcinoma cell line, two units erythropoietin (Boehringer Mannheim; Lewes, East Sussex, UK) in 1.35% (w/v) methylcellulose. Cultures were plated in triplicate and incubated at 37°C in 5% CO2, 5% O2, and 90% N2. At day 14 of culture, BFU-E- and CFU-GM-derived colonies were scored according to established criteria [24].
RNA Extraction
Total cellular RNA was extracted from CD34+ cells by resuspending the cell pellet in a minimal volume of ice-cold PBS. Cells were lysed by addition of five volumes RNA extraction buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate pH7.0, 0.5% [w/v] sarcosyl, 0.1 M 2-mercaptoethanol). One-tenth volume 3 M sodium citrate pH 4.0 and one volume water-saturated phenol were added and the suspension mixed vigorously. After addition of one-tenth volume of chloroform, the suspension was again mixed vigorously and cooled on ice for 15 min before centrifugation at 10,000 x g for 15 min. RNA in the aqueous phase was precipitated with an equal volume of isopropanol at –20°C and sedimented at 10,000 x g for 20 min. The RNA pellet was then resuspended in 400 µl resuspension buffer (0.3 M sodium acetate pH 5.2, 1% [w/v] SDS) and 400 µl chloroform/butanol added. After mixing, the aqueous phase was separated by centrifugation, removed, and precipitated with two volumes of 100% ethanol. Finally, the RNA pellet was washed with 70% ethanol and resuspended in DEPC (diethylpyrocarbonate)-treated water. The OD260 was checked, and an aliquot of RNA was run on a gel to assess amount and purity.
Northern Blot
5-10 µg RNA from CD34+ cells were loaded on a 1% agarose gel containing 1XMOPS (3-(N-morpholino) propane sulfonic acid) pH 7.0 and 2.7% (v/v) formaldehyde. Samples were resuspended in 1XTE (10 mM Tris-HCl, 1 mM EDTA pH 7.6), mixed with sample buffer (7% [v/v] formaldehyde, 50% [v/v] formamide, 1XMOPS) and incubated at 68°C for two min. Heated samples were cooled on ice before loading on the gel. After electrophoresis, RNA was transferred to Biodyne B nylon membrane (PALL) by blotting overnight and then crosslinked by UV illumination.
Probe Preparation and Hybridization
For Northern hybridization, human cDNA probes for CCR1, CCR4, and CCR5 were prepared by random prime labeling. Fifty nanograms of denatured DNA were used for each reaction, and the DNA labeled with 50 µCi [-32P]dCTP using a random prime labeling kit (Boehringer Mannheim). After preparation, probes were incubated with the membranes and hybridized overnight at 65°C. Following hybridization, membranes were washed for 20 min in 2X standard saline citrate (300 mM NaCl, 30 mM sodium citrate pH7.2) containing 1% (w/v) SDS at 65°C followed by a final wash in 0.2X SSC containing 1% SDS at the same temperature. Membranes were then exposed on a phosphor screen (PHOSPHORIMAGER, Molecular Dynamics Ltd., Kemsing, UK) overnight, then scanned and analyzed using ImageQUANT software. The membrane was stripped and rehybridized sequentially with the labeled cDNA probes for all the other chemokine receptors. Finally, a labeled cDNA probe for actin was used as a loading control and also for normalization.
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Results |
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on Clonogenic Potential of CD34+ Cells on CD34+ cells from bone marrow and cord blood, we monitored both CFU-GM and BFU-E in colony assays using a range of concentrations of MIP-1. In Figure 1A, both CFU-GM and BFU-E from bone marrow CD34+ populations were inhibited over the entire concentration range examined, with maximum inhibition at the highest concentration of MIP-1. The maximum inhibitions observed for CFU-GM and BFU-E were 81% (standard error [SE] ± 5.2) and 74% (SE ± 13.2), respectively. When cord blood cells were used ( Fig. 1B), BFU-E colony formation was suppressed at concentrations as low as 15 ng/ml with maximum inhibition at the highest concentration of MIP-1. From Figure 1B, it can be seen that the effect of MIP-1 on the BFU-E appears to plateau between 15 ng/ml and 150 ng/ml, where colony numbers were inhibited by 13.3% to 22.5% (SE ± 5.2 to 9.4, respectively) compared with control numbers. However, total inhibition was not obtained even at 500 ng/ml, and only 43.7% (SE ± 11.9) of BFU-E were inhibited in the cord blood compared with 74% (SE ± 13.2) in bone marrow. In contrast to its inhibitory effect on the BFU-E, MIP-1 consistently stimulated CFU-GM colony formation over the concentration range used. An increase of 31.5% in CFU-GM numbers over control was seen even at the lowest concentration of MIP-1 used. A bell-shaped response curve was obtained with cord blood CFU-GM. This pattern of inhibition of BFU-E and stimulation of CFU-GM was consistent in six experiments.
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on the Percentage of Progenitors in DNA Synthesis on the cell cycle was tested on CD34+ cells isolated from bone marrow and cord blood. Table 1 shows the results obtained from eight experiments. The percentage of CFU-GM progenitors from bone marrow in S phase was 33%, with 54% of BFU-E also in cycle. After incubation with MIP-1, the percentage kill by 3H-TdR of both progenitor cell populations was reduced, indicating that MIP-1 suppresses the number of progenitors actively cycling in bone marrow. Similarly, 52% of cord blood BFU-E were initially in cycle, and this was reduced to 28% in samples preincubated with MIP-1. However, the cord blood CFU-GM in cycle increased significantly from 35% to 54% when cord blood CD34+ cells were preincubated with MIP-1, suggesting that MIP-1 increases the number of cord blood CFU-GM in S phase.
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Receptors Expressed on CD34+ Cells receptors on CD34+ cell populations from both cord blood and bone marrow samples ( Fig. 2). The mean proportion of CD34+ bone marrow cells staining positive for MIP-1 was slightly lower than that of cord blood ( Table 2). Flow cytometric analysis of the CD34+ cells showed a wide spread of staining intensity associated with the labeled MIP-1 ligand, which may reflect a difference in the number of receptors per cell. The fluorescence intensity varied 100-fold for MIP-1 stained CD34+ cells, from both bone marrow and cord blood samples ( Figs. 2C and 2D), but in general, high fluorescence with MIP-1 was always associated with cord blood samples. This is clearly seen by the clustering of cells to the right in Figure 2D. The binding of biotinylated MIP-1 was specific and could be blocked by prior incubation of the biotinylated reagent with a polyclonal anti-MIP-1 antibody provided with the kit.
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Receptor mRNA in CD34+ Cells receptors. The presence of transcripts for these receptors in CD34+ cells was assessed by Northern blot analysis ( Fig. 3). mRNA from the Jurkat cell line served as a positive control for CCR4. Our results indicate that CCR1 is the only receptor expressed by both bone marrow and cord blood CD34+ cells. Although CCR4 was clearly present in the Jurkat cell line, it was not detected in cord blood or bone marrow. Surprisingly, mRNA transcripts for CCR5 were only seen in the bone marrow samples. When sample loading was compared by ethidium bromide labeling, it was clear that more RNA was present in bone marrow samples than in cord blood.
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Discussion |
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on CD34+ cells isolated from cord blood versus those isolated from bone marrow and suggest that these responses may be due to the difference in the type and level of MIP-1 receptor expression on hemopoietic cells from the two sources. Some studies have demonstrated that when purified populations of CD34+ cells from bone marrow were used, inhibition of CFU-GM and BFU-E was observed with recombinant MIP-1 [8, 26]. This is in agreement with our data using CD34+ cells from bone marrow, where the numbers of CFU-GM and BFU-E colonies were significantly reduced in the presence of MIP-1. In addition, we observed a reduction of these progenitors in S phase of the cell cycle and attribute this to the direct action of MIP-1 on the progenitor cells. However, we also noted a clear increase in the numbers of CFU-GM from cord blood CD34+ cells, with a simultaneous suppression of the BFU-E, in clonogenic assays containing MIP-1. The stimulation of cord blood CFU-GM correlated with an increase in the number of these progenitors in S phase ( Table 1). Other studies have indicated that the action of chemokines may be linked to effects on the cell cycle [15, 26], so MIP-1 could exert its effects either by shortening the cell cycle of the cord blood CFU-GM or by bypassing restriction points between G1 and S phase of the cycle. We found a higher percentage of CD34+ cord blood progenitor cells in cycle than figures previously reported [15] and attribute this to a difference in the populations isolated. Lu et al. used a highly enriched subpopulation of CD34+++ cells which are likely to include the more immature and quiescent progenitors.
It has been suggested that chemokines such as MIP-1 have enhancing effects on the more mature hemopoietic progenitors from unfractionated bone marrow, but only in the presence of colony-stimulating factors such as GM-CSF or M-CSF [7, 8]. In our experiments, enriched populations of progenitor cells from both cord blood and bone marrow were cultured in clonogenic assays in the presence of 5637-conditioned medium containing several growth factors, including GM-CSF, G-CSF, and interleukin 1 [27]. Nevertheless, the dual enhancing and suppressive effect of MIP-1 was only observed with CD34+ cells from cord blood. As cord blood is a rich source of stem and progenitor cells and the CD34+ cells in these samples are enriched for the more immature myeloid progenitor cells, we do not conclude that MIP-1 stimulates only the mature progenitor cells. In agreement with the data of Lu et al. (1993), we also noted that cord blood BFU-E were less responsive to the suppressive effects of MIP-1 than BFU-E from bone marrow.
We considered whether the difference in response to MIP-1 of cord blood and bone marrow CD34+ cells may be due to differences in the level of receptor expression. The bell-shaped curve observed in the dose response to MIP-1 of cord blood CFU-GM could be the result of downregulation of the responsive receptors with increasing concentrations of MIP-1. In addition, less than 50% of BFU-E were inhibited in the dose response with cord blood cells, suggesting that not all progenitors were equally sensitive to the inhibitory effects of MIP-1. Altogether, this suggested that the differences may lie at the level of receptor expression. As a first step, we compared the overall expression of MIP-1 receptors on CD34+ cells from cord blood and bone marrow using flow cytometry. The majority of CD34+ cells in both samples expressed receptors for MIP-1, but it was clear from the flow cytometry data ( Fig. 2 ) that not all cells were labeled with equal intensity. This variation may be due to both the number and affinity of the receptors expressed per cell from the two different sources. Indeed, high and low affinity receptors for MIP-1 have been detected on human hemopoietic cells [28]. The presence of these receptors on CD34+ cells in the present study confirmed earlier results indicating that the effects of MIP-1 are direct and do not require accessory cells [12].
At least three receptors which bind to MIP-1—CCR1, CCR4, and CCR5—have been identified and cloned, but the assay technique used here (which utilizes a biotinylated ligand) cannot distinguish which of the three receptors are present on the cell surface of the CD34+ cells. As specific monoclonal antibodies which can bind to the extracellular domain of the receptors were not available, we opted to examine quantitatively mRNA levels in CD34+ cells using Northern blots. We detected CCR1 in both bone marrow and cord blood and noted that it was the only MIP-1 receptor seen in both samples on Northern blots. However, it does not appear to be the receptor involved in stem cell inhibition [20]. Some evidence for its potential role in mediating the enhancing effects of MIP-1 on colony formation came from Broxmeyer [29], who showed that marrow cells from CCR1 null mice (–/–) did not respond to stimulatory signals in the colony assays when compared with cells from control mice (+/+). Further studies with specific monoclonal antibodies, as they become available, will have to be performed to clarify the role of CCR1 in proliferation.
No mRNA transcripts for the CCR4 chemokine receptor were observed in any sample, although it was clearly present in the Jurkat T cell line used as a control. This may be due to mRNA levels of CCR4 being below the threshold of detection of the Northern blotting technique. Alternatively, CCR4 may not be present on CD34+ cells, and there are conflicting reports on the selectivity of CCR4 for MIP-1. CCR4 was originally cloned by Power et al. from a basophilic cell line, and it was presumed to be a functional ligand for MIP-1 [17]. However, a recent report indicated that CCR4 is the specific receptor for the chemokine TARC (thymus- and activation-regulated chemokine) and is strongly expressed on human T cells and T cell lines [30]. When CCR4 was transfected into mammalian cells, they did not respond to MIP-1 or any of the other chemokines tested except TARC, suggesting that CCR4 is not a receptor for MIP-1. Although mRNA transcripts for CCR4 have been reported in bone marrow CD34+ cells using reverse transcriptase-polymerase chain reaction (RT-PCR) [31], we are not aware of any study where transcripts for CCR4 have been identified by Northern blot analysis in either bone marrow or cord blood CD34+ cells.
It is unclear why CCR5 was not present in the cord blood cells. One explanation may be that this receptor is transcribed at very low levels and/or has a high rate of mRNA degradation relative to the corresponding cell population from bone marrow. CCR5 may be downregulated in circulating immature hemopoietic cells, and in this respect, the observations of Deichmann et al. [32] are of interest. This study used a stringent nested RT-PCR protocol with highly purified CD34+ cells to examine CCR5 mRNA expression in a series of leukapheresed cells. Results showed that more than 75% of the samples were negative for the CCR5 receptor.
The present study demonstrates that MIP-1 receptors are present on CD34+ cells from bone marrow and cord blood as shown by flow cytometry. mRNA for the chemokine receptor CCR1 was detected in both bone marrow and cord blood CD34+ cells, although CCR5 was only present in bone marrow samples. It will be of interest to see if these results are reflected at the protein level. Further studies with specific antibodies should confirm both the presence and function of the different MIP-1 receptors on hemopoietic progenitors and should establish whether specific receptor subtypes are responsible for the differences in the growth of CFC reported here.
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Acknowledgments |
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References |
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exert their main inhibitory activity on very primitive CD34++CD38– cells but show opposite effects on more mature CD34+CD38+ human hematopoietic progenitors. Exp Hematol 1996;24:1509-1515.[Medline] and transforming growth factor ß on hematopoietic progenitor/stem cell growth. Blood 1994;84:2175-2181. (MIP-1) abrogates IL-1 and cKit ligand (KL) induced proliferation of murine high proliferative potential (HPP) colonies. Blood 1991;78:259., interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro. J Exp Med 1994;179:643-649., TGF-ß, and MIP-1 on marrow purified CD34+ progenitors. Exp Hematol 1995;23:551-556.[Medline] enhancement of proliferation of mature progenitors in vitro, MIP-1; mobilisation of progenitors in vivo and MIP-1 enhancement of G-CSF induced mobilisation, but not for MIP-1 suppression of immature progenitors in vitro: effects elucidated using CCR-1 knockout mice. Blood 1997;90:571a.
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