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Macrophage-Inflammatory Protein-1 Receptor Expression on Normal and Chronic Myeloid Leukemia CD34⁺ Cells¹

Sian E. Nicholls^*, Guy Lucas, Gerard J. Graham, Nigel H. Russell^§, Rachel Mottram^*, Anthony D. Whetton^* and Anne-Marie Buckle^2,*

^* Leukemia Research Fund Cellular Development Unit, University of Manchester Institute of Science and Technology (UMIST), Manchester, United Kingdom; Department of Clinical Hematology, Manchester Royal Infirmary, Manchester, United Kingdom; The Beatson Institute for Cancer Research, Glasgow, United Kingdom; and ^§ Department of Haematology, Nottingham City Hospital, Nottingham, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have assessed expression of MIP-1 binding sites on the surface of CD34⁺ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34⁺ (15.7 ± 6.2% in NBM and 9 ± 4% in CML), which has specific macrophage-inflammatory protein-1 (MIP-1) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34⁺ Thy⁺ stem cells. However, more than 80% of methanol-fixed CD34⁺ cells do bind MIP-1, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34⁺, MIP-1-R⁺ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1 receptor implicated in HIV infection, CCR5, revealed that very few CD34⁺ cells expressed these receptors and that expression was confined to the CD34⁺ Thy^- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1 are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Macrophage-inflammatory protein-1 (MIP-1)³ is a member of the chemokine family of cytokines that have been shown to elicit a wide variety of effects on cells of the immune system, including adhesion, chemoattraction, and stimulation of Ig synthesis, thereby defining chemokines as important mediators of the inflammatory response (1).

In addition to these shared chemokine activities, MIP-1 has been reported to act as an inhibitor of the proliferation of murine hemopoietic stem cells both in vivo and in vitro (2, 3); indeed MIP-1 was originally described as a murine stem cell inhibitor and has subsequently been shown to inhibit epidermal stem cells (4). Although both inhibitory and stimulatory effects of MIP-1 on the proliferation of human stem cells have been reported according to the specific cytokine conditions of the assay (3), MIP-1 is known to inhibit the formation of methylcellulose colonies from highly purified progenitors (5). Clonogenic studies suggest that MIP-1 is acting directly on progenitors rather than indirectly via accessory cells. Effects of MIP-1 on more primitive stem cell populations have also been described both in inhibition of stem cell proliferation (6) and in maintenance of the stem cell compartment under conditions promoting differentiation (7, 8, 9). However, since stem cells have a requirement for stromal feeder cells, it is not clear whether the action of MIP-1 is directly on the stem cells, or an indirect effect via the stroma.

Chemokines are divided into four groups, C, CC, CXC, and CX3C, based on the number and arrangement of conserved cysteines (10, 11). MIP-1 is a CC chemokine, for which multiple human G protein-coupled seven-membrane-spanning receptors have been cloned, and termed CCR (12). These receptors have complex patterns of ligand binding, whereby they display both specificity and promiscuity. For example, human chemokine receptors CCR1 and CCR5 bind MIP-1, but also bind other members of the CC group, such as MIP-1ß and RANTES. Furthermore, CCR1 additionally binds monocyte-chemotactic protein 3 (12, 13), whereas CCR5 binds monocyte-chemotactic protein 2 (14). It has also been shown that cross-desensitization can occur among the chemokine receptors, although the underlying mechanisms of receptor internalization and signal transduction are not well characterized (15, 16). Recently, an additional high affinity receptor for MIP-1, known as D6, has been identified in the mouse (17) and in humans (18). There is currently much interest in understanding how specific receptor engagement relates to individual responses such as chemotaxis or growth inhibition, although there is now emerging evidence in the murine system that some MIP-1 receptors such as CCR1 and CCR4 may be related to inflammation, whereas others such as CCR5 and the novel receptor, D6, may be important in the control of proliferation (17). The recent finding that several chemokine receptors may act with CD4 as coreceptors for HIV-1 infection suggests that these proteins may play a role in controlling the progression of AIDS (19). CCR5, CCR2b, and to a lesser extent CCR3 have been shown to serve as coreceptors for HIV infection of macrophage-tropic HIV-1 (20, 21, 22), whereas CXCR4 functions as a coreceptor for T cell-trophic HIV strains (23). The detection of CD4 expression on some CD34⁺ cells (24) has raised the possibility that these cells may be a target for HIV-1 infection. Although it is not clear whether primitive stem cells do indeed constitute a potential reservoir for HIV-1, the detection by PCR of the CXCR4 receptor suggests that they may be a target for T cell-trophic strains (25), although no conclusions could be drawn from the PCR data generated in this study about CCR5 expression (25).

Since cancer chemotherapy is dose limited by the damage inflicted on proliferating cells within the bone marrow, members of the chemokine family are under consideration in strategies aimed at the chemoprotection and mobilization of normal bone marrow (NBM) stem and progenitor cells (26). There is now evidence in diseases such as CML (6, 27, 28), acute myelogenous leukemia (29), and some forms of cancer (30) that chemokines may not inhibit proliferation of leukemic or cancer cells. The possibility that growth inhibition is selective for normal cells presents chemokines as particularly attractive candidates for chemoprotective therapies.

In this study, we have further defined the role of MIP-1 and its receptors in the inhibition of stem cell proliferation by analyzing the binding of biotinylated MIP-1 (bMIP-1) to human CD34-positive bone marrow stem cells. The use of biotinylated cytokines to study cytokine receptor expression pattern on rare populations of human stem cells has been successfully demonstrated by Wognum et al. (31, 32). We have now adopted a similar approach using bMIP-1 in a multiparameter staining protocol that enables us to examine the MIP-1-binding ability of specific human hemopoietic progenitor cell populations. This approach not only has the advantage of identifying small subpopulations of MIP-1 receptor-bearing progenitor cells, but also is not restricted to identifying any one class of receptor and will therefore detect all members of the known CCR family that bind MIP-1, as well as novel MIP-1 binding sites. This is of particular significance in the case of hemopoietic stem cells, since there may be novel receptors on these cells yet to be identified. In addition, we have used Ab to the specific receptor CCR5 that is implicated in the control of stem cell proliferation and in the infection of CD34⁺ cells by HIV-1 to define CCR5 expression patterns on CD34⁺ cells.

	Materials and Methods

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Isolation of mononuclear cells from clinical samples

Mononuclear cells (MNC) were prepared from samples of NBM from consenting donors and peripheral blood from patients with CML at presentation using Ficoll-Paque density-gradient separation (d = 1.077) (Pharmacia, Uppsala, Sweden). MNC were washed and kept in HBSS supplemented with 5% heat-inactivated newborn calf serum (HBSS + 5% NCS (HI)) (Life Technologies, Paisley, Scotland) at 4°C for all further cell manipulations.

Cell lines

The TF-1 cell line was maintained in RPMI 1640 (Life Technologies) + 10% FCS + 2 ng/ml GM-CSF (R&D Systems, Minneapolis, MN). The THP-1 cell line was maintained in RPMI 1640 + 10% FCS. HEK293 cells transfected with human CCR5 and the parent HEK293 cells were maintained in DMEM (Life Technologies) and 10% FCS, and were passaged using trypsin EDTA (Life Technologies).

Ab staining

MNC were incubated for 20 min at 4°C with Ab to CD34 conjugated to PE, or allophycocyanin (APC) (Becton Dickinson, Oxford, U.K.). In some experiments, cells were additionally stained with Ab to Thy-1 conjugated to PE (PharMingen, San Diego, CA). Negative controls using isotype-matched Abs conjugated to APC and to PE were routinely performed. After staining, cells were washed and resuspended in HBSS + 5% NCS for staining with bMIP-1 (see below). Rabbit polyclonal anti-human CCR-5 sera and preimmune control sera were kind gifts from Jane McKeating at the University of Reading, and were used in conjunction with FITC sheep anti-rabbit (The Binding Site, Birmingham, U.K.). mAbs to CCR3 and CCR5 were kindly provided by Dr. Charles Mackay (Leukosite, Cambridge, MA) and were used in conjunction with FITC anti-mouse (PharMingen).

Labeling for MIP-1 binding sites

MNC, prelabeled with anti-CD34 APC and anti-Thy-1 PE, were assayed for the presence of MIP-1 binding sites using the method provided with a Fluorokine kit purchased from R&D Systems. Briefly, cells were incubated with a biotinylated human rMIP-1 at 4°C for 60 min. The cells were then incubated (without washing) with streptavidin-fluorescein (SAv-FITC). The kit includes a negative control Ab (a soybean trypsin inhibitor protein that has been biotinylated to the same extent as the MIP-1) and a MIP-1-blocking Ab (a polyclonal goat IgG anti-human MIP-1 Ab). The competable binding of the bMIP-1 was assessed using a 50-fold excess of unlabeled BB-10010 (British Biotech Phamaceuticals, Oxford, U.K.), which is a well-characterized mutant of MIP-1 with a single amino acid substitution (33), or a 50-fold excess of the R&D Systems MIP-1 of the same composition as that which is biotinylated in the Fluorokine kit. Cells were incubated at 4°C for 60 min in bMIP-1 premixed with unlabeled MIP-1, then labeled with SAv-FITC. All samples were labeled with 200 µg/ml propidium iodide (PI) (Molecular Probes, Eugene, OR), for dead cell exclusion, at the end of the bMIP-1 labeling. Using this assay, only cells expressing a specific MIP-1 receptor can be identified, and the intensity of the staining is directly proportional to the receptor density.

Fixation and acid washing of samples

In some experiments, cells were fixed in 70% cold methanol overnight. Cells were then washed and resuspended in HBSS + 5% NCS (HI) for 30 min before Ab labeling. The acid wash procedure consisted of two washes in HBSS + 5% NCS (HI), resuspension of the cells in PBS, pH 3, for 1 min exactly at 4°C, then further washing before resuspending in HBSS + 5% NCS (HI). All of the above cell preparations were then kept at 4°C for Ab labeling.

Flow cytometry

Cells were visualized using a dual laser FACSVantage (Becton Dickinson) flow cytometer with excitation of FITC, PE, and PI from an Argon laser, and of APC using a HeNe laser. Data were collected and analyzed using CellQuest software.

Immunocytochemistry

Cytospin preparations of THP-1 cells were made using a Shandon Cytospin at 500 x g for 5 min. Slides of CD34⁺ cells from NBM and CML peripheral blood were prepared by sorting 3000 cells directly onto microscope slides using the flow cytometer. Slides were air dried before fixation for 10 min in methanol (prechilled to -20°C), then allowed to dry at room temperature.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Validation for specific binding of bMIP-1 to viable human hemopoietic cells

The human erythroleukemic TF-1 cell line and the myelomonocytic cell line THP-1 have both been reported to bind MIP-1 (34). To determine whether the specific binding of bMIP-1 could be quantified using viable cell populations, we investigated the binding of bMIP-1 to TF-1 and THP-1 cells. The histogram plots A and B in Fig. 1 show that there was a significant level of bMIP-1 binding over that seen with the control biotinylated protein for both cell lines. Furthermore, anti-human MIP-1 mixed with bMIP-1 before usage with TF-1 or THP-1 cells totally abrogated the specific binding observed. The upward shift in fluorescence shown in TF-1 cells that bind bMIP-1 indicates that the majority of the homogeneous cell population binds bMIP-1. As a further control, a competable binding experiment was performed in which a 50-fold excess of either BB-10010 or human rMIP-1 was used. The binding of bMIP-1 to TF-1 cells was competed by both forms of MIP-1, confirming the specificity of the binding assay (see Fig. 1C). It is important to note that the binding assay is performed at 4°C, thus minimizing intracellular uptake of labeled receptors. The ability of bMIP-1 to detect CCR5 receptors was confirmed by its ability to bind specifically to the HEK293 cell line transfected with CCR5 (Fig. 1D).

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Validation of the Fluorokine assay using TF-1 and THP-1 cell lines. TF-1 and THP-1 cells were thoroughly washed to remove medium, serum, and growth factors. Cells were labeled with bMIP-1, as described in Materials and Methods. Histogram plots A and B show the level of bMIP-1 binding to TF-1 and THP-1 cells, respectively (solid line), as compared with the negative control (dotted line). Specificity was demonstrated using an anti-MIP-1 Ab (dashed line). These results using the TF-1 cell line are also presented in the dot plots I, II, and III. The upward shift in FL-1 (bMIP-1-SAv-FITC) seen indicates that the majority of cells bound bMIP-1. Plot C shows the competable binding of bMIP-1 (solid line) on TF-1 cells by a 50-fold excess of BB-10010 or MIP-1 (two dashed lines), as compared with the control level (dotted line). Plot D shows the binding of bMIP-1 to HEK293 cells transfected with CCR5 (solid line) as compared with the control level (dotted line) or level in the presence of blocking Ab (dashed line).

MIP-1 binding sites on normal CD34⁺ cells

To study bMIP-1 binding on viable primitive cells, which are extremely rare in bone marrow and peripheral blood samples, and which can be lost during complex purification procedures, we adopted a strategy of using MNC preparations with no further purification. We established a four-color staining procedure in which freshly isolated bone marrow or peripheral blood MNC could be labeled with anti-CD34 Ab conjugated to APC and anti-Thy-1 Ab conjugated to PE, and also, importantly, stained with PI for dead cell exclusion. Cells were assessed for the presence of bMIP-1 binding sites by labeling with bMIP-1, which was detected by SAv-FITC. Fig. 2 presents data from seven NBM MNC preparations (mean ± SEM) showing the percentage of CD34⁺ cells that express bMIP-1 binding sites as compared with the biotinylated negative control. It was found that 15.7 ± 6.2% of the seven normal CD34⁺ cells expressed MIP-1 binding sites (mean ± SEM). The flow-cytometric profile of a typical sample is shown in Fig. 3A. The level of binding site expression over and above the negative control was highly significant (p < 0.001), and binding was specific, as demonstrated by the anti-MIP-1 Ab (Figs. 2 and 3A).

View larger version (38K): [in this window] [in a new window]   FIGURE 2. MIP-1 binding site expression on normal and CML CD34+ cells. Seven samples of NBM MNC and ten samples of CML PBMC were labeled for CD34 expression and bMIP-1 binding. Cells were labeled with anti-CD34 APC and Thy PE, followed by staining with bMIP-1 detected with SAv-FITC. Results are presented as the percentage of CD34+ cells that displayed positive binding (mean ± SEM) when incubated with control Ab, bMIP-1, or bMIP-1, plus blocking Ab. See Fig. 4 for staining patterns of Thy-1 expression. The percentage of CD34+ cells that displayed positive binding, as compared with the negative control, is shown to be significant on both normal and CML CD34+ cells: p < 0.001 and p < 0.00001, respectively; and the percentage of CD34+ cells expressing MIP-1 binding sites is shown to be significantly greater in NBM than in CML (p < 0.025). Data were analyzed using a Student’s t test.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Flow-cytometric demonstration of MIP-1 binding sites on CD34+ cells. Flow-cytometry dot plots show typical labeling of A, NBM CD34+ cells, and B, CML peripheral blood CD34+ cells with negative control Ab (plots a and d), bMIP-1 (plots b and e), and the bMIP-1-blocking Ab (plots c and f). Plots b and e show that only a subpopulation of CD34+ binds bMIP-1, unlike the binding of bMIP-1 to the homogeneous cell lines in Fig. 1.

MIP-1 binding sites on viable CML CD34⁺ cells

Samples of 10 CML peripheral blood MNC were stained with anti-CD34 PE, followed by bMIP-1 labeling, as in the above experiments on NBM samples. As can be seen in Figs. 2 and 3B, there is bMIP-1 binding to CML CD34⁺ cells. The percentage of CML CD34⁺ cells that bind bMIP-1, 9 ± 4% (mean ± SEM), is, however, significantly lower (p < 0.025) than that on normal CD34⁺ cells. As with NBM, the level of binding was significantly above the control value (p < 0.00001), and specificity of MIP-1 binding was demonstrated using a blocking Ab (Figs. 2 and 3B).

MIP-1 binding site expression on CD34⁺ subpopulations

We next characterized further the subpopulation of CD34⁺ cells that express MIP-1 binding sites. Unlike the binding of bMIP-1 by the homogeneous cell lines TF-1 and THP-1, bMIP-1 appeared to bind to a subpopulation of CD34⁺ cells from NBM or CML peripheral blood. Previous studies on the phenotypic characterization of stem cells suggested that CD34⁺ Thy⁺ cells are more primitive than CD34⁺ Thy^- cells. We therefore used a three-color multiparameter analysis of CD34, Thy-1, and bMIP-1 to phenotypically assess whether the CD34⁺bMIP-1⁺ population in NBM expresses the Thy-1 Ag with a view to determining whether MIP-1 expression is dependent upon the stage of development of hemopoietic cells. We can detect Thy-1 expression on CD34⁺ cells from NBM (as has been described previously in 35); however, when Thy-1 and bMIP-1 staining was assessed, it was found that the majority of the bMIP-1-staining population of cells did not coincide with the Thy⁺ population, thereby demonstrating that MIP-1-R⁺ cells are generally Thy^- (see Fig. 4).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. MIP-1 binding site expression on CD34+ Thy+ and CD34+ Thy- cells from NBM and CML peripheral blood. Simultaneous analysis of CD34, MIP-1, and Thy expression on NBM cells (A) and CML peripheral blood (B). Plots show bMIP-1 binding site and Thy-1 expression on CD34+-gated cells.

MIP-1 binding sites on fixed cells

The work described above was performed on freshly isolated MNC from bone marrow or peripheral blood. These data are, however, somewhat different from a previous study in which the expression of MIP-1 receptors on fixed MNC was reported (28) and >90% of CML CD34⁺ cells were shown to express MIP-1 binding sites. We therefore studied the binding of bMIP-1 on fixed MNC compared with unfixed, viable cells. Fig. 5 shows that in a typical experiment in which an aliquot of CML MNC was fixed before Ab labeling, 87.37% of CD34⁺ cells in the fixed sample bound bMIP-1, whereas only 16.9% of the nonfixed CD34⁺ cells expressed MIP-1 binding sites. It is possible that MIP-1 binding sites at the cell surface may be revealed after fixation because they are already occupied in vivo by MIP-1, and that after fixation these sites are vacated, allowing bMIP-1 to bind. We have assessed the possible effect of any residual MIP-1 bound to cell surface binding sites in clinical samples by acid washing of fresh CML and normal MNC before the bMIP-1-binding assay. It was found that this treatment had no effect on the binding of bMIP-1 to CML and normal MNC, in terms of either the percentage of cells binding bMIP-1 or the level of fluorescence intensity (data not shown). These data strongly suggest that bMIP-1 receptor expression at the cell surface is observed in only a fraction of cells, although almost all express bMIP-1 binding sites within the cells, as revealed by permeabilization through fixation.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. MIP-1 receptor staining using fixed CD34 cells from CML peripheral blood. MIP-1 binding was compared on samples before and after fixation. CML PBMC were fixed in cold methanol, as described in Materials and Methods. Histogram A shows the level of bMIP-1 identified on freshly isolated CML MNC (dotted line), together with the negative control (solid line) and the blocking Ab (dashed line). B shows the equivalent bMIP-1 binding to fixed MNC.

Subcellular localization of bMIP-1 binding sites in fixed cells

The subcellular location of the bMIP-1 binding sites observed after fixation of cells was investigated further through the use of deconvolution microscopy. Immunochemical staining of THP-1 cells showed evidence of cell surface binding and also some weak cytoplasmic binding, suggesting that in addition to the cell surface binding sites detected on fresh cells by flow cytometry, there is an intracellular pool of bMIP-1 binding sites (Fig. 6, a and b). The sorted CD34⁺ cells from NBM (data not shown) and CML peripheral blood (Fig. 6, c and d) also gave positive staining by immunocytochemistry. The staining seen on CD34⁺ cells was more patchy than that seen with THP-1 cells, suggesting clustering of the binding moiety. CD34⁺ cells have a high nuclear:cytoplasmic ratio that, combined with the patchy staining, made discrimination between cell surface and cytoplasmic staining less clear. However, as can be seen in Fig. 6, c and d, there was evidence of cell surface staining with binding concentrated in a tight ring at the outer edge of the cell.

View larger version (35K): [in this window] [in a new window]   FIGURE 6. Immunochemical staining of sorted CD34+ cells. THP and CD34+ cells were sorted onto slides and fixed in methanol before staining with the bMIP-1 kit. Staining of THP-1 cells in the presence (a) and absence (b) of blocking Ab is shown. Fig. 6 (c) shows the staining of CD34+ cells isolated from a CML patient. Magnification is x40. A more detailed image of c is shown in d.

Identification of specific MIP-1 receptors

Ab specific for the MIP-1 receptor CCR5 was used to stain MNC isolated from NBM and CML peripheral blood. As shown in Table I, 19 ± 9.8% of CD34^- cells in NBM expressed detectable levels of CCR5. While the majority of CD34⁺ cells were found not to express CCR5, staining was seen on a small subpopulation (4.9 ± 2.6%). As was seen in the previous three-color staining experiments with bMIP-1, these receptor-expressing cells were confined to the CD34⁺ Thy^- progenitor population of cells, and no staining was seen on CD34⁺Thy⁺ cells (Fig. 7). Abs to CCR3 were found to stain a small but distinct subpopulation (2.4 ± 1.4%) of CD34^- cells in NBM, whereas only 1.5 ± 0.8% of the CD34⁺ cells stained positively for CCR3. Analysis of cells from CML patients was also performed, and 8.9 ± 3% of CD34^- cells expressed CCR5, although less than 1% of CD34⁺ cells were positive for this receptor. CCR3 was also detected on 4.1 ± 2.3% of CD34^- cells and on 2.3 ± 1.1% of CD34⁺ cells. As with NBM, three-color analysis showed that any CCR3 or CCR5 staining was confined to the CD34⁺ Thy^- population, with no detectable staining of CD34⁺ Thy⁺ cells (Fig. 7).

View larger version (28K): [in this window] [in a new window]   FIGURE 7. CCR5 and CCR3 expression on CD34+ cells from NBM and CML peripheral blood. Plots show CCR3 or CCR5 expression on CD34+-gated cells in relation to Thy-1+ cells. Receptor-specific Abs were used to detect CCR5 staining on NBM (A) and CML (B), and CCR3 on NBM (C) and CML cells (D).

The Ab specific for CCR5 was shown to recognize a fixation-resistant epitope, as determined by the staining of fresh and fixed HEK293 cells transfected with CCR5. However, CD34⁺ cells from NBM or CML peripheral blood did not stain positively with this Ab after fixation, suggesting that this is not the receptor that binds MIP-1 after fixation (Fig. 8).

View larger version (43K): [in this window] [in a new window]   FIGURE 8. CCR5 expression on fixed cells. HEK293 cells transfected with CCR5 were stained with anti-CCR5 Ab before and after fixation (A). CCR5 staining on unfixed (B) and methanol-fixed (C) CD34+ cells from NBM is also shown. HEK293 CCR5 transfectants labeled with an irrelevant isotype-matched Ab were used as controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have found that, as previously reported, the majority of CD34⁺ cells are able to bind bMIP-1 after permeabilization by fixation in methanol, implying that all CD34⁺ cells have the potential to respond to MIP-1. We have now extended this study to show that unfixed cells have very little capacity to bind bMIP-1, and that only a small subpopulation of CD34⁺ cells demonstrated significant binding. The flow-cytometric approach used in these experiments has an important limitation in that we are unable to directly quantitate the number of receptors per cell. However, THP-1 cells, which we have clearly shown to express MIP-1 receptors by flow cytometry, have previously been shown in radiobinding assays to express a single class of high affinity receptor for monomeric MIP-1, with about 1300 receptors per cell detected (34). Therefore, we may postulate that our detection system is sufficiently sensitive to readily detect less than 1000 receptors per cell.

Abs to CD34 are known to mark a heterogeneous population of hemopoietic progenitor cells encompassing lineage-committed progenitors, more primitive progenitors, and also the small Thy⁺ subpopulation of candidate stem cells (35, 36, 37). To investigate whether the low percentage of unfixed cells that showed MIP-1-binding capacity was associated with the Thy⁺ stem cell compartment, we established a three-color stain for the simultaneous analysis of CD34, Thy-1, and MIP-1 binding site expression. We found that binding ability did not coincide with Thy-1⁺ cells, but rather was confined to a subpopulation of the Thy-1^- population of cells. Thus, binding sites are found on cells with the phenotype of progenitors, but not on stem cells themselves. Presumably the MIP-1-binding CD34⁺ subpopulation has substantial colony-forming potential, since high levels of colony inhibition of CD34⁺ cells have been reported with MIP-1 treatment (5, 28). These data are consistent with that of others (see Ref. 38 for example) who have suggested that MIP-1 inhibits the proliferation of primitive cells that have developed beyond the hemopoietic stem cell compartment, and that the action of MIP-1 is direct rather than via accessory cells.

The differences in staining profiles between fixed and fresh samples of CD34 cells may have important biological implications. One possibility is that there are receptor sites available within the cytosol that are made accessible by the fixation procedure. Immunochemical staining of TF-1 cells revealed evidence of cell membrane staining and some intracellular staining. Immunochemical analysis of CD34⁺ cells confirmed that these cells bind bMIP-1 upon fixation, although since these cells have very little cytoplasm, cell surface expression could not be clearly distinguished from cytoplasmic staining.

Cytosolic receptor binding implies that an as yet unidentified stimulatory event will induce cell surface expression. Little is known about the regulation of MIP-1 receptor expression, although induction on T lymphocytes has been shown to be affected by IL-2 (39). It is possible that combinations of cytokines may prove capable of inducing cell surface expression on stem cells. We addressed this question by investigating whether stimulation by stem cell factor and GM-CSF (growth factors used in one study by Chasty et al. (28), in which MIP-1 inhibited normal CD34⁺ cell proliferation) and also by MIP-1 itself induced cell surface expression on CD34⁺ cells. We were unable to see any changes in cell surface expression.

The lack of binding sites detected on the stem cells is however puzzling, particularly with respect to the reported expression of the MIP receptor CCR1 on the surface of CD34⁺ cells (40, 41), as detected by specific Ab. It is possible that there is a further level of complexity within the chemokine receptor family, whereby a change in conformation of the receptor may be required for effective binding, such as within the integrin family of adhesion molecules. Indeed, the IL-8R CXCR1 has been reported to undergo a conformational change after ligand binding that results in activation of signaling events (42).

Taken together, these observations suggest that CD34⁺ cells have the potential to bind MIP-1, as evidenced by the staining of fixed cells. However, the ability of MIP-1 to bind unfixed stem cells may be subject to regulation of receptor expression or conformation, such that in the resting state there is very limited capacity for these cells to bind MIP-1 at the surface. In contrast to the binding of MIP-1 to human cells, primitive mouse cells appear to bind well with almost all cells staining positively (A. Buckle, unpublished data).

This study has raised the possibility that there may be a more complex mechanism underlying the effects of MIP-1 on primitive progenitor cells than previously considered. Control of MIP-1 activity on these cells may be mediated through the inducibility of active form of receptor expression. It will therefore be important to understand more about the regulation of this receptor family in normal and Ph⁺ primitive cells to exploit fully the clinical potential of MIP-1 as a growth inhibitor.

Understanding the relationship between receptor expression and binding capacity of MIP-1 is hampered by the lack of reagents to detect receptor expression. PCR analysis of CCR5 in sorted CD34⁺ cells was inconclusive, with occasional positive staining being observed (25). In this study, we have investigated, using receptor-specific Ab, the expression of the high affinity MIP-1 receptor CCR5, which is a coreceptor for macrophage-trophic strains of HIV-1 (20, 21, 22). In addition, CCR5 has been implicated in the control of stem cell proliferation (17). Although staining of MNC could be seen with Ab to CCR5 and also with Ab to a further coreceptor for HIV, CCR3, very little expression of either receptor was observed in the CD34⁺ compartment, and in particular there was no evidence of staining on CD34⁺ Thy⁺ stem cells. These profiles suggest that resting CD34⁺ Thy⁺ cells are not potential targets for the macrophage-tropic strains of HIV-1 and that the growth-inhibitory effects of MIP-1 are not mediated through CCR5 on resting primitive stem cells.

	Acknowledgments

 
We thank all clinicians in the North West who supplied us with patient samples, and also Dr. Jane Owen-Lynch and Andrew Pierce for helpful discussions, and Furheen Rafiq for establishing the immunocytochemical staining method for MIP-1.

	Footnotes

 
¹ This work was funded by the Leukaemia Research Fund (U.K.).

² Address correspondence and reprint requests to Dr. Anne-Marie Buckle, Leukaemia Research Fund Cellular Development Unit, Department of Biomolecular Sciences, UMIST, Sackville Street, Manchester, U.K. M60 1QD. E-mail address:

³ Abbreviations used in this paper: MIP, macrophage-inflammatory protein; APC, allophycocyanin; bMIP, biotinylated MIP; CML, chronic myeloid leukemia; HI, heat-inactivated; MNC, mononuclear cell; NBM, normal bone marrow; NCS, newborn calf serum; PI, propidium iodide; SAv, streptavidin.

Received for publication September 16, 1998. Accepted for publication March 1, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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