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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3073-3081
Expression of Macrophage Inflammatory Protein-1 Receptors in Human
CD34+ Hematopoietic Cells and Their Modulation by Tumor
Necrosis Factor- and Interferon-
By
Jan Dürig,
Erika A. de Wynter,
Christoph Kasper,
Michael A. Cross,
James Chang,
Nydia G. Testa, and
Clare M. Heyworth
From the CRC Section of Haemopoietic Cell and Gene Therapeutics,
Paterson Institute for Cancer Research, Christie Hospital NHS Trust,
Manchester, UK.
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ABSTRACT |
Macrophage inflammatory protein-1 (MIP-1) can stimulate growth
inhibitory and potent chemotactic functions in hematopoietic cells. To
investigate whether the action of MIP-1 may be regulated at the
cellular receptor level, we studied the expression and modulation of
MIP-1 receptors on CD34+ cells isolated from normal
bone marrow (NBM), umbilical cord blood (CB), and leukapheresis
products (LP). Expression of MIP-1 receptors on CD34+
cells was analyzed by two-color flow cytometry using a biotinylated MIP-1 molecule. The mean percentage of LP CD34+ cells
expressing the MIP-1 receptors was 67.7 ± 7.2% (mean ± SEM; n
= 22) as compared with 89.9 ± 2.6% (n = 10) and 74.69 ± 7.04%
(n = 10) in CB and NBM, respectively (P = .4).
The expression of the MIP-1 receptor subtypes on LP
CD34+ cells was studied by indirect immunofluorescence
using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5.
Microscopical examination revealed a characteristic staining of the
cytoplasmic cell membrane for all three receptor subtypes. Detailed
analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of
CD34+ cells express CCR-1, CCR-4, and CCR-5,
respectively. Culture of LP CD34+ cells for 24 to 36 hours in the presence of tumor necrosis factor- (TNF-) and
interferon- (IFN-) resulted in a significant increase in MIP-1
receptor expression. TNF- induced MIP-1 receptor upregulation in
a time- and concentration-dependent manner. Our results suggest that
inhibitory cytokines produced by the bone marrow microenvironment are
likely to be involved in the regulation of MIP-1 receptor expression
on hematopoietic cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE PROLIFERATION OF hematopoietic cells
is regulated by a balance between stimulatory and inhibitory signals
normally provided by the bone marrow microenvironment. Over recent
years, macrophage inflammatory protein-1 (MIP-1), which is
constitutively1 and inducibly2 produced by
stromal macrophages and myoid cells3 and is a member of a
family of closely related small (8 to 12 kD) proteins called
chemokines,4-6 has been shown to inhibit cell cycling of
primitive hematopoietic cells in vitro7,8 and in
vivo.9-11 This chemokine affects the proliferative state of
hematopoietic progenitor cells in a complex way depending on the
origin12 and the maturational stage of the target
cells.13
Chemokines elicit their effects on primitive hematopoietic cells via
G-protein-coupled receptors, several of which have recently been
cloned.14-17 MIP-1 has been shown to bind to at least
three of these receptors, designated chemokine receptors CCR-1, CCR-4, and CCR-5.14-18 MIP-1 binding to these receptors is
associated with transient elevations of intracellular calcium levels
[Ca2+]i, which has been used as an indicator
of receptor activation.18,19 The signal transduction
pathways further downstream of the receptors have only been partially
elucidated. Aronica et al20 showed that treatment of Mo7e
cells with MIP-1 inhibits upregulation of Raf-1 kinase activity in
response to granulocyte-macrophage colony-stimulating factor (GM-CSF)
and stem-cell factor (SCF). The same authors recently reported that the
chemokines, interferon-inducible protein 10 (IP-10) and MIP-1, block
the stimulatory effects of GM-CSF and SCF on MAP kinase activity in
MO7e cells.21 Thus, it is 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 of ligand binding.
As the action of MIP-1 may be regulated at the cellular receptor
level, we studied the expression and modulation of MIP-1 receptors
in hematopoietic CD34+ cells. We show that
CD34+ cells, from normal bone marrow, umbilical cord blood,
and leukapheresis products, exhibit comparable receptor levels as
determined by flow cytometric analysis of biotinylated MIP-1 binding
to CD34+ cells. Furthermore, we demonstrate that
CD34+ cells express the three MIP-1-binding chemokine
receptors CCR-1, CCR-4, and CCR-5 on their cytoplasmic cell membranes.
MIP-1 receptor expression on the cell surface of CD34+
cells was found to be associated with cell cycling and can be upregulated by the inhibitory cytokines tumor necrosis factor- (TNF-) and interferon- (IFN-).
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MATERIALS AND METHODS |
Cells.
Human umbilical cord blood (CB) samples were collected from full-term
normal deliveries and normal bone marrow (NBM) samples were obtained
from allogeneic BM donors. Leukapheresis products (LP) were obtained
from 27 patients with hematological malignancies or solid tumours (12 multiple myeloma, 1 acute lymphoblastic leukemia, 1 acute myeloid
leukemia, 4 teratoma, 1 sarcoma, 4 non-Hodgkin's lymphoma, 2 breast
cancer, 1 Hodgkin's lymphoma, 1 chronic lymphocytic leukemia). To
mobilize hematopoietic progenitor cells into the peripheral blood, all
patients received appropriate cytotoxic chemotherapy followed by
recombinant granulocyte colony-stimulating factor (G-CSF;
Lenogastrim [Chugai Pharma, London, UK], 263 µg/d subcutaneously); leukapheresis was performed on these patients when the
rising blood white cell count exceeded 3 × 109/L. At
the time of the leukapheresis, all patients were in remission. The
median age was 48 years (range, 15 to 65 years). The female:male ratio
was 12:15. 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
400g for 25 minutes. The MNC at the interface were collected and washed in phosphate-buffered saline (PBS) containing 0.5% (wt/vol)
bovine serum albumin (BSA).
Isolation of CD34+ cells.
CD34+ cells from CB, NBM, and 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 mmol/L EDTA and 0.5% [wt/vol] 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 minutes 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.
Immunophenotypical analysis of MIP-1 receptor expression.
MIP-1 receptor expression on CD34+ cells was
analyzed using the commercially available Fluorokine hu MIP-1
receptor kit (R&D Systems, Abingdon, UK) according to the
manufacturer's instructions. Briefly, MACS purified CD34+
cells were washed and labeled with human MIP-1 biotin conjugate for
1 hour at 4°C. Avidin-fluorescein and directly-conjugated CD34-PE
(HPCA-2; Becton Dickinson, Cowley, Oxon, UK) were added and the cell
suspension was further incubated at 4°C for 30 minutes. Subsequently, the cells were washed and fixed in 1% (vol/vol) formaldehyde. Receptor expression was analyzed by flow cytometry in a
uniformly set gate comprising lymphoblast-like cells expressing the
CD34 antigen. Results are expressed as the percentage of MIP-1 receptor positive cells in the CD34+ cell population. The
receptor density on these cells was assessed by determining the mean
fluorescence intensity (MFI) for fluorescein isothiocyanate (FITC) in
an identical gate (Fig 1). The specificity of the MIP-1 receptor labeling was tested using a blocking antibody (R&D Systems) in combination with MIP-1 biotin and using a
nonspecific biotinylated protein (soybean trypsin inhibitor) as a
negative control as described by the manufacturer. When tested on LP
CD34+ cells, this blocking antibody reduced the number
stained with the biotinylated MIP-1 to less than 1%. In some
experiments, MIP-1 receptor-labeled cells were fixed in 70%
(vol/vol) ethanol/PBS and stained with propidium iodide (Sigma, St
Louis, MO) to analyze the cell cycle distribution of the different cell
populations as previously described.22 Flow cytometric
analysis was performed on a FACScan flow cytometer (Becton Dickinson)
equipped with an Argon-Ion laser tuned at 488 nm within 24 hours of
sample processing.
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| Fig 1.
Flow cytometric analysis of isolated LP
CD34+ cells from a representative experiment;
two-color immunofluorescence staining with
FITC-avidin-biotin-MIP-1 complex and PE-conjugated anti-CD34
monoclonal antibody. Control cells were stained with a nonspecific
biotinylated protein (soybean trypsin inhibitor) and irrelevant mouse
monoclonal conjugated to PE as described in Materials and Methods.
Gates for the analysis of MIP-1 receptor expression on
CD34+ are defined as shown: R1, lymphocyte gate (A); R2,
CD34+ lymphocytes (B). MIP-1 receptor expression was
assessed as mean fluorescence intensity (C) in R2 and percentage of
positive cells (D). A negative control using a irrelevant biotinylated
protein (shaded histogram) was used to set the quadrant such that at
least 99% of the analyzed cells were negative for MIP-1 receptor.
Sorting gates are indicated in (D): R3, CD34+MIP
R ; R4, CD34+MIP R+, and were
set to include a strongly negative and positive fraction,
respectively.
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Fluorescence-activated cell sorter (FACS) sorting of
CD34+ cells.
Immunomagnetically isolated CD34+ cells were sorted under
sterile conditions into MIP-1 receptor positive and negative
fractions using the Automatic Cell Deposition Unit (ACDU) on a FACS
Vantage flow cytometer (Becton Dickinson). A total of 2 to 3 × 103 cells of each fraction was plated in colony-forming
assays and analyzed for the presence of colony-forming
cells-granulocyte-macrophage (CFC-GM) and burst-forming
unit-erythroid (BFU-E) as previously described.23
Immunophenotypical analysis of MIP-1 receptor subtypes.
Commercially available goat polyclonal antibodies against the MIP-1
binding chemokine receptors CCR-1, CCR-4, and CCR-5 (Santa Cruz
Biotechnology, Santa Cruz, CA) were used to characterize the expression
of MIP-1 receptor subtypes on CD34+ cells.
Indirect immunofluorescent cell staining was performed as described by
the manufacturer. Cells were washed and cytospin preparations were
prepared. Cells were fixed in methanol at  10°C for 5 minutes
and air dried. Nonspecific IgG binding was blocked by preincubating
cells with human AB serum (1:200 dilution in PBS). Primary antibody (1 µg/mL) staining was performed for 1 hour followed by three washes
with PBS. Thereafter, the secondary anti-goat IgG-FITC antibody (15 µg/mL; Autogen Bioclear, Calne, Wiltshire, UK) was added
and incubated for 45 minutes. Slides were washed in PBS and cells were
counterstained with DAPI and mounted in antifade solution (Vectashield;
Vector, Brelton, Peterborough, UK) under glass cover-slips. Control
slides were subjected to the same protocol, with the exception of
substituting PBS for the primary antibody in the first incubation step.
All incubations were performed in a humidified atmosphere at room
temperature. Slides were viewed using a Zeiss Axioskop Fluorescent
microscope (Oberkocken, Germany) equipped with single-band
excitation filters for each fluorochrome mounted in a
computer-controlled filter wheel. Grey levels were captured for
each fluorochrome using a Photometrics (Tucson, AZ) charge-coupled
device (CCD) camera, and final analysis and preparation of figures were
performed using Vysis Quips software (Vysis, London, UK).
In two experiments we quantitated the chemokine receptor subtype
expression on MACS purified CD34+ cells (purity >90%)
using the same antibodies and a single-color flow cytometric approach.
CD34+ cell suspensions were subjected to the same fixation
and staining protocol as outlined for the cytospin preparations. FACS
analyses were performed as described in the previous section.
Colony forming cell assays.
A total of 2 to 3 × 103 sorted cells from each of the
selected populations were plated as previously described.23
Briefly, cells were added to a 1-mL mixture of 30% (vol/vol) fetal
calf serum (FCS), 10% (wt/vol) deionized BSA, 10% (vol/vol)
5637-conditioned medium, 2 U erythropoietin, and 1.35% (wt/vol)
methylcellulose. 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 using standard
criteria after 14 days' incubation.
Suspension cultures.
A total of 1 to 2 × 105 CD34+ cells was
cultured in 24-well flat-bottom plates in Iscove's modified Dulbeccos
medium (IMDM), 15% (vol/vol) FCS plus various concentrations of
TNF- (Sigma), 1,000 IU/mL IFN-, and 2.5 ng/mL (100 pmol/L)
transforming growth factor- (TGF-; both from R&D Systems). Cells
were incubated for 24 hours at 37°C in 5% CO2 and 5%
O2 in nitrogen. At the end of the culture period cells were
washed 2× in PBS, and viability was measured using the Trypan
blue exclusion method. In four experiments, the kinetics of MIP-1
receptor expression in response to TNF- (2 ng/mL) were studied by
obtaining cultures after various time intervals and comparing the
results to the time 0 baseline values.
Statistical analysis.
Data are expressed as mean ± SEM. Statistical significance
was determined using the nonparametric Wilcoxon test for paired samples. The Kruskal Wallis test was used to compare differences in
MIP-1 receptor expression between NBM, CB, and LP CD34+
cells.
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RESULTS |
Purification of CD34+ cells.
The median purity of LP-derived CD34+cells was 82% (range
55% to 96%) after MiniMACS purification. The median numbers of
processed MNC and CD34+ recovered were 2.2 × 108 (range 6 × 107 to 5 × 108) and 2.7 × 106 (range1.2 × 105 to 1 × 107), respectively. The purity
of CB and NBM 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.24 Cell
viability following separation was always greater than or equal to
90%.
Two-color immunofluorescence analysis of CD34+ cells
for MIP-1 receptor expression.
The coexpression of the CD34 antigen and MIP-1 receptors was
assessed on cells isolated from LP, NBM, and CB. MIP-1 receptor expression was assessed as the percentage of MIP-R+ cells
in the CD34+ population and MFI as described in Fig 1. The
median percentage of CD34+ cells coexpressing the MIP-1
receptors (Fig 2) was higher in CB (range
74.2% to 98.1%) than in NBM and LP (range 33.5% to 96.0% and 0.5%
to 99.4%, respectively), but this difference was not significant
(P = .4). None of the patient characteristics including the
quality of the apheresis product (CFC-GM and CD34+ cell
content) and the extent or type of pretreatment with radio/chemotherapy appeared to correlate with MIP-1 receptor expression on the isolated CD34+ cells in the LP samples (data not shown).
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| Fig 2.
Constitutive expression of MIP-1 receptors on human
CD34+ cells isolated from NBM, CB, and LP. Purified
CD34+ cells were double-stained with biotinylated
MIP-1 and antibodies specific for the CD34 antigen. Results are
expressed as the percentage of CD34+ cells coexpressing
MIP-1 receptors. Horizontal bars denote the median of n individual
determinations.
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Colony formation by MIP-1 receptor positive and negative
CD34+ cells.
The colony forming potential of MIP-1 receptor positive
(R+) and negative (R) CD34+
cells is shown in Table 1. In general,
CFC-GM colony forming efficiency in the MIP-1-R+ cell
fraction exceeded those in the MIP-1-R fraction,
but this difference was found to be significant only in LP
CD34+ cells (P < .05). The BFU-E were more evenly
distributed between the two fractions in all cell sources examined.
Cell cycle distribution of MIP-1 receptors in
cytokine-stimulated CD34+ cells.
The cell-cycle distribution of ungated, MIP-1 receptor positive and
negative cells is shown in Table 2.
Significantly more MIP-1-R+ than
MIP-1-R cells were in S/G2/M phase
(P < .05). Furthermore, the MIP-1 receptor density on
cells in S/G2/M was significantly higher than in the
G0/G1 population (MFI: 232.7 ± 50.9 v 181.4 ± 39.8; n = 4, mean ± SEM, P < .05).
Characterization of MIP-1 receptor subtypes expressed by
CD34+ cells.
In some experiments CD34+ cells were spun onto slides and
the expression of the MIP-1 binding chemokine receptors CCR-1,
CCR-4, and CCR-5 was analyzed microscopically using indirect
immunofluorescence. All three receptor subtypes were detected on LP
CD34+ cells, and the staining showed a characteristic
membrane-associated distribution. In a second series of experiments, we
tried to quantify the coexpression of the MIP-1 receptor subtypes
using highly purified CD34+ cells (purity >90% as
analyzed by flow cytometry) and single-color flow cytometry.
Table 3 summarizes the results, and
Fig 3 shows an example of a representative
experiment.
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| Fig 3.
Flow cytometric analysis of isolated LP
CD34+ cells (purity 98%) from a representative
experiment; indirect single-color immunofluorescence staining was
performed with specific antibodies raised against the human chemokine
receptor subtypes CCR-1, CCR-4, and CCR-5 as described in Materials and
Methods. Control cells were stained with secondary FITC-linked antibody
only. Chemokine receptor expression was analyzed in a uniformly set
lymphocyte gate comprising 20,375, 21,503, and 21,659 cells in the
CCR-1, CCR-4, and CCR-5 experiments, respectively. Overlay plots of the
histograms (cell number against MFI) for the different chemokine
receptors and the control are shown.
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Effects of inhibitory cytokines on the expression of
MIP-1 receptors.
To determine whether TNF-, IFN-, and TGF- could modulate
MIP-1 receptor expression, LP CD34+ cells were cultured
in the presence of these cytokines and subsequently analyzed by flow
cytometry.
Figure 4A and
C shows that TNF- significantly increased MIP-1 receptor
expression on CD34+ cells in a time-dependent fashion with
maximum stimulation observed after a 10-hour incubation period
(2.1-fold MFI increase; P < .05) followed by a gradual
decline. However, the MFI was still 1.3-fold over baseline values at 24 hours. In contrast, the control showed a time-dependent decrease in
MIP-1 receptor expression with a reduction of MFI to 20.1 ± 7.4% (P < .05, mean ± SEM, n = 4) of baseline values at
24 hours. The differential kinetics of MIP-1 receptor expression was
also reflected by the decreased percentages of MIP-1-R+
cells in the control but not in the TNF--treated cells (Fig 4B).
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| Fig 4.
Time-dependent expression of MIP-1 receptors on LP
CD34+ in short-term suspension culture. Cells were
cultured at a concentration of 1 to 2 × 105 cells in 1 mL
of IMDM supplemented with 15% (vol/vol) FCS and incubated in 5%
CO2 and 5%O2 in nitrogen for various time
periods (4, 10, and 24 hours) in the absence or presence of TNF- (2 ng/mL). MIP-1 receptor expression was assessed by determining the
mean fluorescence (A) and the percentage of CD34+ cells
expressing MIP-1 receptors (B). (C) Shows a representative
experiment. Overlay plots of the histograms (cell number against MFI)
for the different cell populations at successive time-points are shown.
T = 0 hours, empty area represents the untreated cell population
stained for MIP-1 receptors (FITC). T = 10 hours and T = 24 hours, shaded and empty areas represent MIP-1 receptor expression in
TNF--treated and untreated cells, respectively. Data points in (A)
and (B) are the mean ± SEM of four independent experiments.
Nonspecific binding of avidin-FITC in TNF--treated cultures was not
increased over untreated cells at T = 0 hours. (*) Denotes
significant differences between the TNF--treated and untreated
cells (P < .05). (**) Indicates significant differences in
comparison to control values at 0 hours incubation (P < .05).
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Furthermore, to exclude the possibility that changes in receptor
expression were due to preferential death or survival of CD34+ subpopulations, cell viability was assessed using the
Trypan blue exclusion method. Cell viabilities were similar in all
groups and always within the range 80% to 92%.
In general, TNF- was found to be a potent upregulator of MIP-1
receptor expression (Fig 5A), whereas
INF- elicited only a moderate but statistically significant
stimulatory effect (Fig 5B). In contrast, under the conditions used,
TGF- (100 pmol/L) did not influence MIP-1 receptor expression
(data not shown).
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| Fig 5.
(A) Concentration-dependent increase of MIP-1
receptors on LP CD34+ in response to TNF-. Purified
CD34+ cells were cultured for 24 hours in the absence or
presence of varying concentrations of TNF-, as detailed in the
legend of Fig 3. Thereafter, the expression of MIP-1 receptors on
CD34+ cells was analyzed by flow cytometry. Bars
represent the mean ± SEM of n independent experiments. (*) Denotes
significant differences between the TNF--treated cells and controls
(P < .05). (B) IFN--induced upregulation of MIP-1
receptors on LP CD34+ cells. Purified LP
CD34+ cells were cultured for 24 hours in the absence or
presence of IFN- (1,000 U/mL) as detailed in the legend of Fig 3.
Thereafter the expression of MIP-1 receptors on CD34+
cells was analyzed by flow cytometry. Bars represent the mean ± SEM of four independent experiments. (*) Denotes significant difference
between the IFN--treated cells and the control (P < .05).
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In two experiments CD34+ cells cultured under control
conditions (serum-supplemented medium only) for 24 hours were
stimulated with TNF- at the 24-hour time point. As can be seen in
Table 4, following a further 12-hour
incubation in the presence of TNF-, MIP-1 receptor expression
increased when compared with the 24-hour levels and in one case to time
zero levels. This indicates that the receptor downregulation in the
control group was at least partially reversible.
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DISCUSSION |
We have analyzed the constitutive and induced expression of MIP-1
receptors in human CD34+ cells from normal bone marrow,
cord blood, and leukapheresis products. The majority of
CD34+ cells were found to express MIP-1 receptors
constitutively on their cell surface. On average, MIP-1 receptor
expression in CB was higher than in LP CD34+ and NBM
CD34+ hematopoietic progenitor cells, although differences
were not statistically significant. Perhaps more importantly,
individual variation between different LP samples was substantially
higher than in NBM and CB samples. Individual values could not be
correlated to the patient's disease characteristics, including
pretreatment with radio/chemotherapy or the quality of the apheresis
product. This raises the possibility that heterogeneity in MIP-1
receptor expression may reflect the variability of CD34+
populations mobilized with different regimes. Our results in NBM and CB
are consistent with previous studies by Su et al,25 showing
that most NBM CD34+ cells express the MIP-1 receptor
CCR-1. However, using a similar assay as described in this study,
Chasty et al22 found that greater than 52% of NBM
CD34+ express MIP-1 receptors.
The majority of colony forming cells were observed in the MIP-1
receptor positive fraction, although statistical significance was
reached only in the case of LP CD34+ CFC-GM. Furthermore,
cell cycle analysis of cytokine stimulated LP CD34+ cells
revealed that MIP-1 receptor expression is significantly increased
in cycling cells, which is in line with the results obtained in the
clonogenic assays. Our cell cycle data are in contrast to those
reported by Chasty et al,22 who did not observe a cell
cycle-dependent expression of MIP-1 receptors on NBM
CD34+ cells. However, this discrepancy may be explained by
differences in the experimental systems. We have used growth factor
stimulated hematopoietic progenitor cells to initiate cell cycling
whilst Chasty et al used unstimulated cells.
Because analysis of biotinylated MIP-1 binding to CD34+
cells cannot distinguish between the three different human MIP-1 receptors CCR-1, CCR-4, and CCR-5, we used specific polyclonal antibodies and standard indirect immunofluorescent techniques to
identify these receptor subtypes in highly purified LP
CD34+ cells. All three receptor subtypes were detected in
cytospin preparations of LP CD34+cells. Microscopical
evaluation revealed a characteristic staining of the cytoplasmic
membrane, and this staining pattern was similar for all the three
receptor subtypes, in all cell samples investigated. However, CCR-1 and
CCR-5 were more highly expressed than CCR-4, and this observation was
confirmed and further quantified in a flow cytometric assay. It is as
yet unknown which MIP-1 receptor subtypes relay the different
biological effects elicited by MIP-1. Graham et al26
suggested that MIP-1 uses different receptors to convey
chemoattractant as opposed to stem-cell-inhibitory signals. In
particular, CCR-1 was identified as an important receptor for chemoattraction, whereas no stem-cell-inhibitory effects were observed
following CCR-1 activation. However, using neutralizing CCR-1
antibodies Su et al25 showed that the inhibition of BFU-E formation from NBM CD34+ induced by MIP-1 is at least
partially mediated through CCR-1, suggesting a role of this receptor in
the proliferative control of erythroid progenitor cells.
The more variable surface expression of the MIP-1 receptors in LP as
compared to CB and NBM CD34+ cells led us to hypothesize
that cellular and/or humoral factors could regulate the
MIP-1 receptor status on CD34+ cells. Our first finding
in support of this hypothesis was that CD34+ cells cultured
in serum-supplemented medium exhibited a significant decrease of
MIP-1 receptor expression over an incubation period of 24 hours in
the absence of significant changes in cell viability. We then exposed
LP CD34+ cells to TNF- and IFN-, which have recently
been shown to modulate Fas-receptor and IFN--receptor expression on
CD34+ bone marrow progenitor cells.27 Both
cytokines increased the MIP-1 receptor expression significantly over
24-hour control values. For TNF- this effect was shown to be time-
and concentration-dependent and was detectable at concentrations of 0.2 to 2 ng/mL. Similar levels of TNF- have been observed in the
supernatants of normal LTBMC,28 suggesting that TNF-
could play a physiological role in MIP-1 receptor regulation in
these culture systems. TGF-, which was previously reported to
downregulate MIP-1 receptors on FDCP-Mix cells,29 did
not change receptor levels in our experimental conditions. Analysis of
cell viability and the gating strategy used to analyze MIP-1
receptor expression excluded the possibility that changes in receptor
expression were due to preferential cell death or survival of
CD34+ cell subsets. This was also confirmed by the fact
that receptor downmodulation on cultured CD34+ cells could
be reversed by addition of TNF-.
To our knowledge this is the first report on the upmodulation of
MIP-1 receptor levels on human CD34+ cells by other
inhibitory cytokines. However, the underlying mechanisms remain to be
determined. Evidence for the regulation of chemokine receptors
including CCR-1 at the transcriptional level has been described for T
lymphocytes that were cultured in the presence of interleukin-2
(IL-2).30 The kinetics of IL-2-induced CCR-1 mRNA
expression showed a gradual increase to a maximum after a 4-day culture
period, while our results revealed a peak receptor protein expression
after only 10 hours incubation. Furthermore and importantly, Loetscher
et al 30 reported that CCR-1 upregulation was correlated
with increased cell migration toward MIP-1 and RANTES, indicating
that chemokine receptor levels can regulate the biological
responsiveness of cells to chemokines.
TGF- and MIP-1 play important roles in the cell cycle control of
primitive hematopoietic progenitor cells that are localized in the
adherent layers of long-term bone marrow cultures.31,32 It
is known that MIP-1 is constitutively and inducibly produced by
stromal cell types.3,33 Using immunohistochemistry we
identified macrophages lodged in the stroma as the main producers of
MIP-1 in these culture systems (E.A.D., unpublished
data, August 1997). Furthermore, stromal fibroblasts, macrophages, and
accessory T lymphocytes have been reported to secrete an array of
cytokines including IL-1, TNF-, and IFN-.2,33,34
Importantly, these proinflammatory cytokines have recently been shown
to stimulate the production of MIP-1 in the bone marrow
microenvironment.35,36 Therefore, in view of our data the
inhibitory cytokines TNF- and IFN- not only induce the production
of MIP-1 but could also modulate the chemokine responsiveness of the
hematopoietic target cells through regulation of MIP-1 receptor
expression.
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FOOTNOTES |
Submitted May 18, 1998;
accepted June 25, 1998.
Supported by the Cancer Research Campaign of Great Britain. J.D. was
supported by a grant from ESMO. C.K. was supported by a grant from
"Aktion Kampf dem Krebs," Germany.
Address reprint requests to Clare M. Heyworth, PhD, CRC
Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Rd, Manchester M20 4BX, UK; e-mail: Cheyworth{at}PICR.man.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank M. Hughes and J. Barry for assistance with flow cytometry,
Prof T.M. Dexter for helpful discussions, and the Cancer Research
Campaign for support.
| |
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Abstract
Introduction
Abstract