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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 756-768
CHEMOKINES
Chemokine SDF-1 enhances circulating CD34+ cell
proliferation in synergy with cytokines: possible role in progenitor
survival
Jean-Jacques Lataillade,
Denis Clay,
Catherine Dupuy,
Sylvain Rigal,
Claude Jasmin,
Philippe Bourin, and
Marie-Caroline Le Bousse-Kerdilès
From the Laboratoire d'Immunologie Cellulaire, Centre de
Transfusion Sanguine des Armées Jean Julliard, Clamart Cedex,
France; Institut National de la Santé et de la Recherche
Médicale, Hôpital Paul Brousse, Villejuif, France;
Hôpital d'Instruction des Armées Percy, Clamart Cedex,
France.
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Abstract |
The chemokine stromal cell-derived factor-1 (SDF-1), and its
receptor, CXCR-4, have been implicated in the homing and mobilization of human CD34+ cells. We show here that SDF-1 may also be
involved in hematopoiesis, promoting the proliferation of human
CD34+ cells purified from normal adult peripheral blood
(PB). CXCR-4 was expressed on PB CD34+ cells. The amount
of CXCR-4 on PB CD34+ cells was 10 times higher when
CD34+ cells were purified following overnight incubation.
CXCR-4 overexpression was correlated with a primitive PB
CD34+ cell subset defined by a
CD34high CD38lowCD71lowc-KitlowThy-1+
antigenic profile. The functional significance of CXCR-4 expression was
ascertained by assessing the promoting effect of SDF-1 on cell
cycle, proliferation, and colony formation. SDF-1 alone increased the
percentage of CD34+ cells in the S+G2/M
phases and sustained their survival. In synergy with cytokines, SDF-1
increased PB CD34+ and
CD34highCD38low cell expansion and colony
formation. SDF-1 also stimulated the growth of colonies derived from
primitive progenitors released from quiescence by anti-TGF-
treatment. Thus, our results shed new light on the potential role of
this chemokine in the stem cell engraftment process, which involves
migration, adhesion, and proliferation. Furthermore, both
adhesion-induced CXCR-4 overexpression and SDF-1 stimulating activity
may be of clinical relevance for improving cell therapy settings in
stem cell transplantation.
(Blood. 2000;95:756-768)
© 2000 by The American Society of Hematology.
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Introduction |
Stromal cell-derived factor-1 (SDF-1) is a member of
the chemokine CXC subfamily initially cloned from the murine bone
marrow stromal cell lines ST-2 and PA6,1 then purified from
supernatant from the murine MS-5 cell line.2 It has been
suggested that SDF-1 is involved in immune surveillance because it is a
highly effective lympho-monocyte chemoattractant3 and it
supports B-cell progenitor proliferation.1,4 The
constitutive expression of SDF-1 in various tissues and its highly
conserved nucleotide and amino acid sequences5,6 suggest
that this molecule may play an important biological role. Gene knockout
experiments have confirmed the essential role of SDF-1 in hematopoietic
development.4 SDF-1-deficient mice have fewer myeloid
progenitors than normal mice in bone marrow, but fetal liver
hematopoiesis is unaffected in these mice, suggesting that SDF-1 is
involved in the migration of hematopoietic stem cells between embryonic
hematopoietic sites.4 Other studies have implicated SDF-1
in cell trafficking, in a model in which hematopoietic progenitor
migration and mobilization are caused by a gradient of SDF-1
concentration in the bone marrow microenvironment.7,8 SDF-1
may also be involved in megakaryocytic migration and platelet
formation, by increasing the adhesion of mature megakaryocytes to
endothelium.9,10
SDF-1 has been identified as the ligand for the leukocyte-derived seven
transmembrane domain receptor (LESTR). This G-protein-linked receptor
was originally identified as an orphan receptor with a structure very
similar to that of interleukin (IL)-8 receptors.11,12 Owing to the similarity of its sequence to that of other CXC chemokine receptor genes, LESTR was also named CXCR-4.13 It was also
called fusin because it mediates HIV-1-CD4 T lymphocyte cell
fusion.14 Several authors have reported CXCR-4 expression
on mature blood cells,15 including lymphocytes, monocytes,
megakaryocytes, and platelets.9,10 It has also been
detected on CD34+ progenitors purified from bone marrow
(BM), mobilized peripheral blood, and cord blood.10,13,16
In vitro transmigration assays have shown that circulating
CD34+ cells from healthy adults are sensitive to SDF-1, but
expression of its cognate receptor on these progenitors has not been
reported.7,8
All of these observations provide evidence that SDF-1 is critical for
mobilizing and homing hematopoietic progenitor cells. However, nothing
is known about the role of SDF-1 in progenitor proliferation. With
respect to the stem cell engraftment process in which trafficking and
proliferation are both involved, we investigated a possible role for
SDF-1 in hematopoiesis. We found that CD34+ cells isolated
from adult peripheral blood (PB) expressed the CXCR-4 receptor and that
SDF-1 increased the proliferation of human circulating
CD34+ cells. Both the percentage of CXCR-4+
cells and the level of CXCR-4 expression were much higher if PB
CD34+ cells were purified following overnight incubation on
a plastic support (Inc+). CXCR-4 overexpression was
correlated with the presence of a primitive
CD34highCD38lowCD71lowc-KitlowThy-1+
cell subset in PB. CXCR-4 was functional, as shown by the increase in
PB CD34+ and CD34highCD38low cell
expansion and colony formation caused by SDF-1, in synergy with
cytokines. This stimulatory effect was even stronger with Inc+ CD34+ progenitors released from quiescence
by an anti-TGF- antibody and was correlated with a large percentage
of CD34+ cells in the S+G2/M phases. Thus, the
SDF-1/CXCR-4 couple seems to have a novel biological function in
increasing primitive hematopoiesis.
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Materials and methods |
Cell sample collection
Buffy coats were obtained from healthy adults during the preparation
of transfusion products. Peripheral blood was collected (450 mL) in a plastic bag (MacoPharma, Tourcoing, France)
containing 80 mL of preservative-free anticoagulant
(citrate-phosphate-dextrose) and centrifuged at 2300g for 20 minutes at 20°C in a cryofuge 8500 (Heraeus Sepathec, Prolabo,
Briare le Canal, France).
BM cells were collected from normal donors undergoing hip prothesis
surgery. Spongy bone and femoral fluid were removed and rapidly stored
in RPMI 1640 (Biological Industries, ATGC Biotechnologies, Noisy le
Grand, France) containing heparin (100 U/mL, Roche). BM
cells nested in spongy bone were recovered with the use of a Potter
grinder and several washes with RPMI 1640.
All samples were obtained, with informed consent, from donors at the
Centre de Transfusion Sanguine des Armées Jean-Julliard, the
Services d'Hématologie Clinique et de Chirurgie
Orthopédique de l'Hôpital d'Instruction des Armées
Percy (Clamart, France), and the Service de Chirurgie
Orthopédique du CHR d'Aulnay-Sous-Bois (France).
Mononuclear cell preparation
PB and BM cell suspensions were diluted 1:2 in Dulbecco's
phosphate-buffered saline (Biological Industries, ATGC Biotechnologies) containing 0.5% human serum albumin (HSA) and 5% citrate
(PBS/HSA/ACD). Mononuclear cells were isolated by centrifugation on a
Ficoll density gradient (d = 1.077 g/mL; Seromed, ATGC
Biotechnologies) at 700g for 30 minutes at 20°C. The
mononuclear cell layer was collected, washed twice, and resuspended in
PBS/HSA/ACD. Two gentle centrifugation steps (91 g for 7 minutes) were
performed to remove platelets from PB samples.
Immunomagnetic purification of CD34+ cells
PB and BM mononuclear cells expressing the CD34 antigen were
immunomagnetically selected with the use of the MACS system (Miltenyi Biotech, Tebu) directly after density gradient separation
(Inc ) or after an overnight incubation (18 to 20 hours) (Inc+). We incubated 5 × 106
cells/mL in Iscove's modified Dulbecco's medium (IMDM)
containing 2% HSA in 75 cm2 plastic culture flasks
(Falcon, Becton Dickinson, Le Pont de Claix, France) at 37°C in a
5% CO2/95% air atmosphere. Nonadherent cells, collected
by washing the flask 3 times with PBS/HSA, or mononuclear cells,
recovered directly after Ficoll centrifugation, were incubated for 15 minutes at 4°C with 50 µL of blocking reagent (human
IgG) and 50 µL of CD34 antibody (QBEND/10, mouse IGg1) per 108 cells. Cells were washed once in
PBS/5mmol/L EDTA/0.5% HSA (PEH) and incubated for 15 minutes at 4°C with colloidal MACS microbeads (50 µL/108 cells) directed against the
haptenized QBEND/10. Cells were washed, resuspended, and passed through
a cell strainer (Falcon) to remove clumps. The labeled cells were added
to a sterile positive separation column (VS+) placed in a
magnetic field. CD34+ cells were flushed with PEH outside
the column, removed from the magnetic field, and collected by
centrifugation (700g). Cell numbers and viability (> 97%)
were assessed with a hemacytometer and trypan blue. For both the
Inc+ and Inc procedures, MACS
purification produced a 90% to 98% pure CD34+ cell
preparation. The Inc and Inc+
CD34+ cells accounted for 0.2% ± 0.05% and 0.18% ± 0.07% respectively of the low-density cells.
Cell sorting
PB Inc+ CD34+ cells were used to sort the
CD34highCD38low subpopulation. Cell suspensions
were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse
antihuman CD38 (T16, Coulter-Immunotech, Marseilles, France) and
phycoerythrin (PE)-conjugated mouse antihuman CD34 (HPCA2,
Coulter-Immunotech) for 30 minutes at 4°C, and washed twice. Then
the CD34highCD38low cells were separated with
the use of a Coulter Elite flow cytometer (Coulter Electronics,
Margency, France). About 10% of CD34high cells with the
lowest CD38 labeling were sorted as
CD34highCD38low.
Cycloheximide treatment
PB mononuclear cells were treated with cycloheximide (Sigma, Saint
Quentin Fallavier, France), a protein synthesis inhibitor, before the
overnight incubation step, as follows. Mononuclear cells
(5 × 106 cells/mL) were incubated in
IMDM 2% HSA with or without cycloheximide (10 µg/mL),
at room temperature for 30 minutes. Cells were then washed twice with
PBS/HSA and incubated overnight as described above.
Monoclonal antibodies and secondary reagents
The monoclonal antibodies (mAbs) used for the flow cytometry were
peridinin chlorophyll protein (PerCP)-conjugated mouse antihuman CD34 (8G12) and matching IgG1 isotype control purchased
from Becton Dickinson (Le Pont de Claix, France). FITC- or
PE-conjugated mouse antihuman CD38 (T16), CD71 (transferrin receptor
YDJ1.2.2), CD117 (c-Kit, 95C3), phosphotyrosine (6D12) and
IgG isotype control were obtained from Coulter-Immunotech.
PE-conjugated mouse antihuman CDw90 (Thy-1, 5E10), PE- or
biotin-conjugated CXCR-4 (12G5) mAbs, and biotin-conjugated IgG isotype
control were purchased from Pharmingen (Becton Dickinson). The
secondary reagent for CXCR-4 labeling was a
streptavidin-allophycocyanin (APC, Becton Dickinson).
Immunophenotyping
We performed 3- and 4-color flow cytometry assays using a
FACScalibur flow cytometer (Becton Dickinson) and CellQuest data acquisition software (Becton Dickinson). The instrument was calibrated with the use of beads (Becton Dickinson) according to the
manufacturer's instructions.
Surface antigen labeling.
We carried out a multiparameter analysis of the purified cells for cell
surface antigen expression. To minimize nonspecific antibody binding,
we incubated cells in a flow cytometry buffer containing PBS/2%
HSA/0.5% polyvalent human immunoglobulins. Cells (5 × 104 cells/40 µL PBS/0.5% HSA)
were then incubated in 96-well culture plates containing 10 µg/mL (saturating concentration) of directly conjugated
or unconjugated mAbs. We used biotin-conjugated anti-CXCR-4 mAb, or if
it was combined with intracellular labeling, we used PE-conjugated
anti-CXCR-4 mAb. Labeling with biotin-conjugated anti-CXCR-4 mAb was
followed by a final incubation step with 10 µL of
streptavidin-APC. Incubations were performed on ice for 20 minutes in
the dark and were followed by 2 washes with ice-cold PBS/0.5% HSA.
Immunostained cells were either kept on ice and immediately analyzed by
flow cytometry or fixed in 1% formol PBS and analyzed within a week.
The percentage of stained cells was calculated by comparison with each
isotype control. In multiple staining, we adjusted compensation using
the single-stained cell samples.
Intracellular antigen labeling.
Intracellular CXCR-4 and phosphotyrosine staining were
performed after permeabilization. CD34+ cells
(5 × 104) were incubated in a permeabilizing solution
(OrthoPermeafix, Ortho Diagnostic System, Issy les Moulineaux, France)
according to the manufacturer's instructions, before staining by a
PE-conjugated anti-CXCR-4 or an FITC-conjugated
anti-phosphotyrosine. Before intracellular CXCR-4
staining, cells were incubated in 40 µL PBS/0.5% HSA
containing an unconjugated anti-CXCR-4 antibody at saturating concentration for 20 minutes in the dark and were washed twice with
ice-cold PBS/0.5% HSA. The percentage of stained cells was calculated
by comparison with the isotype control.
For each sample, images from 1 × 104 cells
were acquired in listmode. Forward (FS) and side (SS) scattering
and 2 to 4 fluorescence signals were stored in listmode data files and
analyzed on a computer with WinMDI software (Trotter J; The Scripps
Research Institute [TSRI], La Jolla, California). Mean fluorescence
intensity (MFI) was expressed in arbitrary units (AU).
Clonogenic cell assay
Erythroid (BFU-E), granulocytic (CFU-G), monocytic (CFU-M),
granulo-monocytic (CFU-GM), and multilineage (CFU-Mix)-derived
colonies.
Progenitors were assessed in methylcellulose medium with the use of a
slightly modified version of the Metcalf technique.17 Freshly purified or cultured CD34+ cells (500 cells/mL) and sorted
CD34+CD38 cells (1000 cells/mL) were plated on methylcellulose culture medium
(Stem  ID, Tebu, Le Perray en Yvelines, France) containing a
cocktail of 7 recombinant human (rh) cytokines: IL-3 (0.8 ng/mL), IL-6 (10 ng/mL), IL-11 (2 ng/mL), SCF (3 ng/mL), G-CSF (0.75 ng/mL), GM-CSF (0.75 ng/mL), and Epo (2 U/mL). In some experiments, we replaced the cocktail by
similar concentrations of IL-3, GM-CSF, SCF (all cytokines were
purchased from R&D Systems), alone or in combination (Stem , Tebu).
Duplicate 35 mm2 tissue culture dishes containing 1 mL of cell suspension were incubated at 37°C in a
4.5% CO2/95.5% air atmosphere. BFU-E, CFU-G, CFU-M,
CFU-GM, and CFU-Mix were scored on day 14 with the use of an inverted
microscope and standard morphological criteria.18 For
erythroid progenitors, 2 differentiation stages were distinguished, based on colony size on day 14. Large bursts containing 16 or more
clusters with low hemoglobin content were defined as immature BFU-E,
whereas mature BFU-E with a higher hemoglobin content were small bursts
containing fewer than 16 clusters.19
Megakaryocyte (MK)-derived colonies.
Progenitors were assessed in collagen matrix with the use of the
Easymega kit (Hemeris, Grenoble, France) according to the manufacturer's instructions.20,21 We incubated
1 × 104 CD34+ cells in a 1 mL serum-free Easymega medium supplemented with the
following cytokines: 2.5 ng IL-3, 10 ng IL-6, and 50 ng thrombopoietin (TPO) (PeproTech Inc, Tebu). Duplicates of the culture
were incubated at 37°C in a humidified atmosphere containing 5%
CO2. Colonies were scored with the use of an inverted
microscope after 10 to 14 days of culture, according to criteria
described elsewhere.21 We distinguished 2 MK
differentiation stages on the basis of clone size on day 14. Colonies
containing more than 10 cells were defined as BFU-MK, whereas CFU-MK
contained fewer than 10 cells. Standard cytology staining (May
Grünwald Giemsa) or immunocytochemistry with a CD41 mAb (clone
CS3) followed by alkaline phosphatase monoclonal anti-alkaline
phosphatase detection was also performed for reliable and accurate
identification of megakaryocytic colonies.
The biological effect of SDF-1 on colony formation was evaluated in
both culture systems by adding free endotoxin rh SDF-1 at various
concentrations (0.01, 0.05, 0.1, 0.5, 5, and 10 ng/mL; R&D
Systems, Abingdon, UK) to the semisolid control media, in the presence
or absence of various cytokines (individually or in combination). In
some experiments, anti-rh SDF-1 monoclonal antibody
(5 ng/mL, R&D Systems) was added to the culture medium.
Anti-TGF- antibody treatment
The effect of SDF-1 on high proliferative potential-quiescent cells
was evaluated with the use of the model of Hatzfeld et al.22 CD34+ cells (2 × 105
cells/mL) were incubated for 48 hours in a
cytokine-free/serum-free liquid medium (Stem A) with or without a
polyclonal anti-TGF- neutralizing antibody (5 µg/mL)
or its IgY isotype control (R&D Systems, Abingdon, UK). Cells were then
plated on a semisolid medium, with or without SDF-1 as described
above, and colonies were scored on day 18.
Liquid cultures
Freshly purified PB CD34+ cells
(1 × 105 cells/mL) were cultured for
72 hours in either a cytokine-free medium (Stem A, Tebu) or in a
medium containing rh IL-3 (0.8 ng/mL), IL-6
(10 ng/mL), IL-11 (2 ng/mL), SCF (3 ng/mL), G-CSF (0.75 ng/mL), GM-CSF (0.75 ng/mL), and Epo (2 U/mL) (Stem A1). The
cells were incubated in the presence or absence (control) of rh
SDF-1 (0.05 ng/mL). At various time
points (0, 48, and 72 hours), cells were harvested and counted. We
assessed the possible priming effect of SDF-1 by treating cells with
SDF-1 (0.05 ng/mL) in a cytokine-deprived culture
medium (Stem A) for 24 hours before culture.
Stroma-free long-term liquid cultures
Freshly purified PB Inc+ CD34+
(3 × 104 cells/mL) or sorted PB
Inc+ CD34highCD38low cells
(1 × 103 cells/mL) were cultured in
quadruplicates in flat-bottomed 24- or 96-well plates for 5 weeks, in 1 mL or 200 µL, respectively, of a serum-free
liquid culture medium (Stem A) containing a combination of the
following cytokines: SCF (50 ng/mL), FLT3-ligand (50 ng/mL; R&D Systems), TPO (10 U/mL; PeproTech,
Tebu), added twice a week,23 with or without SDF-1 (0.5 ng/mL). At the onset of the culture, the total number of
BFU-E, CFU-G, CFU-M, CFU-GM, and CFU-Mix was determined by a
methylcellulose assay. Every week, the wells were semidepopulated by
the removal of one half of the culture volume, which was replaced with
fresh medium and cytokines. Every 1 or 2 weeks, harvested cells were
counted, and suitable aliquots were assayed for BFU-E, CFU-G, CFU-M,
CFU-GM, and CFU-Mix and for CD34+ cell numeration. Only
viable cells incubated with propidium iodide were analyzed by flow cytometry.
Cell cycle analysis
Cell cycle studies were performed by means of DNA-propidium iodide
(PI) binding and analysis with a FACScalibur flow cytometer. CD34+ cells (1 × 105
cells/mL) were incubated for 72 hours in cytokine-free
liquid medium (Stem A) with or without SDF-1 (0.05 ng/mL). At each time point (0, 24, 48, and 72 hours),
cells were harvested by centrifugation, counted, and prepared for cell
cycle analysis. Cells (5 × 104) were suspended in
200 µL of PI diluted 1:50 with permeabilizing solution24 and were incubated in the dark at 4°C for 24 hours before analysis. The cell histogram FL-2 was divided into 3 regions according to cell cycle phase: G0/G1,
S, or G2/M. Doublets were eliminated by gating on a
peak/area plot of PI fluorescence. The data were analyzed with WinCycle
software (Phoenix Flow Systems, San Diego).
Tyrosine phosphorylation analysis
BM and PB Inc+ CD34+ cells
(5 × 104 cells/mL) were incubated for
20 hours in a cytokine-free liquid medium (Stem A) with or without
SDF-1 (0.05 ng/mL and 10 ng/mL). The cells were
collected by centrifugation and were further stimulated with 10 ng/mL SCF or IL-3 for 5 to 15 minutes at 37°C. The
time course of total tyrosine phosphorylation was detected by flow
cytometry after intracellular anti-phosphotyrosine
labeling (as described above).
Statistical analysis
Data were expressed as means ± standard deviation (SD). The
significance of differences between groups was determined by Student t test for paired samples. A P value of < .05 was
considered statistically significant.
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Results |
Distinct expression of CXCR-4 on normal adult BM and PB
CD34+ cells
We analyzed CXCR-4 expression on human BM and PB CD34+
cells using 3-color cytometry. All samples were analyzed identically. We produced 4 dot plots (Figure 1): the
lymphomonocytic cell population was gated (R1) on an FSC versus SSC dot
plot excluding dead cells (plot 1); a second dot plot (plot 2),
displayed on R1, showed the CD34-PerCP versus CD38-FITC profile. An R2
region was further set around the CD34+ population,
excluding CD34neg/low cells. Dot plots 3 and 4, displayed
on additive regions R1 plus R2, showed CD34-PerCP versus CXCR-4-APC
(plot 3) and CD38-FITC versus CXCR-4-APC (plot 4).
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| Fig 1.
CXCR-4 expression is heterogeneous on PB and BM
CD34+ cells and is up-regulated on PB Inc+
CD34+ cells.
BM and PB CD34+ cells were purified immediately after
density gradient separation (Inc , plots a and c) or after
incubation on a plastic support (Inc+, plots b and d).
Cells were stained with CD34-PerCP, CD38-FITC, and CXCR-4-biotin-APC
antibodies and analyzed by 3-color cytometry. An R1 region was first
drawn by selecting the lymphomononuclear cells and excluding dead cells
on an FSC/SSC dot plot (plot 1). A second region R2
corresponding to CD34+ cells was drawn on a second
CD34-PerCP/CD38-FITC dot plot gated on R1 (plot 2). Expression of
CXCR-4-APC versus CD34-PerCP (plots 3) and of CXCR-4-APC versus
CD38-FITC (plot 4) was then assessed on the additive R1 plus R2 gates.
The arbitrary quadrants were drawn on the basis of isotype-matched
negative control profiles (top dot plot). The results shown are for 1 experiment representative of the 5 to 8 performed.
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PB CD34+ cells expressed CXCR-4, but at a lower level than
BM CD34+ cells. If PB CD34+ cells were directly
purified after gradient separation (Inc), fewer of
them (8.8% ± 3.6%) coexpressed CXCR-4 than was the case for BM
Inc CD34+ cells (56% ± 12.3%; P < .01) (Table 1;
Figure 1, plots 3c, 3a). The MFI was also lower in PB than in BM
Inc CD34+ cells (50.2 ± 29.3 AU and
91.2 ± 47.4 AU, respectively; P < .05) (Table 1).
We further characterized the CD34+CXCR-4+
subpopulation by analyzing the coexpression of the CD38 antigen, which
is rare or absent on early hematopoietic progenitors. We defined the
CD34+CD38low subpopulation as the 8% to 10%
of CD34+ cells with the lowest level of CD38; the quadrants
on plots 2 and 4 of Figure 1 were drawn on the basis of this
definition. Regardless of the origin of PB and BM, a small percentage
of CD34+CXCR-4+ cells (6.3% ± 2.9% and
14.2% ± 8.3%, respectively) coexpressed low levels of CD38 (42.3 ± 13.7 AU and 48.2 ± 22.5 AU, respectively) (Figure 1, plots
4c, 4a; Table 1).
Up-regulation of CXCR-4 expression on CD34+
cells purified after incubation on a plastic surface
The overnight incubation in a plastic flask of PB CD34+
cells before purification (Inc+) resulted in a much larger
percentage of CXCR-4+ cells and a much higher level of
CXCR-4 expression (73.6% ± 12% and 460 ± 150 AU) than was
recorded for Inc cells (8.8% ± 3.6%,
P = .0006, and 50.2 ± 29.3 AU, P = .001; n = 8) (Table 1; Figure 1, plots 3d, 3c). In contrast, the percentage of CXCR-4+ cells from BM Inc+ CD34+
cells was not significantly different from that for
Inc CD34+ cells (78.4% ± 17.7% and
56% ± 12.3%) (Table 1), but the level of CXCR-4
expression was significantly higher in Inc+ cells (338 ± 110 AU and 91.2 ± 47.4 AU, P = .003, n = 5)
(Table 1; Figure 1, plots 3b, 3a).
Surface overexpression of CXCR-4 on PB Inc+
CD34+ cells is correlated with a decrease in the
CXCR-4 intracellular pool and protein synthesis
We explored the mechanism of CXCR-4 up-regulation at the PB
Inc+ CD34+ cell surface and found that PB
CD34+ cells had an intracellular CXCR-4 pool (Figure
2). Purification of CD34+ cells
after overnight incubation (Figure 2B) resulted in higher surface
CXCR-4 levels (27.2 ± 9.1AU) and correspondingly lower levels of
intracellular CXCR-4 expression (187 ± 8.4 AU) than were recorded
for PB Inc (4.8 ± 5.4 AU, P = .01,
n = 3, and 310 ± 10.4 AU, P < .05, n = 3,
respectively) (Figure 2A). We investigated the contribution of protein
synthesis to CXCR-4 up-regulation during incubation by treating
mononuclear cells with cycloheximide. This resulted in a significant
inhibition of the increase in extracellular CXCR-4 (6.2 ± 3.6 AU,
P < .01, n = 3) (Figure 2C).
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| Fig 2.
CXCR-4 surface overexpression on PB Inc+
CD34+ cells is correlated with a decrease in the CXCR-4
intracellular pool and protein synthesis.
PB CD34+ cells were purified (A) without
(Inc) or (B) with (Inc+) overnight
incubation on a plastic support. (C) PB mononuclear cells were treated
with cycloheximide before overnight incubation and CD34+
purification. We studied CXCR-4 expression at the surface by labeling
cells with PE-conjugated anti-CXCR-4 mAbs. CXCR-4 expression within the
cell was analyzed after saturation with unconjugated CXCR-4 mAbs,
permeabilization, and labeling using PE-conjugated anti-CXCR-4 mAbs.
Cytometry analysis was performed on the FSC/SSC gated population as
indicated in Figure 1. The histogram represents 8000 to 10 000 events
in the total ungated population. The isotyped matched negative controls
are shown in the overlay (light lines). Data from at least 3 donors
were analyzed, with similar results. Histograms from a typical donor
are presented. The mean percentage and MFI of positive cells for CXCR-4 ± SD are shown for each histogram.
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PB Inc+ CD34+CXCR-4high
cells coexpress low levels of Thy-1, CD38, c-Kit, and CD71 antigens
The increase in CXCR-4 expression on PB Inc+
CD34+ cells especially affected the
CD34+/CD38low subset (Figure 1, plot 4 d)
(Table 1). CXCR-4 levels were also much higher on the PB
Inc+ CD34+/CD38low cell subset than
on PB Inc cells (605 ± 110 AU and 42.3 ± 13.7 AU, P = .0005, respectively, n = 8) (Table 1). In PB Inc+
CD34+ cells, the highest levels of CXCR-4 were detected on
the CD38low subpopulation (Figure 1, top left quadrant of
plot 4d). In contrast, in BM Inc+ CD34+ cells,
the highest levels of CXCR-4 were observed on CD38high
cells (Figure 1, top right quadrant of plot 4b).
We further analyzed the antigenic profile of the
CXCR-4highCD38low subset of PB Inc+
CD34+ cells. We carried out phenotypic characterization by
studying the coexpression of antigens such as Thy-1,25,26
CD71 (transferrin receptor)27 and CD117
(c-Kit),28 low levels of which indicate immaturity. The
CD34+CXCR-4highCD38low population
defined on the R3 gate (Figure 3) also
expressed high levels of CD34 (plot 2), low levels of CD71 and c-Kit
(plots 5 and 6, respectively), and various levels of Thy-1 (plot 4). If a reciprocal gate was drawn on the
CD34highCXCR-4highThy-1+ subset,
this population had a similar phenotypic profile based on CD38, CD71
and c-Kit coexpression (data not shown). Thus, we identified a
CXCR-4highCD34high subset in PB
Inc+ CD34+ cells exhibiting antigenic
characteristics of primitive hematopoietic progenitors, ie, low levels
of CD38, c-Kit, CD71 and heterogeneous expression of
Thy-1.29
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| Fig 3.
PB Inc+
CD34+CXCR-4high cells have a primitive
progenitor phenotype.
PB CD34+ cells were purified after incubation on a plastic
support (Inc+), stained for CD34-PerCP, CD38-FITC, CD71-PE,
c-Kit-PE, or Thy-1-PE and CXCR-4-biotin-APC and analyzed by 4-color
cytometry. A first dot plot (FSC versus SSC) was produced to select the
lymphomonocytic cell population in a first gate (R1). A second dot plot
(CD34-PerCP versus SSC) was displayed on the R1 gate. The
CD34+ population was then selected by a second R2 gate. Dot
plots 3 to 6, displayed on the additive regions R1 plus R2, show
CXCR-4-APC versus CD38-FITC, Thy-1-PE, CD71-PE, and c-Kit-PE
expression. On plot 3, a blue gate (R3) was drawn around the cell
population coexpressing a high level of CXCR-4 and a low level of CD38.
Cells corresponding to the R3 gate
(CXCR-4highCD38low subset) are shown in blue on
each dot plot. The results shown are from 1 experiment out of the 8 performed.
|
|
SDF-1 increases PB CD34+ colony formation in
presence of cytokines
The evidence, in PB, for a primitive CD34+ cell subset
expressing high levels of CXCR-4 led us to assess the effects of SDF-1 on hematopoietic progenitor proliferation. We therefore evaluated the
effect of SDF-1 on colony formation by CD34+ progenitors
purified from normal adult peripheral blood. In all experiments,
results are expressed as differences in the percentage of colonies
relative to that for the controls (CT).
In combination with cytokines, SDF-1 (0.5 ng/mL) gave,
respectively, 42% ± 25% and 60% ± 26% more BFU-E and CFU-G
colony formation by Inc CD34+ progenitor
cells than was recorded for untreated control cells (P < .05, n = 5) (Figure 4).
This stimulatory effect of SDF-1 was dose-dependent. The sizes of BFU-E
and CFU-G were not affected by the addition of SDF-1. No significant
effect was observed on CFU-M, CFU-GM, and CFU-Mix colony formation
(Figure 4). SDF-1 alone had no effect on the growth of PB
Inc CD34+-derived colonies (data not
shown).
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| Fig 4.
SDF-1 increases BFU-E and CFU-G colony formation by PB
Inc CD34+ progenitor cells.
PB CD34+ cells were purified without incubation on a
plastic support (Inc) and plated in duplicate at a
density of 500 cells/mL on semisolid Stem ID medium
containing IL-3, IL-6, IL-11, SCF, G-CSF, GM-CSF, Epo, and various
concentrations of SDF-1. Colonies were scored on day 14, and results
are expressed as a percentage of SDF-1 untreated control (CT) cells
(mean ± SD). Control colony numbers were 53.7 ± 7 (BFU-E); 15.5 ± 4.2 (CFU-G); 5.4 ± 1.6 (CFU-M); 0.75 ± 1.3 (CFU-GM); and
1.5 ± 0.4 (CFU-Mix). The control plating efficiency (calculated on
the total number of colonies) was 16.1% ± 3.6% (n = 5
independent experiments).* indicates significant difference from
control values (P < .05);  , SDF-1 (0.01 ng/mL;  , SDF-1 (0.05 ng/mL;  , SDF-1 (0.1 ng/mL);  , SDF-1 (0.5 ng/mL).
|
|
PB Inc+ CD34+ cells (Figure
5) treated with SDF-1, in combination with
the cocktail of cytokines, produced significantly more colonies than
did PB Inc CD34+ cells (154.4% ± 5.4% and 122.3% ± 14%, respectively, for SDF-1 0.05 ng/mL, P < .05, n = 5). This
increase affected mainly the number of CFU-M (301% ± 124% versus 38.2% ± 26.6%, P = .0003, n = 5), CFU-G (110.6% ± 43.8% versus 31.5% ± 17.8%, P = .02, n = 5), and large immature
BFU-E (440.4% ± 61.4% versus 15.5% ± 2.3%,
P = .000 01, n = 5) (Figure 5A, 5B for
Inc+ CD34+ and Figure 4 for
Inc CD34+). Unlike for BFU-E, the sizes
of the other types of colonies were not affected. The stimulatory
effect of SDF-1 on colony formation by Inc+
CD34+ cells, unlike that in Inc
CD34+ cells, was maximal at 0.05 ng/mL,
resulting in a bell-shaped curve (Figure 5A).
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| Fig 5.
SDF-1 increases CFU-G, CFU-M, and immature BFU-E colony
formation by PB Inc+ CD34+ progenitor
cells.
PB CD34+ progenitor cells were purified after incubation on
a plastic support (Inc+) and plated in duplicate, at a
density of 500 cells/mL, on semisolid Stem ID medium
containing IL-3, IL-6, IL-11, SCF, Epo, G-CSF, GM-CSF, and various
concentrations of SDF-1. Colonies were scored on day 14. (A) Results
for total BFU-E, CFU-G, CFU-M, CFU-GM, and CFU-Mix are expressed as a
percentage of SDF-1 untreated control (CT) cells (mean ± SD).
Control colony numbers were 91.7 ± 5.9 (BFU-E); 16 ± 3.1 (CFU-G); 2.8 ± 1.3 (CFU-M); 0.4 ± 0.2 (CFU-GM); and 1.75 ± 1.3 (CFU-Mix). The control plating efficiency (calculated on the total
number of colonies) was 23.1% ± 2.1% (n = 5 independent
experiments). (B) Results concerning mature and immature BFU-E are
expressed as a percentage of untreated control (CT) cells (mean ± SD). BFU-E maturity was determined on the basis of clone size: large
bursts containing 16 or more clusters with low levels of hemoglobin
were classed as immature BFU-E, whereas small bursts with a higher
hemoglobin content, containing fewer than 16 clusters, were defined as
mature BFU-E. Control colony numbers for mature BFU-E and immature
BFU-E were 77.7 ± 9.2 and 14 ± 3.5, respectively. An asterisk
indicates significant difference from control values: *
.001 < P < 0.05; ** .0001 < P < .001; ***
P < .0001.
|
|
We further assessed the effects of SDF-1 on PB megakaryocytic
progenitors. As for the other myeloid progenitors, SDF-1 alone had no
effect on MK progenitor-derived colony growth (data not shown).
However, in combination with cytokines, SDF-1 (0.1 ng/mL) significantly increased total MK colony formation by PB
Inc (99.8% ± 36.7% more than SDF-1 untreated
control cells, P = .003, n = 3) and PB Inc+
CD34+ cells (92.1% ± 12% more than untreated control
cells, P = .006, n = 3) (Figure
6A and 6B). The stimulatory effect of SDF-1
on MK progenitors was dose-dependent and maximal at 0.1 ng/mL. SDF-1 (0.1 ng/mL) treatment of PB
Inc+ CD34+ cells gave 130% ± 23.6% more
BFU-MK than untreated control cells (P = .006, n = 3)
(Figure 6B).
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| Fig 6.
SDF-1 increases the number of megakaryocytic
progenitor-derived colonies from PB Inc and
Inc+ CD34+ cells.
PB CD34+ cells were purified (A) without incubation on a
plastic support (Inc) or (B) after overnight
incubation (Inc+) and plated in duplicate at a density of
1 × 104 cells/mL in a serum-free
Easymega medium containing IL3, IL6, and TPO in the presence or absence
of SDF-1 (0.05 ng/mL) or anti-SDF-1 antibody (5 ng/mL) or isotype control (5 ng/mL). Colonies
were scored after 10 to 14 days of culture. Two differentiation stages
were distinguished on the basis of clone size on day 14. Colonies
containing more than 10 cells were classed as BFU-MK, whereas colonies
containing fewer than 10 cells were classed as CFU-MK. Results are
expressed as mean percentage of SDF-1 untreated control cells (CT) ± SD. Control colony numbers for CFU-MK and BFU-MK from PB
Inc cells were 118.7 ± 43 and 35.6 ± 12.1, and for PB Inc+ cells, 149 ± 27.7; 46.4 ± 15.3, respectively. Control plating efficiency calculated on the total number
of MK colonies derived from PB Inc and PB
Inc+ CD34+ cells was 1.5% ± 0.6% and
2% ± 0.75%, respectively (n = 3 independent
experiments). * indicates significant difference from control values,
P < .05.
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|
The stimulatory effect of SDF-1 on erythroid, granulo-macrophagic, and
megakaryocytic colonies derived from PB CD34+ cells was
totally abolished by treatment with neutralizing anti-SDF-1 antibody,
demonstrating the specificity of SDF-1 biological activity (Figures 6
and 7). Addition of either an anti-SDF-1
antibody alone or its isotype IgG control had no significant effect on
colony formation.
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| Fig 7.
Anti-SDF-1 treatment inhibits the stimulatory effect of
SDF-1 on colony formation by PB Inc+ CD34+
progenitor cells.
PB Inc+ CD34+ cells, purified after incubation
on a plastic support, were plated in duplicate at a density of 500 cells/mL on semisolid Stem ID medium containing IL3,
IL6, IL11, SCF, G-CSF, GM-CSF, and Epo, in the presence or absence of
SDF-1 (0.05 ng/mL) or anti-SDF-1 antibody (5 ng/mL), or isotype control (5 ng/mL).
Colonies were scored on day 14. Results are expressed as mean
percentages of SDF-1 untreated control cells (CT) ± SD. Control
colony numbers were 90.2 ± 8.6 (BFU-E); 12.2 ± 5.4 (CFU-G); 3.5 ± 1.8 (CFU-M); 0.8 ± 0.3 (CFU-GM); and 1.2 ± 1.2 (CFU-Mix).
The control plating efficiency (calculated on the total number of
colonies) was 19.3% ± 5.4% (n = 3 independent experiments). *
indicates significant difference from control values: 0.001 < P < .05.
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|
We further evaluated the effects of SDF-1 on colony formation from BM
CD34+ cells. Addition of various concentrations of SDF-1
(from 0.05 up to 10 ng/mL) did not increase either the
number (Table 2) or the size of any type of
colony from Inc+ and Inc
CD34+ cells.
SDF-1 acts in synergy with SCF in promoting colony formation and
tyrosine phosphorylation in PB CD34+ cells
We investigated the capacity of SDF-1 to promote PB
CD34+ colony formation in synergy with various cytokines
individually or in combination. SDF-1 alone had no effect on PB
Inc or Inc+ colony formation (data not
shown). The addition of SCF significantly increased the number of
CFU-G, CFU-M, and CFU-GM colonies derived from Inc+
CD34+ cells (Figure 8A). This
effect is strengthened if IL-3 and GM-CSF are added to SCF. In
contrast, addition of IL-3 or GM-CSF alone did not increase the
promoting effect of SDF-1 on CFU-G, CFU-M, and CFU-GM colony formation.
In these experimental conditions, the promoting effect of SDF-1 on
BFU-E was not observed (Figure 8A), suggesting that the cytokine
combinations used were ineffective in immature BFU-E formation.
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| Fig 8.
SDF-1 increases CFU-G, CFU-M, CFU-GM, and CFU-Mix colony
formation by PB Inc+ CD34+ progenitor cells
in synergy with SCF.
PB CD34+ progenitor cells purified after overnight
incubation on a plastic support (Inc+) were incubated for
48 hours in serum- and cytokine-free liquid culture medium (Stem A)
(A) without or (B) with anti-TGF- antibody (5 µg/mL). Cells were harvested, counted, and plated in
duplicate, at a density of 500 cells/mL, on semisolid Stem
medium containing Epo (control medium) and various combinations or
individual cytokines (GM-CSF, IL-3, SCF, IL-3 + SCF, IL-3 + SCF + GM-CSF); SDF-1 (0.05 ng/mL) was added or not to the
culture medium. Results of 2 independent experiments are expressed as
the number of colonies scored on day 14. * indicates significant
difference from control values: P < .05.
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|
We further analyzed the effects of SDF-1-pretreatment on global
tyrosine phosphorylation of intracytoplasmic proteins in response to
various cytokines. Pretreatment with SDF-1 markedly increased the
SCF-induced tyrosine phosphorylation in PB Inc+
CD34+ cells (Figure 9). This
phosphorylation was time-dependent and was not detected after
incubation with IL-3. In contrast, in BM Inc+
CD34+ cells, the SCF signaling was not altered by
SDF-1-pretreament even if SDF-1 was used at 10 ng/mL
(data not shown).
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| Fig 9.
SDF-1 increases SCF-induced tyrosine phosphorylation in
PB Inc+ CD34+ cells.
PB and BM CD34+ cells purified after incubation on a
plastic support (Inc+) were pretreated without (control) or
with SDF-1 (0.05 ng/mL) for 20 hours in serum- and
cytokine-free medium (Stem A) at a density of 5 × 104
cells/mL. Cells were harvested, washed by
centrifugation, and stimulated, with SCF (10 ng/mL) or
IL-3 (10 ng/mL), for 5 to 15 minutes at 37°C. The time
course of total tyrosine phosphorylation was detected by flow cytometry
after intracellular anti-phosphotyrosine labeling. Histograms from a
typical donor are presented. The MFI (AU) of positive cells for
phosphotyrosine are shown for each histogram.
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SDF-1 stimulates PB CD34highCD38low cell
expansion and increases colony formation by primitive hematopoietic
progenitors released from quiescence by anti-TGF- antibody
treatment
In an effort to investigate the possible effects of SDF-1 on
primitive hematopoietic progenitors contained in PB Inc+
CD34+ cells, we set up a stroma-free liquid culture
containing early acting cytokines according to the in vitro culture
system described by Piacibello et al.23 In synergy with
TPO, SCF, and FLT3-ligand, SDF-1 increased the expansion of PB
CD34+ cells, CD34highCD38low cells,
and committed progenitors (CFC). After liquid culture for 5 weeks, the
total number of CD34highCD38low and
CD34+ cells generated in the presence of SDF-1, was,
respectively, 1.5 times (412.5 versus 628 cells) and 2.4 times (486 versus 1161 cells) higher than for the untreated cells (Table
3). The output of total CFC (BFU-E, CFU-G,
CFU-M, and CFU-GM) generated by SDF-1-treated CD34highCD38low and CD34+ cells
after 4 to 5 weeks of culture was 2.5 times (1280 versus 3264 CFC) and
9.7 times (980 versus 9525 CFC) higher than for the SDF-1-untreated
control cells (Table 3). In preliminary experiments, with the use of a
cocktail of cytokines at low concentrations (0.1 to 1 ng/mL) in addition to TPO and FLT3-ligand, the output of
total CFC generated by SDF-1-treated
CD34highCD38low after 4 weeks of culture was
greatly increased, since it was 10.5 times (912 versus 9570 CFC) higher
than for SDF-1-untreated cells. SDF-1 alone had no effect either on
CD34+ and CD34highCD38low cell
expansion or on CFC generation (Table 3).
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|
Table 3.
SDF-1 stimulates expansion of PB CD34+ and
CD34highCD38low cells in stroma-free long-term
liquid culture in synergy with cytokines
|
|
We also assessed whether SDF-1 affected primitive progenitors released
from quiescence by anti-TGF- antibody treatment. PB or BM
Inc+ CD34+ cells were incubated with an
anti- TGF- antibody or its isotype IgY control for 48 hours in a
serum- and cytokine-free liquid culture medium, then plated
on methylcellulose.
Treatment of PB Inc+ CD34+ cells with an
anti-TGF- antibody in the presence of either SCF (alone or in
combination with IL-3 and GM-CSF) (Figure 8B) or a cocktail of
cytokines (Figure 10) potentiated the
effect of SDF-1 on CFU-GM and CFU-Mix colony formation. The number of
such colonies was 425% ± 175% (P = .002, n = 3, for
SDF-1 0.01 ng/mL) and 181.2% ± 18.7%
(P = .04, n = 3, for SDF-1 0.05 ng/mL) higher
than for cells not treated with the anti-TGF- antibody (Figure
10A). Anti-TGF- antibody treatment also resulted in a greater
increase (by 75% ± 19%) in the number of CFU-G
(P < .05, n = 3) and in the number of CFU-M (404% ± 79.1%, P = .001, n = 3) caused by SDF-1 (0.05 ng/mL). In the presence of an anti-TGF- antibody,
SDF-1 also increased the CFU-M and CFU-Mix colony sizes (Figure 10B and
10C). In these experimental conditions, SDF-1 activity was maximal for
concentrations from 0.05 ng/mL to 0.1 ng/mL, and followed a bell-shaped curve (Figure 10A).
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| Fig 10.
SDF-1 stimulates colony formation by PB
Inc+ CD34+ progenitor cells released from
quiescence by anti-TGF- antibody treatment.
PB Inc+ CD34+ cells, purified after overnight
incubation on a plastic support were incubated for 48 hours in
serum-free liquid culture medium without cytokine (Stem A)
containing or not containing anti-TGF- antibody (5 µg/mL) or its isotype IgY control (5 µg/mL). Cells were harvested, counted, and plated in
duplicate at a density of 500 cells/mL on methylcellulose medium
containing IL3, IL6, IL11, SCF, G-CSF, GM-CSF, and Epo (Stem ID) in
the presence or absence of SDF-1 at various concentrations. Colonies
were scored on day 18. (A) Data are expressed as mean percentages of
SDF-1 untreated control cells (CT) ± SD calculated by subtracting
the percentage of colonies obtained with control CD34+
cells not treated with anti-TGF- antibody from that for
anti-TGF--treated CD34+ cells. Control (not treated
with SDF-1) colony numbers were 25.4 ± 2.5 (CFU-G); 4.3 ± 1.2 (CFU-M); 1.2 ± 0.6 (CFU-GM); 6.6 ± 1.2 (CFU-Mix); 66 ± 1.6;
and 16.6 ± 2 (mature and immature BFU-E, respectively). The control
plating efficiency, calculated on the total number of colonies was
37.9% ± 3.3% (n = 3 independent experiments). Photomicrographs
of (B) CFU-M and (C) CFU-Mix from anti-TGF- antibody-treated
CD34+ cells incubated (B1 and C1) without or (B2 and C2)
with SDF-1, × 40. An asterisk indicates significant differences
from control values: * .001 < P < .05; ** .0001 < P < .001;
*** P < .0001.
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|
Treatment with anti-TGF- antibody had no effect on colony formation
from the BM Inc+ CD34+ cells in response to
SDF-1 (data not shown). In every experiment, the addition of the
isotype IgY control had no significant effect on CD34+
colony formation (data not shown).
SDF-1 increases the percentage of PB CD34+ cells in
the S-phase of the cell cycle
We investigated the stimulatory effects of SDF-1 on progenitor
proliferation by evaluating cell-cycle status and the number of PB
CD34+ cells cultured in a liquid medium with or without
SDF-1 and cytokines.
In the absence of cytokine, a 48-hour incubation with SDF-1 (0.05 ng/mL) significantly increased the percentage of PB
Inc+ CD34+ cells in the S+G2/M
phases (33.1% ± 1.6%) in comparison with untreated control cells
(12.1% ± 0.6%, P = .004, n = 3) and PB Inc CD34+ cells (8.6% ± 0.8%,
P < .0001, n = 3) (Table 4).
SDF-1 alone did not stimulate the proliferation of PB CD34+
cells, but did result in a smaller decrease in the PB Inc+
CD34+ cell number than for untreated cells (86.1 ± 0.8 × 103 and 78 ± 1.4 × 103, respectively, at 72 hours,
P < .05, n = 3) (Table 5). If
PB Inc+ CD34+ cells were primed before culture
with SDF-1 to stimulate cell-cycling, the effect of SDF-1 on the cell
number was even greater (88.8 ± 2.3 × 103 and
62.4 ± 2.9 × 103, respectively, at 72 hours,
P < .05, n = 3) (Table 5).
In the presence of cytokines, SDF-1 treatment significantly resulted in
more PB Inc+ CD34+ cells if the cells had been
previously primed with SDF-1 than if the cells had not been primed
(288.1 ± 14.7 × 103 and 142.9 ± 11.2 × 103, respectively, at 72 hours,
P = .002, n = 3) (Table 5) or untreated (200.8 ± 13.9 × 103, P < .05, n = 3) (Table
5). Priming with SDF-1 for 24 hours itself significantly increased the
total number of PB Inc+ CD34+ cells, (200.8 ± 13.9 × 103 versus 136.9 ± 5.1 × 103, for unprimed control cells, at 72 hours,
P = .004, n = 3) (Table 5).
| |
Discussion |
We present here evidence of a role for the chemokine SDF-1 as an
enhancing factor for human circulating CD34+ progenitor
proliferation, in synergy with cytokines. Several studies have reported
CXCR-4 expression on CD34+ cells purified from
BM,10,30 mobilized peripheral blood,13,16,30 and cord blood,30,31 but this is the first
demonstration of CXCR-4 expression on CD34+ cells purified
from adult unmobilized peripheral blood. The percentage of
CXCR-4+ cells and receptor levels were both lower for
CD34+ cells isolated from PB than for CD34+
cells from other sources.10,13,16 The presence of
CD34+ cells in the liquid phase (PB, cord blood, or
mobilized peripheral blood) rather than in organized tissue (BM), in
which CD34+ cells are intimately associated with
stroma,7,30,32 may account for such differences. The
involvement of the CXCR-4/SDF-1 couple in hematopoietic progenitor
migration and homing is now well documented.8 It has been
suggested that changes in the concentration gradient of
chemoattractants such as SDF-1 and SCF favor the homing of
CD34+ progenitors to BM or their peripheralization.
According to this model, SDF-1 may inhibit mobilization of BM
progenitors.7 The observed differences in CXCR-4 levels in
BM and PB CD34+ cells are consistent with this notion. The
low level of CXCR-4 expression on PB CD34+ cells is also
consistent with their circulating status.
The expression of CXCR-4 at the cell surface was significantly
increased by purifying progenitors after incubation on a plastic surface (Inc+). This up-regulation affected mostly PB
Inc+ CD34+ cells because CXCR-4 levels were
about 10 times higher on these cells. Various factors may be involved
in CXCR-4 up-regulation during incubation, including cell-surface
adhesion, cell-cell contact between CD34+ and low-density
mononuclear cells, growth factor production, and adhesion molecule
interactions. Cell-surface adhesion was recently suggested as being
involved in the CXCR-4 increase on BM progenitors and PB
granulocytes.33,34 The lower level of CXCR-4 expression for
PB CD34+ cells purified after incubation in an
adhesion-free flask than for PB Inc+ CD34+
cells (21.5 ± 2.2% and 73.6 ± 12%, respectively; experiment
not described in this paper) is consistent with this notion. The higher level of CXCR-4 expression for CD34+ cells initially
purified without adhesion and then incubated on a plastic surface (38.6 ± 8.7%) than for Inc
CD34+ cells (8.8 ± 3.6%) also supports this
hypothesis. However, in these experimental conditions, CXCR-4 levels
were lower than those of Inc+ CD34+, suggesting
that other events, such as interactions between progenitors and
mononuclear cells, are also involved in CXCR-4 up-regulation. Lapidot
et al35 recently reported that SCF and IL6 can regulate CXCR-4 expression. It is possible that these growth factors are released by mononuclear and/or CD34+ cells during
incubation.36 Surface CXCR-4 up-regulation may also involve
integrins.35 Our preliminary results showed no differences
in VLA-4, VLA-5, and LFA-1 levels in PB CD34+ cells,
purified with and without incubation (data not shown); however, we
cannot exclude the possibility that they were activated.
We further investigated the mechanisms of CXCR-4 overexpression on PB
Inc+ CD34+ cells. The correlation between the
increase in surface CXCR-4 and the decrease in the intracellular pool
is consistent with possible translocation of the receptor from the
cytoplasm to the surface membrane. Cycloheximide treatment indicated
that protein synthesis was involved in the up-regulation of surface
CXCR-4.
In PB Inc+ CD34+ cells, we identified for the
first time a CXCR-4highCD34high subpopulation
with the antigenic characteristics of primitive pluripotent
progenitors, ie, CD38low, CD71low,
c-Kitlow, and Thy-1+. In contrast, in BM, most
of the CXCR-4highCD34+ cells were also
CD38high. This CXCR-4highCD38high
population also coexpressed high levels of CD19 antigen (data not
shown), suggesting that these cells constitute a pre-B lymphocytic progenitor subset. The significance of the primitive
CD34highCXCR-4highCD38lowCD71lowc-KitlowThy-1+
subpopulation detected in PB after incubation is not clear. The higher
CD34 expression level of PB Inc+ CD34+ cells
than of Inc cells (44.6 ± 9.5 AU versus
63.4 ± 10.7 AU) (Figure 1, plots 2c, 2d) may facilitate
the recovery of a higher proportion of CD34high cells,
including this primitive subpopulation.
Some chemokines have different effects on hematopoiesis, depending on
progenitor maturity, but most have a more general effect on suppressing
progenitor cell proliferation.31,37 We found high levels of
CXCR-4 expression on a primitive subset of PB Inc+
CD34+ cells. This led us to assess the effect of SDF-1 on
hematopoietic progenitor proliferation and differentiation. Our results
show for the first time that SDF-1, a known chemotactic factor, also modulates hematopoietic progenitor cell proliferation. SDF-1 increased colony formation by PB CD34+. This effect was significantly
greater if CD34+ cells were purified after an incubation
step, and it applied to all types of myeloid progenitors. A combination
of cytokines is necessary to increase colony formation in the presence
of SDF-1, because SDF-1 alone has no activity.38 SCF, alone
or in combination with at least IL-3 and GM-CSF, is involved in this
synergistic promoting effect. An increase in the
SCF-induced tyrosine phosphorylation in response to SDF-1 is consistent
with this notion and suggested that SDF-1 enhances downstream signaling
of SCF.39
The stimulatory effect of SDF-1 on colony formation was dose-dependent
and was totally inhibited by the addition of a neutralizing antibody,
demonstrating its specificity. If PB CD34+ cells were
incubated on a plastic surface, the SDF-1 concentration effective in
colony formation was 1000 times lower than that effective in migration
tests.7 Such a difference in dose range is observed for
other chemokines acting on migration and
hematopoiesis.7,8,37,40 Hematopoietic progenitor
proliferation is regulated by short-range factors produced in tiny
quantities within the microenvironment.41 The bell-shaped
curve is a common feature of chemokine biological activity,42 and its sharpness for low SDF-1 concentrations
is consistent with a subtle and accurate hematopoiesis regulation.
Despite high levels of CXCR-4 expression on BM CD34+ cells,
SDF-1 (from 0.05 up to 10 ng/mL) had no significant
effects on either the number or the size of colonies from BM
CD34+ cells even if they were treated by anti-TGF-
antibody. The inability of SDF-1 to increase the cytokine-induced
tyrosine phosphorylation in BM CD34+ cells may elucidate
why these cells do not respond to SDF-1. These data are also consistent
with the results of Gotoh et al32 and Majka et
al43 and with the possible lack of correlation between
CXCR-4 expression and the functional activity of SDF-1.44 It is possible that CXCR-4 desensitization occurs in BM progenitors in
an SDF-1-enriched microenvironment; such a regulation process is
typical of G-protein-coupled receptors.45 Also, we could not exclude the possibility that such an SDF-1-responsive progenitor subset is present in the BM, but at too low a proportion to be detected
by our clonogenic assay.
Our results demonstrating a stimulatory effect of SDF-1 on PB colony
formation by erythroid, granulocytic, macrophagic, and megakaryocytic
progenitors, suggest that SDF-1 acts on pluripotent progenitors. Such
cells, thought to be mainly dormant, are released from quiescence by a
neutralizing anti-TGF- antibody, according to the "high
proliferative potential-quiescent cells" model.46 We
assessed the effect of SDF-1 on CD34+ cells treated by
anti-TGF- antibody and showed that SDF-1 increased the number of
CFU-Mix colonies by up to 180%. SDF-1 also dramatically increased the
number of CFU-GM and CFU-M over that in untreated cells (425% ± 175% and 404% ± 79.1%, respectively). This positive effect on both the number and the size of colonies, associated with the
evidence for a
CD34highCXCR-4highCD38lowCD71lowc-KitlowThy-1+
subpopulation, strongly suggests that SDF-1 acts on primitive hematopoietic progenitors. Such a hypothesis is strengthened by our
results showing a stimulatory effect of SDF-1 on PB CD34+
and CD34highCD38low cell expansion and myeloid
progenitors amplification, in stroma-free liquid culture containing
SCF, FLT3-ligand, and TPO.
We further evaluated the effect of SDF-1 on the cycle status of PB
Inc+ CD34+ cells in serum- and cytokine-free
suspension culture. Our results, showing that 98.6% ± 0.8% of PB
CD34+ cells were in the G0/G1
phases, confirm that human steady-state PB progenitors are cell-cycle
dormant.47,48 After culture for 48 hours, the percentage of
cells in S+G2/M was higher in PB Inc+
CD34+ cells than in Inc cells,
suggesting a relationship between CD34+ cell-cycle status
and adhesiveness, consistent with the data of Yamaguchi et
al.49 The addition of SDF-1 alone significantly increased
the proportion of PB Inc+ CD34+ cells in the
S+G2/M phases but had no effect on Inc
cells. The effect on Inc+ cells was not detected before 48 hours of culture, suggesting that most PB Inc+
CD34+ cells were in early G0/G1
phase. Thus, SDF-1 may interfere with cell cycling by shortening the
time required to go through the G1 phase, as has been
suggested for other early-acting growth factors.50 Such a
decrease in the duration of G1 might increase the relative
proportion of responding cells in S-phase, as reported by Roberts et
al.47 SDF-1 alone increased the percentage of CD34+ cells in S+G2/M phases. It supported cell
survival but did not induce cell proliferation. This is consistent with
the role of SDF-1 in preventing cell death, as proposed by Gotoh et
al.32 Our SDF-1 priming studies indicate that SDF-1
potentiated the stimulatory effect of cytokines on PB Inc+
CD34+ cell proliferation. Thus, SDF-1 may act as a
sensitizing factor, rendering progenitors responsive to cytokines.
In conclusion, we report evidence for a primitive PB
CD34high cell subset that overexpresses CXCR-4 after
overnight incubation. The experimental procedures used, which involved
cell-cell interactions, growth factor production, and cytoadhesion,
might mimic the physiological processes occurring during progenitor
cell engraftment. The presence of such primitive progenitors is
consistent with heterogeneity in stem cell engraftment potential
depending on CXCR-4 expression, as reported by Peled et
al.51 Our data also provide evidence that SDF-1 functions
as a promoting factor for circulating progenitor proliferation, as
demonstrated by its stimulatory effect on cell cycling, colony
formation, and progenitor expansion, in synergy with cytokines. Thus,
our results provide new insight into the role of the CXCR-4/SDF-1
couple in hematopoiesis and progenitor cell engraftment, which involve
migration, adhesion, and proliferation. Furthermore, our demonstration
of adhesion-induced CXCR-4 overexpression associated with SDF-1
stimulatory activity may be of clinical relevance for improving
cellular therapy settings for stem cell transplantation.
| |
Acknowledgments |
We thank Pr Nedellec, Dr Dishino, and Dr Masse for
continuously supplying bone marrow samples. We are indebted to Dr M. C. Martyré and Dr D. Dormont for their critical reading of this article.
| |
Footnotes |
Supported by grants from the DRET (Direction des Recherches Etudes et
Techniques, Ministère de la Défense, No 957) and the ANRB
(Association Nouvelles Recherches Biomédicales).
Submitted April 9, 1999; accepted September 10, 1999.
Reprints: M. C. Le Bousse-Kerdilès, INSERM U268,
Hôpital Paul Brousse, 14, Avenue Paul Vaillant Couturier, 94800 Villejuif, France; e-mail: lebousse{at}infobiogen.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction