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Blood, 1 May 2001, Vol. 97, No. 9, pp. 2587-2595
HEMATOPOIESIS
Stromal cell-derived factor 1 increases
polyploidization of megakaryocytes generated by human hematopoietic
progenitor cells
Raffaella Guerriero,
Gianfranco Mattia,
Ugo Testa,
Cristiana Chelucci,
Giampiero Macioce,
Ida Casella,
Paola Samoggia,
Cesare Peschle, and
Hamisa Jane Hassan
From the Department of Clinical Biochemistry and the
Department of Hematology-Oncology, Istituto Superiore di Sanità,
Rome, Italy; and Thomas Jefferson University, Philadelphia, PA.
 |
Abstract |
The alpha chemokine receptor CXCR4 has been shown to be expressed
on human hematopoietic progenitor cells and during the
megakaryocytic differentiation pathway. Stromal cell-derived factor 1 (SDF-1) is the ligand for CXCR4. In this study, the role of SDF-1 in megakaryocytopoiesis was investigated. CD34+ progenitors
purified from peripheral blood were grown in serum-free liquid
suspension culture supplemented with thrombopoietin to obtain a
virtually pure megakaryocytic progeny. In this condition, the addition
of SDF-1 gives rise to megakaryocytes (MKs) showing an increased DNA
content and a rise of lobated nuclei, as compared with untreated cells:
at day 5, approximately 20% of the cells already showed the presence
of more than one nuclear lobe versus fewer than 5% in the control
cells; at day 12, approximately 85% of the cells were of large size
and markedly polyploid, whereas approximately 60% of the control cells
were polyploid, showed fewer lobes, and were a smaller size. This
effect was dose-dependent and did not affect the megakaryocytic
proliferation. Experiments with the mitogen-activated protein kinase
(MAPK) inhibitor PD98059 suggested a role for MAPK pathway on
SDF-1-induced endomitosis. Furthermore, SDF-1 induced a
significant increase in the number of proplatelet-bearing MKs and
promoted the migration of megakaryocytic cells. Treatment with SDF-1
caused reduction in CXCR4 abundance on the plasma membrane, seemingly
owing to receptor internalization. Furthermore, the presence of
SDF-1 did not affect the expression of megakaryocytic markers,
indicating that differentiation and polyploidization are independently
regulated events.
(Blood. 2001;97:2587-2595)
© 2001 by The American Society of Hematology.
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Introduction |
Chemokines are peptides of 70 to 100 amino acids
secreted by different cell types in response to injury and infection.
Chemokines activate and induce migration of leukocytes in a
cell-specific fashion to sites of inflammation.1 Stromal
cell-derived factor 1 (SDF-1) is a CXC chemokine defined on the basis
of a typical sequence of cysteine residues.2 Characterized
as a pre-B stimulatory factor at the beginning,3 SDF-1
is constitutively expressed by many tissues.4,5 Initially
cloned from bone marrow stroma,6 it has been identified as
a highly efficient chemotactic factor for T cells, monocytes, and
CD34+ human progenitors4,7 and, more recently,
as a potent chemotactic factor for mature megakaryocytes
(MKs).8 Furthermore, it has been shown that it inhibits
the colony formation of the myeloid progenitor cell line 32D, which
suggests also a myelosuppressor role for this
molecule.9
The signaling of SDF-1 is mediated by CXCR4, a G-protein-coupled
receptor.10,11 The intracellular signals induced in
myeloid precursor cells were monitored by increases in the level of
intracellular Ca++.9 Ligand binding induced
rapid internalization and down-modulation of the receptor on the cell
surface.12 Data on CEM cell line and leukocytes indicated
that, after removal of exogenous SDF-1, CXCR4 was again localized on
cell surface as a consequence of recycling from the internalized
pool.13,14 Internalization by SDF-1 does not involve
protein kinase C activation.12
Knockout of the SDF-1 gene15 in mice is lethal with severe
abnormalities in B-cell lymphopoiesis and bone marrow myelopoiesis; similar defects were observed in knockout of the CXCR4
gene.16,17 Particularly, the defect in myelopoiesis
also involved the megakaryocytic lineage, resulting in a markedly
reduced number of these cells in the bone marrow of
CXCR4 / animals.16 The CXCR4
expression on MKs at different stages of differentiation and maturation
was demonstrated by flow cytometry and reverse
transcriptase-polymerase chain reaction (RT-PCR).18-20 Wang et al18 found that the percentage of CXCR4 expression
in CD34+ and CD61+ cells was different from one
donor to another. The coexpression of CXCR4 along with CD34 or CD61
ranged from 13% to 93% and from 13% to 89%, respectively.
Rivière et al19 demonstrated a close correlation
between CD41a and CXCR4 expression on cell surface: the level of CXCR4
increased in parallel with the CD41a antigen during megakaryocytic
differentiation. We recently analyzed protein and messenger RNA (mRNA)
expression of CXCR4 on CD34+ and monocytic, megakaryocytic,
erythroid, granulocytic-derived progenies: in particular, in
megakaryocytic-lineage CXCR4 expression is sustained up to late stages
of maturation, with an increase in the percentage of positive cells
from 50% in undifferentiated hematopoietic progenitor cells (HPCs) to
90% in mature MKs.20 The expression of CXCR4 on platelets
suggests that SDF-1 might also modulate platelet
functions.8,18
The role of SDF-1 on human megakaryocytic cells at early or late stages
of differentiation has been investigated with controversial results.8,18,19 Wang et al18 showed that
megakaryocytic populations with large forward- and side-scatter
properties, which correspond to MKs of larger size and higher ploidy
levels, exhibited stronger staining for CXCR4 than MKs with relatively
smaller ploidy levels. Rivière et al19 showed a
preferential attraction of immature MKs by SDF-1, whereas Hamada et
al8 suggested a potent chemotactic effect on mature MKs.
In spite of these discrepancies, all these studies reported the CXCR4
expression on MK and suggested a role for SDF-1 on megakaryocytic function.
Recently Hodohara et al21 demonstrated that SDF-1
exerts a direct growth-promoting effect on MK progenitors (MK
colony-forming units [CFU-MK]) purified from murine bone marrow
cells, when combined with thrombopoietin (TPO).
CXCR4 receptor is known as one of the major CD4 coreceptors that allow
the T-tropic human immunodeficiency virus (HIV) strains entry by
cell-membrane fusion.22,10 We previously demonstrated T-tropic HIV infection of HPC-derived megakaryocytic
precursors23 and the involvement of CXCR4, showing that
SDF-1-treated cells were essentially resistant to HIV
infection.20
In the present report, we have analyzed the effect of SDF-1 on
CD34+ cells obtained from peripheral blood (PB) and induced
to megakaryocytic differentiation in serum-free liquid suspension
culture in the presence of saturating amounts (100 ng/mL) of TPO.
| |
Materials and methods |
Hematopoietic growth factors and culture media
Recombinant human interkeukin 3 (rhIL-3) (2 × 106
U/mg), granulomonocytic colony-stimulating factor (rhGM-CSF)
(1.7 × 105 U/mg), and IL-6 (rhIL-6)
(2 × 106 U/mg) were supplied by the Genetics Institute
(Cambridge, MA); erythropoietin (rhEPO) (5 × 104
U/L) by Amgen (Thousand Oaks, CA); flt3-ligand (rhFL)
(1.9 × 106 U/mg) and kit-ligand (rhKL)
(1 × 105 U/mg) by Immunex (Seattle, WA); granulocytic
colony-stimulating factor (rhG-CSF) (1 × 108 U/mg) and
monocytic colony-stimulating factor (rhM-CSF) (6 × 107
U/mg) from R&D Systems (Minneapolis, MN); and rhTPO
(1 × 106 U/mg) from Peprotech (Rocky Hill, NJ). A
synthetic preparation of SDF-111 was kindly provided by
Dr Ian Clark-Lewis, Biomedical Research Centre, University of British
Columbia, Vancouver, BC, Canada; a recombinant preparation of human
SDF-1 was purchased from R&D Systems; and Iscove modified Dulbecco
medium (Gibco, Grand Island, NY) was prepared weekly before
each purification experiment.
HPC purification
Adult PB was obtained from 20- to 40-year-old healthy male
donors after informed consent. HPCs were purified as described in
Gabbianelli et al24 and Labbaye et al.25 In
particular, a negative selection of low-density cells was performed
with a cocktail of anti-T, anti-B, and anti-natural killer cell
lymphocytes and antimonocyte, antigranulocyte monoclonal antibodies
(mAbs) supplemented with anti-CD45, anti-CD11a, and anti-CD71 mAbs
(Becton Dickinson, Mountain View, CA).
HPC clonogenetic assay was performed as previously
described.26 Briefly, HPCs were seeded
(1 × 102 cells per milliliter dish, in triplicate) and
cultured in 0.9% methylcellulose in the presence or absence of fetal
calf serum (FCS). In FCS culture, FCS was substituted by
bovine serum albumin (BSA), pure human transferrin, human low-density
lipoproteins, insulin, sodium pyruvate, L-glutamine (2 µM),
rare inorganic elements supplemented with iron sulfate
(4 × 108 mM), and nucleosides. Both FCS+
and FCS cultures were supplemented with FL (100 ng/mL),
KL (100 ng), IL-3 (100 U), TPO (100 ng), GM-CSF (10 ng), EPO (3 U),
M-CSF (250 U), and G-CSF (500 U).
Granulocytic/erythroid/monocytic/megakaryocytic colony-forming unit,
erythroid burst-forming unit, and granulocytic/macrophage colony-forming unit colonies were scored on days 14 to 15 and 16 to 17 in FCS+ and FCS cultures, respectively.
MK cultures
Liquid suspension culture.
Purified HPCs were grown in FCS liquid culture at
4 × 104 cells per milliliter,27 in the
presence of a saturating dose of TPO (100 ng/mL) alone or with SDF-1
(1 µg/mL). Cells were incubated in a fully humidified atmosphere of
5% CO2, 5% O2, 90% N2. The cells
were counted and the concentration was adjusted to
1 × 105 cells per milliliter twice a week.
In some experiments, HPCs were grown in the presence of PD98059
(Calbiochem, La Jolla, CA), a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated protein kinase (ERK) pathway.28 PD98059 was dissolved in
dimethylsulfoxide (DMSO), cell culture grade (Sigma, St Louis, MO), and
added to cells 20 minutes before stimulation by TPO or TPO + SDF-1 at final concentration of either 25 or 50 µM/L.28 In the mock culture, an equivalent amount of
DMSO was added.
Detection of proplatelet-forming MKs.
From day 9 onward, the cultures were examined daily for the emergence
of proplatelets. An MK bearing one or more cytoplasmic processes (whose
length was longer than the cell body diameter) was considered a
proplatelet-displaying MK.29 The percentage of MKs
displaying one or more cytoplasmic processes was determined by visual
examination of video prints of the culture wells.
Platelet analysis.
Platelets were isolated from culture supernatants by centrifugation at
room temperature for 10 minutes at 120g, washed with Tyrode-Hepes buffer (Sigma; 136.9 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose, 10 mM Hepes, pH 7.4), pelleted at room temperature for 20 minutes at 900g, and then counted in a Bürker
camera with a contrast phase microscopy. In parallel, platelets were
stained with an anti-CD61 mAb (Becton Dickinson) and analyzed by flow
cytometry with a setting optimized for platelet analysis. The
functionality of platelets released from cultured MKs was evaluated by
the study of P-selectin (CD62) expression following 1 U/mL thrombin
stimulation, according to Choi et al.29 CD62 expression
was assayed by flow cytometry by means of a phycoerythrin (PE)-labeled
anti-P-selectin mAb (Becton Dickinson).
MK characterization
Morphological analysis.
Cells collected at different days of culture were cytocentrifuged onto
glass slides, stained with May-Grünwald Giemsa (Sigma), and then
identified by morphology analysis.
Flow cytometric analysis.
The following mAbs directly conjugated with fluorescein isothiocyanate
(FITC) or PE were used to characterize the membrane phenotype of cell
samples: anti-CD34, anti-CD61, anti-CD62, anti-CD42b (Becton
Dickinson), and anti-CD41a (Serotec, Oxford, UK). PE-labeled anti-CXCR4
mAb (clone 12G5, PharMingen, San Diego, CA) was used for the study of
the chemokine receptor expression. Cells were washed twice in
phosphate-buffered saline (PBS) and then incubated for 45 minutes at
4C° in the presence of appropriate amounts of specific mAbs. After 3 washes with cold PBS containing 1 g/L BSA, cells were
resuspended in 0.2 mL PBS/2.5% formaldehyde (Sigma) and analyzed by
FACScan (Becton Dickinson) by means of the Lysis II
program for fluorescence intensity analysis.26
DNA staining.
MKs' ploidy was analyzed by flow cytometry after DNA staining with
propidium iodide (PI) (Sigma) according to the procedure described in
Dolzhanskiy et al.30 Cells were washed and resuspended in
medium containing 0.5% Tween-20 for 30 minutes to permeabilize the
cell membranes. Then, an equal volume of medium containing 0.5%
Tween-20 and 2% paraformaldehyde was added. After 5 minutes at 4°C,
the cells were pelleted, and freshly prepared PI was added. The
suspension was stored overnight in the dark at 4°C. The following day, 50 µg/mL of RNAse A was added for 30 minutes at room temperature in the dark, and the cells were analyzed by flow cytometry.
Chemotaxis.
We used 5-µm pore filter (Transwell 24-well cell clusters; Costar,
Cambridge, MA) for migration study.8,18 We loaded
1 × 105 MKs (200 µL in FCS medium) into
each Transwell filter pretreated with RPMI 1640 (Gibco) containing
0.3% human serum albumin (Sigma). Filters were then transferred to
another well containing SDF-1 at a concentration of 200 ng/mL in
serum-free medium. After 3 hours' incubation at 37°C in 5%
CO2, cell migration was calculated by counting the recovered cells from the lower chamber. The viability of the cells was
assessed by trypan blue dye exclusion. Cytospin preparations of the
migrated cells were stained with May-Grünwald Giemsa.
Immunolabeling for confocal microscopy.
Cells derived from liquid culture were washed in PBS and immobilized on
poly-D-lysine-coated glass coverslips, fixed in PBS/3.7% paraformaldehyde, quenched in 0.1 M glycine, and permeabilized by incubation with 0.05% saponin-PBS/0.2% BSA for 15 minutes.13 The cells were then incubated for 45 minutes at
room temperature with anti-CXCR4 mAb and labeled with a secondary
FITC-conjugated goat antimouse immunoglobulin G antibody (Dako,
Glostrup, Denmark). After 3 washes in PBS, some samples were
double-stained with a PE-conjugated anti-CD41a mAb. Cells were again
washed and mounted in 50% glycerol and examined by means of a TCS 4D
confocal microscope (Leica, Nussloch, Germany) interfaced with
argon/krypton lasers. Single fluorescence was analyzed with a 488-nm
laser line for FITC dye excitation; simultaneous double-fluorescence
acquisition was performed by means of 488- to 568-nm laser lines to
excite FITC-PE dyes.
RT-PCR analysis.
RT-PCR was performed on megakaryocytic cells as previously
described.31 Briefly, 1 to 3 × 104 cells
were lysed in 200 µL of 4 M guanidine isothiocyanate, and total RNA
was extracted by the CsCl gradient technique in the presence of 12 µg
rRNA of Escherichia coli as carrier. Samples were reverse
transcribed according to the manufacturer instructions (Boehringer,
Mannheim, Germany). RT-PCR products were normalized for Sp26;
amplification within the linear range was achieved by 20 PCR cycles:
denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 45 seconds. To evaluate the
expression of CXCR4 gene, aliquots of RT-RNA were amplified within the
linear range by 30 cycles: denaturation at 95°C for 30 seconds,
annealing at 56°C for 30 seconds, and extension at 72°C for 45 seconds. PCR products were then electrophoresed on agarose gel,
transferred onto nylon membranes, and hybridized with the specific
probes. The following primers and probes were used:
Sp26: forward 5'-GCCTCCAAGATGACAAAG-3'; reverse
5'-CCAGAGAATAGCCTGTCT-3'; and probe
5'-GAGCGTCTTCGATGCCTATGTGCTTCCCAA-3'.
CXCR4: forward 5'-CCTCTATGCTTTCCTTGG-3'; reverse 5'-CCTGAAGACTCAGAC
TCA-3'; and probe 5'-AGCAGAGGGTCCAGCCTCAAGATCCTCT-3'.
Statistical analysis.
The significance of differences in mean value was determined by means
of the Student t test.
| |
Results |
HPC characterization and MK differentiation and maturation
HPCs purified from PB as described in "Materials and methods"
were characterized by 93% ± 1% CD34+ cells (mean ± SEM from 8 separate experiments), as evaluated by flow cytometry and
91% ± 1% HPC frequency (mean ± SEM from 8 separate
experiments), as evaluated by clonogenetic assay.
Purified HPCs grown in liquid suspension culture in the presence of TPO
(100 ng/mL) undergo a gradual wave of differentiation and maturation
along the MK lineage (Figure 1, left
panel), giving rise to a virtually pure MK population (98% to 99% of
the cells were CD61+).23 The differentiation
stages were characterized during the whole culture by morphologic and
phenotypic analysis: at day 0, cells were essentially composed of small
undifferentiated blasts; at day 5, most cells were larger than at day 0 and had one nuclear lobe, representing MK precursors. At day 8, a
significant proportion of cells were 2N and 4N and showed a more dense
chromatin. At day 12, a high proportion of MKs (60%) showed lobulated
polyploid nuclei with a highly granular cytoplasm. Platelets were
produced at the end of the culture as evaluated with contrast phase
microscopy. However, a significant proportion (approximately 40%) of
MKs at this day still displayed only one nuclear lobe, suggesting that factors other than TPO are required for optimal polyploidization of MK
precursors.
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| Figure 1.
Morphology of megakaryocytic cells grown in presence of
TPO alone or combined with SDF-1.
Cell morphology of HPCs, derived from PB, cultured in serum-free liquid
suspension medium in the presence of TPO (100 ng/mL) or TPO + SDF-1 (1 µg/mL) (representative results). Cells at different days
of culture are presented (May-Grünwald staining; original
magnification × 400).
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Effect of SDF-1
At day 0 after purification, HPCs were treated with different
amounts of synthetic SDF-1: 0.1; 0.25; 0.5; 1 µg/mL in the presence of saturating amounts of TPO (100 ng/mL).
The morphological analysis and DNA content of the cells cultured in the
presence of both TPO and SDF-1 revealed a significant increase in
the number of nuclear lobes (Figure 1) and ploidy (Figure
2) compared with the control cells grown
in the presence of TPO alone (Figure 1, right panel). Cell
polyploidization started earlier in culture supplemented with SDF-1
(1 µg/mL) than in the controls: at day 5, 20% of the resulting cells
were already tetraploid (4N) or even more highly lobated, whereas in
the control cultures almost all the cells were mononuclear; in
SDF-1-treated MKs, an increased polyploidization was maintained
until the end of the culture, where, at day 12, more than 85% of the
cells were larger than untreated control cells and showed a significant
increase in the number of nuclear lobes per cell (Figure 1). This
increase was dose dependent: at 0.1 µg, we did not observe any
differences in respect to the untreated cells; at 0.25 and 0.5 µg, a
clear increase in the number of polynucleated cells was evident, with a
prevalence of 4N (Figure 3). Additional
experiments were performed with a preparation of rhSDF-1. Optimal
stimulation of MK ploidization was observed at a concentration of 100 ng/mL (data not shown). In cytospin preparations, the presence of
proplatelets was more consistent when SDF-1 was used (Table
1), and the number of platelets counted
at day 12 in the supernatant from SDF-1-supplemented cultures was
increased compared with the control (5 × 105 vs
18 × 105 platelets per 105 MKs). These
platelets were functional according to standard criteria, ie,
thrombin-induced increase of membrane CD62 expression (data not shown).
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| Figure 2.
DNA content of MKs.
Polyploidy was evaluated by flow cytometry analysis, at day 9 of
culture, on MKs grown either in the absence (A) or the presence (B) of
SDF-1 (1 µg/mL). MKs were stained with PI as described in
"Materials and methods." A representative experiment from 3 separate experiments is shown.
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| Figure 3.
MK polyploidization increase after treatment with
SDF-1.
Effect of SDF-1 dose-response treatment on the polyploidization of
the HPC-derived MK cells. Data relative to day 9 of culture are
presented as mean ± SEM of 3 independent experiments. Asterisks
denote a significant difference (P < .05) in comparison
with control.
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The effect of SDF-1 on megakaryocytic cultures was relevant when
SDF-1 was added in the HPC culture medium at day 0 and abolished if
it was added at day 5 of culture, when the cells were already
differentiated to MK precursors (data not shown).
When we used lower levels of TPO (1 to 10 ng/mL), we observed a
decrease in the total number of cells although the relative percentage
of MKs was not affected and remained around 99%: in these conditions,
the addition of synthetic SDF-1 (1 µg/mL) still induced an
increase in the ploidy of the cells (Table
2). However, cell growth was not
significantly affected by the presence of the chemokine, at any
concentration used, and no MK growth was observed when SDF-1 was
used alone. In Figure 4, the growth
curves of the cells during the whole culture (0 to 12 days) in the
presence or absence of 1 µg/mL SDF-1 are reported.
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Table 2.
Effect of graded amounts of thrombopoietin in the presence
or absence of stromal cell-derived factor 1 (1 µg/mL) on
megakaryocyte percentage, proliferation, and polyploidization
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| Figure 4.
The MK proliferation is not affected by the presence of
SDF-1.
Proliferation of HPCs along the megakaryocytic lineage in the presence
of TPO (100 ng/mL) alone ( ) or in combination with SDF-1 (1 µg/mL,  ). Data are expressed as the mean ± SEM of 9 separate
experiments. No significant differences were observed when statistical
analysis was performed by means of the Student t test for
paired data.
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When we characterized the cells for their membrane phenotype during the
differentiation and maturation process, we observed the same level of
expression for the specific megakaryocytic markers, ie, CD61 and CD42b
for cells grown in either the absence or the presence of SDF-1. In
both culture systems, direct immunofluorescence for the expression of
CD34, CD61, and CD42b antigens confirmed gradual decrease of CD34 and
an inverse increase of both CD61 and CD42b, which are expressed on more
than 98% and 90% of cells respectively at day 12 of the culture (data
not shown).
Effect of SDF-1 on the migration of differentiating MKs
We examined the capacity of differentiating MKs to migrate in
response to a positive concentration of SDF-1 through a 5-µm Transwell cell filter.
As shown in Figure 5A, these cells
migrated in response to SDF-1 in a dose-dependent manner. The
concentration of 200 ng/mL of SDF-1 in the lower chamber resulted in
significant migration of day-10-culture-derived MKs: on average, 18%
of MKs added to the upper chamber had the capacity to migrate.
Megakaryoblastic cells at day 4 of culture also showed a migration but
at a level of 10% (data not shown).
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| Figure 5.
SDF-1 migration experiments of HPC-derived MKs at day
10 of cultures.
(A) Migration of MKs at different doses of SDF-1. (B) Migration of
MKs under different stimuli: no addition; TPO (100 ng/mL) in the lower
well; SDF-1 (200 ng/mL) in the lower well; and SDF-1 in both the
upper and lower wells.
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Neutralization of SDF-1 gradient by adding the chemokine to both
upper and lower chambers completely abrogated the migration; replacement of SDF-1 with TPO also failed to induce cell migration (Figure 5B).
Analysis of CXCR4
The effects of SDF-1 are mediated by the membrane receptor
CXCR4. We evaluated the CXCR4 expression in quiescent HPCs and in
MK-differentiating cells in the presence or absence of SDF-1. Immunofluorescence studies with anti-CXCR4 mAb showed that about 50%
of HPCs were CXCR4+, and this percentage increased to 90%
at the end of the culture (day 12). In the presence of SDF-1, CXCR4
was rapidly and markedly down-modulated (day 2) and remained scarcely
expressed at membrane level up to day 5; at day 7 of culture, CXCR4
expression was partially recovered and then became only moderately
lower than that observed in control cultures (Figure
6). On the contrary, RT-PCR analysis performed on RNA obtained from the same cells showed similar CXCR4 mRNA
levels in both control and SDF-1-supplemented cultures (Figure 7).
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| Figure 6.
SDF-1 affects CXCR4 expression.
Cells were treated at day 0 after HPC purification with TPO (100 ng/mL)
(thin line) or with TPO + SDF-1 (1 µg/mL) (broad line) and
analyzed at intervals during the culture (days 2 through 12) by flow
cytometry with the use of PE-conjugated CXCR4 mAb as described in
"Materials and methods." The staining with negative control
antibody is shown, as solid profile, at day 0, and appears similar in
the following days. Representative results from 1 out of 5 independent
experiments are shown.
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| Figure 7.
CXCR4 mRNA expression during MK differentiation.
Representative results of CXCR4 mRNA expression analyzed by RT-PCR in
HPCs after purification (97% CD34+) and during MK
differentiation in the presence or absence of SDF-1. Total RNA from
CEM cell line and from UT-7 cell line was used as a positive
(C+) and negative (C) control, respectively.
RT-PCR samples were normalized for Sp26.
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Confocal laser microscopic analysis was performed to investigate
cellular distribution of CXCR4 in MKs grown in the absence or presence
of SDF-1 (Figure 8). Cells maintained
in the presence of TPO alone or of TPO combined with SDF-1 were
compared for spatial distribution of CXCR4 receptor, ie, internalized
after treatment with its specific ligand. CXCR4 receptor endocytosis was monitored on the control MKs and at different times after SDF-1
addition (ie, 30 minutes, 1 hour, 3 days, and 8 days). Untreated cells
showed a homogeneous expression of CXCR4 on their surfaces (Figure 8A).
Internalization of CXCR4 was evidenced as early as 30 minutes after
SDF-1 addition, as suggested by a decrease of membrane reactivity,
coupled with the appearance of internal endosomic spots (Figure 8B).
This phenomenon became more evident after 1 hour of SDF-1 treatment
(Figure 8C). At day 3, cells were double-stained with anti-CXCR4 and
anti-CD41 mAbs: most of the SDF-1-treated cells showed the presence
of intracytoplasmic CXCR4 (Figure 8D), whereas the membrane
megakaryocytic-specific marker CD41 was localized on cell surface
(Figure 8E); the 2 markers do not colocalize (Figure 8F). Control
double-stained cells displayed both the CXCR4 (Figure 8G) and the CD41a
(Figure 8H) exclusively on the cell membrane, with colocalization
pointed out by numerous yellow areas (Figure 8I).
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| Figure 8.
Internalization of CXCR4 after exposure to SDF-1.
Internalization of CXCR4 was analyzed by confocal laser scanning
microscopy in HPCs. (A) Untreated cells. (B) Cells after exposure to 1 µg/mL of SDF-1 for 30 minutes. (C) Cells after exposure to 1 µg/mL of SDF-1 for 1 hour. (D-I) Double immunofluorescence for
CXCR4 (green) and CD41 (red) on day 3 cells, cultured in the presence
of SDF-1 (D-F), compared with control cells (G-I). The yellow area in
panel I indicates the colocalization of both the receptors on the
membrane (bar = 10 µm).
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MAPK pathway contributes to the stimulatory effect of SDF-1 on
megakaryocytic endomitosis
To evaluate a possible functional role of MAPK activation in
mediating the effects of SDF-1 on MK endomitosis, purified HPCs stimulated with TPO alone or in combination with SDF-1 were cultured in either the absence or the presence of the specific MEK inhibitor PD98059. These experiments showed that the inhibitor significantly reduced the MK endomitosis elicited both by TPO alone and by TPO in
combination with SDF-1 (Figure 9), as
evaluated through morphological analysis of the number of nuclear
lobes. This result was also confirmed by flow cytometric analysis of
DNA content (data not shown). In these experiments, PD98059 was used at
a concentration of either 25 or 50 µM/L, both of which were reported
to inhibit phosphorylation of ERKs in cultured MKs.28 The
low amount of DMSO used in the culture had no adverse effects on MK
ploidy as compared with untreated culture.
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| Figure 9.
MK polyploidization decrease after treatment with
PD98059.
The effect of MAPK inhibitor PD98059 was evaluated on differentiating
MKs grown in the presence of TPO (100 ng/mL) alone or in combination
with SDF-1 (1 µg/mL). Percentage of polylobated MKs derived from
cultures grown in the presence of either 25 µM/L ( ) or 50 µM/L ( ) PD98059 was evaluated by morphological analysis.
An equal volume of diluent (DMSO) added to mock culture ( ) had no
adverse effects on polyploidization as compared with the untreated
culture. Data from day 9 culture are presented as mean values ± SEM of 3 independent experiments.
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Discussion |
In this study, we investigated the effect of the chemokine
SDF-1 on megakaryocytopoiesis. The expression of CXCR4 receptor on
HPCs, MKs, and circulating platelets18-20 indicates a
potential role for its ligand on these cells. We observed that the
polyploidization process was clearly enhanced when PB-purified
CD34+ cells were induced to differentiate into MK lineage
in serum-free liquid suspension culture in the presence of TPO and
SDF-1, as compared with TPO alone. The stimulatory effect of cell
maturation induced by SDF-1 was apparently specific for MKs: this is
shown by the absence of any effect of the cytokine on HPCs induced to differentiate along the granulocytic pathway, where the CXCR4 receptor
was expressed at high level during the whole differentiative and
maturative process (Chelucci et al20 and our unpublished results, December 2000).
SDF-1 accelerated the process of megakaryocytic polyploidization: at
day 5, a significant percentage of these cells already showed the
presence of more than one nuclear lobe, and at day 12, most of the
cells were of large size and markedly polyploid, with a consistent
percentage (20%) of cells more than 16N. At the end of the culture,
the SDF-1-supplemented MKs were huge, with large cytoplasm
displaying a granular appearance and lobulated polyploid nuclei; we
also observed a significant increase of proplatelet-bearing MKs.
Finally, the stimulatory effect of SDF-1 was more pronounced in MK
culture supplemented with very low doses of TPO.
Previous studies indicated that TPO supports full MK differentiation in
vitro, including proplatelets and platelet formation,27,32 but is unable to induce an optimal polyploidization.33,34
Hypothetically, additional MK-active cytokines are essential for
maximal differentiation and polyploidization of human
MKs.34 Using a synthetic preparation of SDF-1, we
observed optimal effects on MK ploidization at a relatively high
concentration (1 µg/mL). However, using recombinant SDF-1, we
observed a comparable stimulation of MK ploidization at a concentration
(100 ng/mL) near to the levels released by stromal cells under
physiological conditions.5
This is the first report showing that the chemokine SDF-1 enhances
the polyploidization of MKs driven by TPO.
CXCR4 is known as the only receptor for SDF-111 and
presumably could mediate different MK functions at different maturation stages of the megakaryocytic lineage from hematopoietic progenitors to
platelets. Cytofluorimetric analysis indicated that treatment of
CD34+ cells with SDF-1 reduced CXCR4 cell surface
expression35: we observed that this reduction was
maintained during the early stage of MK differentiation, but the
initial expression level was almost completely restored starting from
day 7 of culture. In line with these findings, confocal microscopy
analysis showed that 1 hour after SDF-1 addition, a significant
CXCR4 reactivity was found as intracellular spots, seemingly
representing endocytic vesicles; however, at day 8 of culture, CXCR4
reactivity reappeared on the MK cell membrane. Although it is well
established that MK cells express CXCR4 and bind SDF-1 during all
differentiation and maturation steps,36 the specific role
of the cytokine in the different stages of megakaryocytopoiesis is
unclear. Recent studies on the effect of SDF-1 on migration and
proliferation of MK progenitor cells8,18 indicate a role on
the migration of MK progenitors and on cell adhesion of mature marrow
MKs to endothelium, whereas only a moderate effect is reported on MK proliferation. More recently, Hodohara et al21
demonstrated direct proliferative effect of SDF-1 on purified
CD41bright/ c-kitbright CFU-MK derived from
TPO-treated mice. In our liquid culture system, SDF-1 did not
significantly affect MK proliferation. The discrepancy between our
results and those reported by Hodohara et al21 on MK
proliferation could be related to the different hemopoietic progenitors
used in these 2 studies (total CD34+ human HPCs in our
study and murine CD41bright/ c-kitbright
cells in the study of Hodohara et al). Furthermore, in the study by
Hodohara et al,21 the hemopoietic progenitors were
isolated from mice treated in vivo with TPO, and one cannot exclude the possibility that this treatment could sensitize MK
progenitors/precursors to the stimulatory effect of SDF-1 on MK proliferation.
The mechanism responsible for the enhanced megakaryocytic maturation
elicited by SDF-1 in cooperation with TPO is unclear. Recent studies
showed that SDF-1, in addition to its effect on stimulation of
Ca++ influx, induced stimulation of the Janus kinase/signal
transducers and activators of transcription (JAK/STAT)
pathway, particularly of STAT3 and STAT5,37 and
activated ERK1 and ERK2 and its upstream kinase MAPK
kinase.38 These observations were also confirmed by recent
studies performed directly on human MK precursors.39 In
addition, it was shown that PD98059, a specific MEK inhibitor, reduced
the TPO-induced MK polyploidy, thus suggesting an important role for
MAPK in MK endomitosis.28,40 In line with these studies, we observed that the addition of PD98059 to MK cultures stimulated by
TPO alone or by TPO in combination with SDF-1 inhibited the MK
polyploidy, suggesting a role for MAPK in the stimulatory effect of
SDF-1 on ploidy. This conclusion is also supported by a previous study showing that SDF-1 clearly potentiated the ERK-1/ERK-2 phosporylation induced by TPO.28
Our results demonstrated an effect on the polyploidization of the MK
cells only when SDF-1 was added to purified CD34+
cells; no effect was observed on the morphology of the cells when
SDF-1 was added in the TPO-culture medium at day 5 on MK precursors.
However, at this time, the CXCR4 coreceptor is expressed and SDF-1
was able to block the HIV entry in the same MK
population,20 thus confirming the existence of a specific
binding between CXCR4 and SDF-1 also at the MK- precursor stages.
Hamada et al8 demonstrated that SDF-1 induced rapid
transmigration of intact polyploid MKs through bone marrow endothelial cells, followed by fragmentation of MKs into platelets within 12 to 24 hours after migration. On the basis of the effect of SDF-1 on MK
ploidy, we suggest that the cytokine not only affects transmigration,8 but also induces an increase in the
ploidy of MK cells. Kowalska et al36 reported that only
the more immature cells responded to the chemotactic stimulus of SDF-1.
In line with this observation, our transmigration results indicate that differentiating MKs respond to a positive gradient of SDF-1: 10% of
MKs were already able to transmigrate at day 4 of culture, when about
60% of the cells were positive for CXCR4; this percentage grew to 18%
at day 10, when 90% of the cells expressed CXCR4 on the membrane.
Morphological analysis on day-10-transmigrated MKs indicated that only
monolobated or bilobated cells were able to pass through the membrane
into the lower chamber.
The presence of the CXCR4 receptor on platelet surface has not been
correlated with a specific function. However, a recent study suggests
that SDF-1 acts as a weak agonist of platelet activation when added
alone and as an enhancer of platelet activation in response to low
doses of adenosine 5'-diphosphate.41
Our data suggest that the increased number of the nuclear lobes per
cell induced by SDF-1 is coupled with an increase in platelet
formation. Indeed, the number of proplatelet-bearing MKs is higher in
SDF-1-supplemented cultures than in controls. The number of
generated platelets was also more elevated in the presence of SDF-1.
Mechanisms of platelet production and release by mammalian MKs are
poorly understood. Mice lacking transcription factor NF-E2 have a late
arrest in megakaryocytic maturation, resulting in profound
thrombocytopenia.42 Our results on NF-E2 mRNA analyzed on
megakaryocytic culture supplemented with SDF-1 and TPO or with TPO
alone did not show any significant difference in the expression of this
transcription factor (unpublished data, 1999).
Since the expression of the phenotypical markers analyzed (ie, CD61,
CD62, CD41, CD42b) was similar in both TPO- supplemented and
TPO + SDF-1-supplemented cultures, we suggest that acquisition of membrane differentiation markers and polyploidization are
independently regulated events, as was already proposed when the
megakaryocytic differentiation process was investigated with the use of
the UT-7 cell line.43
In conclusion, our results indicate that the chemokine SDF-1,
combined with TPO, is involved not only in migration but also in
polyploidization of MKs and in proplatelet and platelet production, providing a novel insight into the physiological regulation of megakaryocytopoiesis.
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Footnotes |
Submitted March 20, 2000; accepted December 27, 2000.
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.
Reprints: Hamisa Jane Hassan, Dept of Clinical Biochemistry,
Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome,
Italy; e-mail: j.hassan{at}iss.it.
| |
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Activa |
Top
Abstract
Introduction