|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1511-1523
Phenotypic and Functional Evidence for the Expression of CXCR4
Receptor During Megakaryocytopoiesis
By
Christel Rivière,
Frédéric Subra,
Karine Cohen-Solal,
Véronique Cordette-Lagarde,
Remi Letestu,
Christian Auclair,
William Vainchenker, and
Fawzia Louache
From the INSERM U 362, Institut Gustave Roussy; and CNRS URA 147, Institut Gustave Roussy, Villejuif, France.
 |
ABSTRACT |
The identification of stromal cell-derived factor (SDF)-1 as a
chemoattractant for human progenitor cells suggests that this chemokine
and its receptor might represent critical determinants for the homing,
retention, and exit of precursor cells from hematopoietic organs. In
this study, we investigated the expression profile of CXCR4 receptor
and the biological activity of SDF-1 during megakaryocytopoiesis. CD34+ cells from bone marrow and
cord blood were purified and induced to differentiate toward the
megakaryocyte lineage by a combination of stem-cell factor (SCF) and
recombinant human pegylated megakaryocyte growth and development factor
(PEG-rhuMGDF). After 6 days of culture, a time where mature and
immature megakaryocytes were present, CD41+ cells were
immunopurified and CXCR4mRNA expression was studied. High transcript
levels were detected by a RNase protection assay in cultured
megakaryocytes derived from cord blood CD34+ cells as
well as in peripheral blood platelets. The transcript levels were about
equivalent to that found in activated T cells. By flow cytometry, a
large fraction (ranging from 30% to 100%) of CD41+
cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on
peripheral blood platelets. SDF-1 acts on megakaryocytes by inducing
intracellular calcium mobilization and actin polymerization. In
addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1 is a potent attractant for immature megakaryocytic cells but is less active on
fully mature megakaryocytes. This hypothesis was further supported by
the observation that SDF-1 induced the migration of colony forming
unit-megakaryocyte progenitors (CFU-MK) and the expression of
activation-dependent P-selectin (CD62P) surface antigen on early
megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is
expressed by human megakaryocytes and platelets. Furthermore, based on
the lower responses of mature megakaryocytes and platelets to SDF-1
as compared with early precursors, these data suggest a role for this
chemokine in the maintenance and homing during early stages of
megakaryocyte development. Moreover, because megakaryocytes are also
reported to express CD4, it becomes important to reevaluate the role of
direct infection of these cells by the human immunodeficiency virus
(HIV)-1 in HIV-1-related thrombocytopenia.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
MATURE BLOOD CELLS are derived from
hematopoietic precursor cells that are present in specific
(hematopoietic) tissues. Within this environment, hematopoietic cells
lodge, proliferate, and differentiate. Upon maturation, cells must
traverse specialized venus sinus walls to enter the
circulation.1 Cellular interactions between precursor and
stroma cells are thought to serve multiple functions, including
retention of precursor cells within the hematopoietic tissue and
regulation of the release of mature hematopoietic cells into the
circulation, although the exact cellular and molecular mechanisms
involved are not well understood.2 Like other blood leukocytes, platelets are continuously generated from precursors cells
in the bone marrow.3,4 Megakaryocyte maturation proceeds by
a series of steps characterized by the appearance of cell surface markers, including platelet glycoproteins.5
In the past few years, a new superfamily of chemotactic cytokines named
chemokines has been described. These chemokines have been implicated in
many aspects of leukocyte behavior, including regulation of leukocyte
adhesion, locomotion, and chemotaxis.6 Depending on the
number and spacing of conserved cysteines, the chemokine superfamily
can be divided into four groups designed C, CC, CXC, and
CX3C. The C and CX3C chemokines include only
one known member, whereas the CC and CXC groups each have several members. CXC chemokines mainly target neutrophils but also show some
action on T cells. CC chemokines exert their action on multiple leukocyte populations, including monocytes, eosinophils, basophils, T
cells, natural killer (NK), and dendritic cells with variable selectivity, but in most cases they do not target neutrophils. Stromal
cell-derived factor 1 (SDF-1), also called pre-B stimulating factor,
is a CXC chemokine cloned from mouse bone marrow stromal cells.7 Besides its powerful chemoattactant effect on T
cells,8,9 SDF-1 has been shown to be a chemoattractant for
early hematopoietic cells including uncommitted and committed
progenitor cells.10 It has also been shown that SDF-1 is a
chemoattractant for early B-cell precursors and may partially replace
the need for stromal cells for in vitro generation of B
cells.8,10,11 Moreover, inactivation of the SDF-1 gene
induces a marked defect in B-cell lymphopoiesis and bone marrow
myelopoiesis, although normal numbers of myeloid progenitors were
observed in fetal liver, suggesting that SDF-1 is the major
chemoattractant of hematopoietic cells into the marrow.12
SDF-1 was found to be the ligand for a previously identified orphan
receptor called HUMSTR or LESTR.8,9,13 Following the
conventions established for the nomenclature of chemokine receptors,
this receptor has been renamed CXC chemokine receptor 4 (CXCR4). This
receptor, also called fusin, has been identified as one of the major
coreceptors that in association with CD4 allows the entry into cells of
lymphocytotropic HIV-1 strains.14-18
Knowledge of the expression and regulation of this receptor is of prime
importance for understanding what drives the homing, retention, and
exit of megakaryocytes and their precursors from hematopoietic organs
and for understanding the mechanisms involved in HIV-1 related thrombocytopenia.
Until recently, expression of CXCR4 chemokine receptors on cells of the
megakaryocyte/platelet lineage had not been studied. The goal of this
study was to investigate whether CXCR4 was expressed on cells of the
megakaryocyte/platelet lineage. We could demonstrate, as also recently
shown,19,20 that megakaryocytes expressed very high level
of CXCR4. CXCR4 expression increased with maturation and was also found
in platelets. Our results also show that this receptor is only
functional in a subset of megakaryocytes expressing both low levels of
CD41a and CD42a, suggesting that SDF-1 action predominates during early
stages of human megakaryocytic differentiation.
| |
MATERIALS AND METHODS |
Cord Blood, Bone Marrow Cells, and Platelets
Cord blood samples from normal full-term newborn infants were obtained
from a cord blood bank (Dr Van Nifderick, Hopital St Vincent de Paul,
Paris, France). After informed consent, bone fragments from adult
patients undergoing hip surgery were collected in heparin-containing
medium. Marrow cells were collected by vigorous shaking of bone
fragments in  -minimum essential medium (MEM) supplemented with 100 µg/mL of deoxyribonuclease (Sigma, St Louis, MO; DNase type I). Low-density mononuclear cells (LDMC) were prepared by centrifugation on Lymphoprep (Nyegaard, Oslo, Norway) and were used
for immunomagnetic bead separation.
Primary megakaryocytes were enriched from bone marrow using
fractionation over a discontinuous Percoll (Pharmacia, les Ulis, France) density gradient as previously reported.21 Blood
platelets were purified by gel filtration on Sepharose 2B in a buffer
containing NaCl 129 mmol/L, Na3 citrate 13.6 mmol/L,
glucose 11.1 mmol/L, KH2PO4 1.6 mmol/L, and
NaH2PO4 8.6 mmol/L, pH 7.3.
Antibodies
Directly conjugated monoclonal antibodies (MoAb) R-phycoerythrin
(PE)-HPCA2 (anti-CD34), PE-anti-CD62 (anti-P-selectin), and fluorescein isothiocyanate (FITC) anti-CD41a and CD42a were obtained from Becton Dickinson (Mountain View, CA). FITC-TAB (anti-CD41b) was
provided by Dr R. McEver (Oklahoma Medical Research Foundation). PE-12G5 (anti-CXCR4) and a PE-CD41a MoAbs were obtained from Pharmingen (San Diego, CA) and FITC- and PE-conjugated IgG1 and
IgG2a MoAb controls from Becton Dickinson. Unconjugated
antibodies directed against CXCR4 (12G5) and an isotype-control MoAb
were obtained from R&D Systems (Minneapolis, MN).
Human Cytokines
Recombinant human stem cell factor (rhuSCF) and PEG-rhuMGDF (gifts of
Amgen Corp, Thousand Oaks, CA) were usually used at a final
concentration of 50 ng/mL and 10 ng/mL, respectively. Recombinant human
SDF-1 was obtained from R&D Systems.
Isolation of CD34+ Cells or Cultured Megakaryocytes
Mononuclear cells were separated using a magnetic cell sorting system
(miniMACS; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in
accordance with the manufacturer's recommendations. The purity of
CD34+ cells recovered was determined by flow cytometry
using PE-HPCA2 and was over 90%. Cultured megakaryocytes were purified
by the same technique using an anti-CD41b MoAb. Purity after
purification was over 95% as attested by labeling with a
PE-anti-CD41a MoAb.
Cell Cultures
Liquid cultures.
CD34+ cells were cultured in Iscove's modified Dulbecco's
medium (IMDM) with penicillin/streptomycin/glutamine and
11.5 mmol/L -thioglycerol (Sigma). Cultures were performed in
serum-free conditions in which IMDM was supplemented with 1.5% bovine
serum albumin (BSA; Cohn's fraction V; Sigma), sonicated lipids, and iron-saturated human transferrin and stimulated by the combination of
PEG-rhuMGDF and SCF (MK medium).22
Semisolid cultures.
Cultures were performed in serum-free clot in the presence of
PEG-rhuMGDF and SCF. Ingredients for serum-free cultures were similar
to those of liquid culture in which were added bovine plasma fibrinogen
(1 mg/mL, Sigma), 0.01 mol/L  amino caproic acid and horse thrombin
(6 mU/mL; Stago, Asnières, France). Target cells were either
CD34+ cells (1 × 103 cells/mL) or
cultured cells before and after in vitro migration. Cultures were
incubated at 37°C in a fully humidified atmosphere containing 5%
CO2 in air and scored after 10 to 12 days. Colonies were
quantitated by an indirect immuno-alkaline phosphatase labeling technique using an anti-GPIIIa MoAb (CD61, Y2-51) as previously described.23 Dishes were scanned in toto under an inverted
microscope at 4× or 100× magnification.
Flow Cytometry Analysis
After washing, cells were stained with an appropiate dilution of the
antibody. Double staining was performed using differently conjugated
IgG1 as a control. Cells were suspended in phosphate buffer (PBS), kept
at 4°C, and analyzed on a FACsort (Becton Dickinson) with the Cell
Quest software package.
Chemotactic Assay
To analyze megakaryocyte migration, CD34+ cells were
cultured for 6 to 12 days in MK medium. Unless otherwise specified, the cells were used directly for migration assays. In a limited number of
experiments (n = 6), cultured cells were immunopurified on the basis of
CD41a expression before the migration assay. The cells were then
resuspended in serum-free medium to a final concentration of 2.5 × 106 cells/mL, and 100 µL of the cell suspension
was placed into the upper chamber, whereas 600 µL of medium with or
without SDF-1 was introduced in the lower chamber. The migration
assay was performed using 5-µm or 8-µm pore filters (Transwell,
24-well cell clusters; Costar, Cambridge, MA). SDF-1 at different
concentrations was diluted in serum-free medium and placed in the lower
wells. Cells (2.5 × 105) were suspended in the same
medium and added to upper wells. The chambers were incubated for 1 hour
at 37°C in 5% CO2 and 95% air. The cells in the upper
chamber were recovered. The upper chamber was then carefully recovered
and cells in the bottom chambers were recovered in the same volume for
counting. The different cell fractions were then labeled with a
PE-anti-CD41a and FITC anti-CD42a MoAbs and analyzed by flow
cytometry. For the experiment using pertussis toxin, cells were
preincubated for 2 hours at 37°C with pertussis toxin (100 ng/mL;
Sigma) before migration. All assays were done in triplicate. Data are
presented as the chemotaxis index calculated by the following ratio:
number of cells migrating to SDF-1/number of cells migrating to
medium. For morphological studies, the different cell fractions
obtained were cytospun on slides and observed at light microscopy after May-Grunwald Giemsa staining. Cell diameter of cells in suspension was
measured using a microscope equipped with an ocular micrometer. For
each group, 300 cells were analyzed.
Actin Polymerization
To analyze actin polymerization in megakaryocytes, cultured cells were
washed once in PBS and incubated at 37°C between 30 seconds and 2 minutes in the presence of either SDF-1 (300 ng/mL) or thrombin
(1U/mL; Stago) and fixed for 10 minutes with an equal volume of 0.5%
paraformaldehyde. After washing and permeabilization in 0.01% triton
solution in PBS for 10 minutes, the cells were incubated with a
PE-anti-CD41a MoAb and either 4.10 7 mol/L
FITC-conjugated phalloidin (Sigma) or Oregon green-conjugated DNase1
(Molecular Probes, Eugene, OR). Data were analyzed by flow cytometry
after gating the CD41+ cells.
Calcium Efflux Assay
To analyze the cellular calcium mobilization in megakaryocytes,
cultured cells were washed twice in Hanks' balanced salt solution (HBSS) and labeled with a PE-anti-CD41a MoAb. Cells were
then suspended in HBSS and incubated at 37°C for 30 minutes in
presence of 5 µg/mL Fluo-3/AM cell permeant (Molecular Probes). The
cells were then washed twice with HBSS and resuspended at 1 × 106 cells/mL and stored in the dark at 22°C until
analysis. A sample was analyzed on FACSort for basal level of
fluorescence, then either SDF-1 (300 ng/mL) or thrombin (1 U/mL) was
added. After stimulation, fluorescence changes were monitored during 10 minutes. Data were analyzed by gating the CD41+ cells and
calculating the mean fluorescence intensity every 10 seconds.
Detection of the Activation-Dependent Antigen P-Selectin (CD62) on
Megakaryocytes and Platelets
Cultured cells or platelets were stimulated at 37°C with either
SDF-1 (300 ng/mL) or thrombin (1 U/mL). After 10 minutes of
stimulation, samples were incubated for 15 minutes at 4°C with both
R-PE-anti-CD62 (2 µg/mL) and FITC anti-CD41 MoAbs. Cells were then
fixed for 1 hour with an equal volume of 0.5% paraformaldehyde. Control cells were fixed in the same manner without prior activation. Cells were subsequently resuspended in PBS.
Ribonuclease Protection Assay
The coding region of human CXCR4 was cloned by polymerase chain
reaction (PCR) as previously described,16 as a 1.1-kb
HindIII-Xho I fragment in pCDNA3
(PCD-CXCR4). The BamHI-Dra I fragment, containing nucleotides 566 to 1009 of the cDNA of CXCR4, was transferred from
pCD-CXCR4 into the pBluescript SK+ vector in the
EcoRV polylinker site (pSK-CXCR4). A
32P-UTP-labeled antisense probe was transcribed from the
T7 promoter of the BamHI linearized pSK-CXCR4 plasmid. As an
internal control for each reaction sample, a 32P-labeled
antisense human actin probe was transcribed from the T7 promoter of the
BamHI-digested pSK-actin plasmid. This plasmid was constructed
by inserting a Nar I-Bsa I actin fragment, containing nucleotides 147 to 413, into the polylinker site of the pBluescript SK+ vector. Total RNA (20 µg) prepared from peripheral
blood mononuclear cells, phytohemagglutinin (PHA)-activated mononuclear
cells, CD41+ cord blood cells, and platelets was hybridized
with radioactive probes at 50°C overnight. Nonhybridizing RNA was
digested with RnaseA(10 µg/mL) and RnaseT1 (1,000 U/mL) for 1 hour at
37°C. To stop RNase action, sodium dodecyl sulfate (SDS; 0.6%) and
proteinase K (145 µg/mL) were added for 15 minutes at 37°C.
Protected fragments were extracted in the presence of 15 µg of
carrier transfer RNA with phenol/chloroform/isoamyl alcohol and
precipitated with absolute ethanol at  20°C. Fragments were
resolved on a 4% polyacrylamide, 7 mol/L urea gel, and
autoradiographed on hyperfilm MP (Amersham). The size of the protected
fragments was determined using labeled Msp I-digested pBR322
(New England Biolabs, Beverly, MA).
Determination of Platelet Numbers Produced in Culture
SDF-1 at different concentrations was added after 6 days of culture
of cord blood-derived CD34+ cells in serum-free conditions
in presence of stem cell factor (SCF) and PEG-rhuMGDF. Six days after
SDF-1 addition, platelet production was assessed by flow cytometry
as previously described.24 After collection and rinsing
with PBS/EDTA, cultured cells were centrifuged at 350g for 15 minutes, incubated with the R-PE-anti-CD41a MoAb for 30 minutes, and
fixed with 0.5% paraformaldehyde (Serva, Heidelberg, Germany) for 20 minutes. Cells from each culture condition were diluted to the same
volume (400 µL). For each sample, the acquisition rate was 1 mL/s for
100 seconds. Events were collected using an analytical gate based on
scatter properties of normal blood platelets treated similarly using a
log scale for forward light scatter (FSC) and side scatter (SSC). This
gate excluded large contaminating cells (MKs) and small debris or
microparticles. Samples were analyzed with a FACsort flow cytometer
(Becton Dickinson).
Statistics
Results of experimental points obtained from multiple experiments were
reported as the mean ± SD. Statistical analysis was performed using
the two-tailed Student's t-test for paired data.
| |
RESULTS |
Presence of CXCR4 Transcripts in Megakaryocytes and Platelets
CXCR4 expression was investigated using the RNAse protection assay. To
obtain a large number of megakaryocytic cells, CD34+ cells
were seeded in serum-free liquid culture containing SCF and PEG-rhuMGDF
for up to 6 days. After 6 days in culture at a time where a significant
proportion (25%) of CD41+ cells were present,
CD41+ cells were purified by the immunomagnetic bead
technique with a purity exceeding 95% and total mRNA were extracted.
This time point was chosen for these experiments because few
megakaryocytes could be analyzed at earlier time points. Later in the
cultures, megakaryocytes were more fragile and their immunoselection
using immunomagnetic beads resulted in substantial cell clumping, which is associated with lower purity and viability.
Freshly isolated peripheral blood mononuclear cells and PHA-activated
cells as well as platelets were also analyzed. As shown in
Fig 1, a protected fragment of the
predicted size, which comigrated with that found in PBMNC, was detected
at high levels in purified megakaryocytes and platelets.
View larger version (36K):
[in this window]
[in a new window]
| Fig 1.
Detection of CXCR4 transcripts by RNase protection assay.
mRNA was extracted from peripheral blood mononuclear cells,
PHA-activated mononuclear cells, CD41+ cells, and
platelets. tRNA served as a negative control. Samples of mRNA were
hybridized with a specific CXCR4 riboprobe and a specific actin
riboprobe, then digested with RNase T1. Protected fragments were
analyzed on denaturing acrylamide gels. Sizes of the fragments were
determined using labeled Msp I-digested pBR322.
|
|
Flow Cytometric Expression of CXCR4 by Megakaryocytes and Platelets
We next investigated whether cell-surface expression of CXCR4 could be
detected by multiparameter flow cytometric analysis. Cells derived from
CD34+ cell liquid cultures at different days of culture
were dually labeled with a FITC anti-CD41a MoAb and PE-12G5 MoAb to
detect CXCR4 protein from day 6 to day 12 of culture.
In the representative experiment shown in
Fig 2A and B, CXCR4 was expressed by the
majority of CD41a+ cells at all days of culture. A positive
correlation was found between CXCR4 and CD41a expression. CXCR4 was
also detected but at a lower level on a minority of
CD41a cells present in the culture. A mean of 74 ± 25% (range 30% to 100%, n = 12) of cord blood-derived
CD41a+ cells showed CXCR4 staining. Similar results were
obtained with megakaryocytes derived from adult marrow
CD34+ cells with a mean of 90 ± 5% (range 72% to
100%, n = 5).
View larger version (49K):
[in this window]
[in a new window]
| Fig 2.
CXCR4 expression on megakaryocytes and platelets. (A, B)
Representative data of flow cytometric analysis of CD41a and CXCR4 on
megakaryocytes grown in culture are shown. Cord blood
CD34+ cells were cultured for 6 days in the presence of a
combination of SCF and PEG-rhuMGDF. Cells were stained using an
anti-CD41a MoAb in combination with either an isotype control antibody
(A) or the anti-CXCR4 MoAb (B). (C) CXCR4 expression on freshly
isolated megakaryocytes after percoll enrichment. (D) Platelets,
depleted in leukocytes, were labeled using a similar procedure.
Labeling with control MoAb is shown by the thin line. The thick line
shows the staining with the anti-CXCR4 MoAb.
|
|
We also studied the expression of CXCR4 during megakaryocyte
differentiation in vivo. Bone marrow megakaryocytes were enriched using
a percoll gradient and tested by two-color fluorescence. The expression
pattern of CXCR4 on primary CD41a+ cells is shown in Fig
2C. A mean of 88% (range 82% to 100%, n = 3) of primary
CD41+ cells expressed CXCR4. Moreover, CXCR4 was expressed
on the surface of freshly isolated platelets CXCR4 (Fig 2D). These
experiments allowed us to establish that CXCR4 expression on
megakaryocytes was not simply related to the culture conditions.
Short-Term Effects of SDF-1 on Megakaryocytes and Platelets
On the basis of expression data and because primary megakaryocytes were
difficult to obtain, the following experiments were performed on
cultured cells derived from cord blood CD34+ cells after 6 to 12 days of culture. Because similar results were obtained at day 6, day 9, and day 12 of culture, unless otherwise stated, the data shown
were those obtained on day 6 of culture. At day 6 of culture, repeated
experiments showed that the percentage of CD41a+ cells
averaged 25% CD41a+ cells (n = 13), with a range of 12%
to 33% including some mature megakaryocytes. Most of the cells present
in day 6 cultures were CD34+ blast cells, whereas less than
2% expressed lineage markers (T cells, B cells, neutrophil-macrophage,
and erythroid lineage). Later in the culture at day 12, the percentage
of CD41a+ cells slightly increased with the appearance of
CD15+ cells.
To address the functional role of CXCR4 on megakaryocytes, we first
tested the ability of SDF-1 to induce calcium mobilization. As shown
in Fig 3, we observed a transient calcium
flux in all of the CD41a+ cells, but this effect of
SDF-1 was much weaker than that elicited by thrombin (not shown).
Additional experiments were designed to determine whether SDF-1
influences intracellular actin reorganization. This effect is thought
to be a prerequisite for cell movement. After labeling by a
PE-anti-CD41a MoAb, cells were treated with an optimal concentration
of SDF-1 (500 ng/mL) or thrombin (2 U/mL) for 5 minutes at 37°C.
After fixation, permeabilization, and labeling with fluorescent
phalloidin or DNase I, cells were analyzed by flow cytometry. As shown
in Fig 4, these two reagents augmented the
expression of filamentous actin and decreased expression of actin G. This effect is similar to that reported in T cells.
View larger version (22K):
[in this window]
[in a new window]
| Fig 3.
Calcium flux response of megakaryocytes to SDF-1.
After CD41a labeling, cells were loaded with Fluo-3 and exposed to
SDF-1 (300 ng/mL). Changes in fluorescence were monitored over time
by flow cytometry after SDF-1 addition. The results are derived from
one representative experiment of a total of three separate
experiments.
|
|
View larger version (20K):
[in this window]
[in a new window]
| Fig 4.
Actin polymerization in megakaryocytes after SDF-1
addition. Cultured cells were incubated at 37°C for 30 seconds and
2 minutes in the presence of either SDF-1 (300 ng/mL) or thrombin
(Stago, 1 U/mL). Intracellular F actin (phalloidin) or G actin (oregon
green conjugated DNase 1) was determined by flow cytometry after gating
the CD41a+ cells. Results of one representative
experiment are shown. Two other experiments gave similar results.
|
|
In these experiments, we also sought to determine if SDF-1 induces
actin polymerization directly or indirectly via a secondary effect on
accessory cells. To address this question, day-6 CD41a+
cells were purified using immunomagnetic beads. Purity of the CD41a+ cells in this population consistently exceeded 96%.
Purified cells were then exposed to SDF-1 or thrombin, and actin
polymerization was analyzed by flow cytometry. Results were highly
reproductible with respect to unseparated cultures supporting the
hypothesis that SDF-1 exerted a direct effect on megakaryocytes
(data not shown).
Because thrombin and SDF-1 have common properties, we tested to see
if SDF-1 induced activation of megakaryocytes and platelets by
studying translocation of CD62 (P-Selectin) on the cell surface. As
illustrated in Fig 5A, B, C, D, E, and F,
SDF-1 increased cell surface expression of CD62 in
CD41alow (Fig 5D) cells but not on CD41ahigh
(Fig 5E) cells and platelets (Fig 5F).
View larger version (43K):
[in this window]
[in a new window]
| Fig 5.
Effects of SDF-1 on the expression of CD62 by
megakaryocytes. (A,B,C) Examples of double-color staining with
anti-CD62 in combination with anti-CD41a (A) or with control antibodies
(B) and (C). Primary cultured cells were either left unstimulated
(thick line) or stimulated for 10 minutes with either thrombin (1 U/mL,
thin line) or SDF-1 (300 ng/mL, broken line). CD62 staining in
combination with CD41a staining was then compared by FACS analysis as
described in Materials and Methods. The analysis was performed in
CD41alow (D) and CD41ahigh (E) gates as defined
in (A) and in platelets (F). These data are representative of four
experiments.
|
|
Chemotactic Activity of SDF-1 on Megakaryocytes
We subsequently investigated the ability of SDF-1 to induce
migration of megakaryocytes in vitro. Previous studies have established that this method is sensitive and reliable in the evaluation of chemokine receptor function.10
Figure 6A shows that SDF-1 induced a
significant migration of megakaryocytes in vitro with a typical bi-modal dose-response curve and with a maximum effect at 500 ng/mL.
Pretreatment of the cells with pertussis toxin completly inhibited the
SDF-1-induced migration.
View larger version (16K):
[in this window]
[in a new window]
| Fig 6.
Chemotaxis assay and cell surface expression of CD41a.
(A) Chemotaxis responses of the CD41a+ cell population.
CD34+ cells were seeded in serum-free liquid culture
containing SCF and PEG-rhuMGDF for up to 6 days. The cells were
subjected to chemotaxis through 5-µm pores to various concentrations
of SDF-1 and stained with an anti-CD41a MoAb (black bars). Checkerboard
analysis was performed by adding SDF-1 (500 ng) both in the bottom
and in the top well (white bar). This migration is inhibited by
preincubating the cells with pertussis toxin (PTX; hatched bar). Data
are expressed as chemotaxis index and represent the mean for one
representative experiment done in triplicate. (B) Effect of a blocking
MoAb against CXCR4 (12G5). The cells were preincubated either with
increasing concentrations of 12G5 MoAb (white squares) or an
isotype-control MoAb (black squares) before the migration assay. (C)
Chemotaxis responses of megakaryocytes. After migation in response to
SDF-1 (500 ng/mL), the cells were stained with an anti-CD41a MoAb.
The percentage of CD41+cells was determined in starting
and migrated cells. The results shown are the mean and SD of five
experiments.
|
|
To prove the specificity of the effects of SDF-1, cells were
preincubated either with increasing concentrations of a blocking MoAb
against CXCR4 (12G5)25 or an isotype-control MoAb. Figure 6B shows that the megakaryocyte chemotactic response to SDF-1 could
be inhibited by 12G5 MoAb indicating that CXCR4 is the relevant receptor.
To distinguish between SDF-1-induced chemokinesis (increased random
migration) and chemotaxis (directed movement of cells along a
chemotactic gradient), we performed checkerboard analysis. Cultured
cells were resuspended in medium containing various concentrations of
SDF-1 (10 ng/mL to 1,000 ng/mL) just before transferring the cells
to the upper chamber. As shown in Table 1,
a gradually increasing concentration gradient of SDF-1 between the
lower and the upper compartment led to increased migration of
CD41a+ cells toward the lower compartment. Hence, the
megakaryocyte response to SDF-1 is a chemotactic response rather
than the result of an increase in random migration.
Characterization of SDF-1 Responsive Megakaryocyte
Subsets
CD34+ cell-derived megakaryocytes grown in the presence of
PEG-rhuMGDF and SCF represent an asynchronous population of cells at
various stages of maturation with different ploidy and
sizes.26 Analysis of the cells migrating to the bottom well
showed that the relative proportion of CD41a+ cells did not
change significantly as compared with their relative proportion in the
starting population (Fig 6C), although these cells expressed the
highest level of CXCR4 receptors among the cultured cells. Due to this
lack of enrichment, we thought that it was possible that only a
subpopulation of CD41a+ cells responded to SDF-1. To
examine this possiblity, we compared the starting population to the
cells that migrated in response to SDF-1 and to the cells that
migrated to medium alone for their FSC versus SSC properties as well
for their morphology after May-Grunwald-Giemsa staining. As shown in
Fig 7A,B,and C, the SSC and FSC properties of the migrated cells and the starting cells differ greatly. On average, migrated cells exhibited a low SSC properties as compared with
the starting population or to the population that migrated spontaneously. It has been shown that megakaryocyte maturation is
associated with marked increase of SSC.27 Furthermore, as shown in Fig 7E, no morphologically identifiable megakaryocytes as
defined on the basis of their large size and a polylobulated nucleus
were seen in the cell fraction that migrated in response to SDF-1 in
contrast to the starting population (Fig 7D), suggesting either that
mature megakaryocytes cannot fit through the pores of the transwell due
to their large size or that the megakaryocyte migration in response to
SDF-1 is downregulated during their maturation. However, the use of
8-µm pore size transwells did not modify these results (data not
shown), suggesting that cell size was not the limiting parameter.
View larger version (74K):
[in this window]
[in a new window]
| Fig 7.
Morphological characteristics of cells migrating in
response to SDF-1. After migration, cultured cells were analyzed for
their morphological characteristics. Dot plot analysis (FSC versus SSC)
of starting population (A), cells migrated in response to SDF-1 (B),
and cells migrated in absence of SDF-1 (C) are shown.
May-Grunwald-Giemsa staining of the starting cell fraction (D) and the
cell fraction that migrated in response to SDF-1 (E) are shown.
|
|
To further characterize the properties of the cells that respond to
SDF-1 and to investigate whether megakaryocyte migration was
regulated during their maturation, we compared the starting population
with the cells that migrated in response to SDF-1 for the expression
of CD41a and CD42a. The rationale for this experiment was based on
previous studies showing that CD41a appears at an earlier stage of
megakaryocyte-lineage development than CD42a. In these experiments,
large-sized cells were specifically excluded by analyzing the cells
with low SSC properties as defined in gate R1 (Fig 7). As illustrated
in Fig 8A,B, and C, a direct relationship
between CD41a and CD42a was observed. However, two populations of
CD41a+ cells could be determined:
CD41alow/CD42alow cells (region R6) and
CD41ahigh/CD42ahigh cells (region R5).
CD41a+ cells that chemotaxed to SDF-1 were both
CD42alow and CD41alow (Fig 8D and E). Thus,
when compared with the starting cell population, a marked enrichment in
the proportion of the CD41alow/CD42alow was
observed after migration: 34 ± 2% versus 67.6 ± 5.4%,
respectively (Fig 8H). Moreover, the staining was compared between the
cells that migrated in response to SDF-1 to the cells that migrated spontaneously and indicate a specific migration of CD42alow
and CD41alow cells to SDF-1 (Fig 8F and G). To confirm
this higher response of CD41alow/CD42alow to
SDF-1, we carried chemotaxis assays with isolated megakaryocyte subsets after 10 days of culture. To obtain sufficient number of cells,
cultured cells were first immunopurified as described in Materials and
Methods. The enriched fraction was then stained and separated using the
gate R1 defined in Fig 7 and the gates R5 and R6 defined in Fig 8A.
Purified CD41alow/CD42alow cells showed twofold
to threefold higher migrating ability to SDF-1 (chemotaxis index
22.5 ± 8; mean ± SD of three experiments ) as compared with
purified CD41ahigh/CD42ahigh (chemotaxis index
6 ± 2; mean ± SD of three experiments). To investigate whether
these observations may relate to a different size between these two
subsets in two experiments, we evaluated the mean size of
megakaryocytes in the upper and the lower wells after migration using a
micrometer. This analysis revealed that the mean size of the cells was
8.2 ± 0.6 µm and 9.6 ± 1.8 µm in the lower and
upper well, respectively. This difference in size was weak but
statistically significant (P > .0005).
View larger version (49K):
[in this window]
[in a new window]
| Fig 8.
Flow cytometric analysis of cell surface expression of
CD41a and CD42a on starting and migrated cell populations. After
migration, cultured cells were labeled with an R-PE-conjugated
anti-CD41a MoAb and a FITC-conjugated anti-CD42a MoAb. (A, B, C) An
example of double-color fluorescence beween CD41a and CD42a (A) and the
respective controls (B, C). The cells within the lymphoid blast window
(R1, Fig 7) were analyzed for the expression of CD42a (D) and CD41a
(E). The thick line shows the staining of cells that migrated in
response to an optimal concentration of SDF-1 (500 ng/mL). Broken
lines represent the staining of the starting cell population, and
dotted lines represent the staining with control antibodies. In two
experiments, CD42a (F) and CD41a (G) staining was compared between the
cells that migrated in response to SDF-1 (thick line) and to control
media (thin line). (H) Migration of
CD41alow/CD42alow cells. The mean and SD
percentages of CD42alow/CD41alow were
determined by gating on the entire population of CD41a+
cells.
|
|
Finally, the presence of colony forming unit-megakaryocyte progenitors
(CFU-MK) among the cultured cells that migrated in response to SDF-1
was investigated by performing CFU-MK assays on the cells before and
after in vitro migration. Figure 9 shows that SDF-1 induced a significant migration of CFU-MK among cultured cells in vitro with a typical bimodal dose response curve and a maximum
effect at 500 ng/mL. The proportion of CFU-MK migrating in response to
SDF-1 ranged between 22% and 30% (n = 3). Similar results were
obtained when the migration of CFU-GM and burst-forming unit-erythroid
(BFU-E) was studied (data not shown). These data directly show that
CFU-MK as other hematopoietic progenitors respond efficiently to
SDF-1.
View larger version (28K):
[in this window]
[in a new window]
| Fig 9.
Chemotaxis responses of CFU-MK in response to various
concentrations of SDF-1. CD34+ cells were seeded in
serum-free liquid culture containing SCF and PEG-rhuMGDF for up to 6 days. After 6-day culture, the cells were subjected to chemotaxis
through 5-µm pores and plated in CKU-MK assay. Results are shown of
one representative experiment performed in triplicate. Two other
experiments gave similar results.*P < .05, **P < .01 when compared with control.
|
|
Effects of SDF-1 on Megakaryopoiesis and Platelets
Production
In a first set of experiments, SDF-1 (10 to 1,000 ng) was added at
the onset of the culture in the CFU-MK assays stimulated by a
combination of PEG-rhuMGDF and SCF at optimal concentrations. No effect
of SDF-1 was observed on MK colony growth
(Fig 10).
View larger version (29K):
[in this window]
[in a new window]
| Fig 10.
Effects of SDF-1 on CFU-MK growth. After
CD34+ selection, 1 × 103cells were assayed
in fibin-clot cultures in presence of increasing concentrations of
SDF-1. Data are expressed as the mean and SD of two experiments
performed in triplicate.
|
|
We then studied whether SDF-1 modified platelet production in vitro.
For this purpose SDF-1 (500 ng/mL) was added after 6 days of culture
of cord blood-derived CD34+ cells stimulated by SCF and
PEG-rhuMGDF. Six days later (12 days of culture), platelet production
was assessed by flow cytometry after CD41a labeling. SDF-1 did not
modify platelet production in the cultures stimulated by PEG-rhuMGDF
alone, whatever the concentration of PEG-rhuMGDF that was used
(Fig 11A). Similarly, no effect of
SDF-1 on platelet formation was observed when the cells were
cultured in the presence of a combination of PEG-rhuMGDF at saturating
concentration (10 ng/mL) and SCF at different doses (Fig 11B), or in
the presence of SCF at saturating concentration (25 ng/mL) and
PEG-rhuMGDF at different doses (Fig 11C).
View larger version (17K):
[in this window]
[in a new window]
| Fig 11.
Effects of SDF-1 on platelet production.
CD34+ cells were seeded in serum-free liquid culture
containing SCF and PEG-rhuMGDF for up to 6 days. After two washes,
SDF-1 was added at optimal concentration (500 ng/mL) in a second
phase of the culture in the presence of increasing concentrations of
PEG-rhuMGDF without any other factor (A) or in presence of an optimal
concentration of SCF (25 ng/mL) (B). Conversely, platelet production
was tested in the presence of an optimal concentration of
PEG-rhuMGDF(10 ng/mL) and increasing concentrations of SCF (C). Six
days after SDF-1 addition, platelet production was assessed by flow
cytometry after anti-CD41a labeling. Cells from each culture condition
without SDF-1 (white bar) and in presence of SDF-1 (black bar)
were distributed in the same volume (400 µL), and for each sample the
acquisition rate was 1 µL/s for 100 seconds. Data represent the mean
for two experiments done in triplicate.
|
|
| |
DISCUSSION |
The aim of this study was to determine the expression and the function
of CXCR4 chemokine receptors that would have important implications
both in understanding what drives the homing, retention, and exit of
precursor cells from hematopoietic organs and the mechanisms involved
in HIV-1-related thrombocytopenia. CXCR4 is a member of the
G-protein-coupled seven transmembrane domain receptor family.18 It is expressed in many cell tissues including
brain, spleen, and lung.13,28 Among hematopoietic cells,
CXCR4 is differentially expressed during T-cell
differentiation29,30 and is present on mature T cells, B
cells, and monocytes. Recently, it was shown by reverse-transcription
(RT)-PCR that CXCR4 transcripts were detected in CD34+
cells.31 It also seems very likely that CD34+
cells express a functional CXCR4 receptor because its ligand SDF-1 is a
chemoattractant for all CD34+ cell subsets.10
Using RNase protection assays, CXCR4 transcripts were readily detected
in purified megakaryocytes derived from cord blood and adult bone
marrow. Transcript levels were quite similar to that detected in
PHA-activated T cells. This was not the consequence of transcript
upregulation by culture conditions, because primary megakaryocytes and
peripheral blood platelets also strongly expressed the CXCR4 transcript.
This expression study was greatly facilitated by the availability of an
anti-CXCR4 MoAb.25 The expression pattern of the membrane
protein correlated with the CD41a antigen. Among cultured myeloid
cells, megakaryocytes were those that expressed the highest protein
level. It is noteworthy that the protein could also be detected on the
platelet surface. Similar results were recently obtained by two other
groups.19,20 The only other chemokine receptors that have
been previously found on platelets are the interleukin-8 (IL-8)
receptor A and a CC chemokine receptor named K5.5.32
The role of chemokines in the regulation of megakaryocytopoiesis has
been extensively studied. It was shown that several CXC or CC
chemokines inhibited in vitro megakaryocytopoiesis.33 Studies were only performed on the functions of PF4 and
 -thromboglobulin that are produced by
megakaryocytes.34-36 These chemokines may induce a specific
inhibition of megakaryocytopoiesis, suggesting a negative autocrine
loop of regulation.34-36 SDF-1, the ligand of CXCR4, has
not yet been involved in the regulation of cell proliferation and
apoptosis. In contrast, it is a powerful chemoattractant for
lymphocytes, monocytes, and hematopoietic
progenitors.8,10,11 Results obtained with the
SDF-1/ mice as well as with in vitro
migration assays on CD34+ cells suggest that SDF-1 is
the physiological chemoattractant of hematopoietic cells into the
marrow and regulates their migration into the blood.10,12
In the present work, we have shown that SDF-1 is also a potent
chemoattractant for megakaryocytes. It is, as yet, the first chemokine
that is involved in this function. As for lymphocytes and
CD34+ cells, this migratory function is preceded by an
intracellular calcium mobilization.8 It is associated with
an increase in actin polymerization, which leads to the reorganization
of intracellular actin necessary for cell movement.8 The
CXCR4 expression by peripheral blood platelets was unexpected because
platelets are devoid of chemotaxis. However, despite a high level of
expression of CXCR4, only a fraction (30%) of megakaryocytes were
induced to migrate by SDF-1. The megakaryocytes that responded to
SDF-1 were immature and included CFU-MK progenitors. In addition, a majority of CD41a+ blasts were present in cells that
migrated, suggesting either that mature megakaryocytes cannot fit
through the pores of the transwell due to their large size or that the
megakaryocyte migration in response to SDF-1 is downregulated during
their maturation. Nevertheless, phenotypic studies of a cell population
with similar scatter properties (with about similar size) showed that
CD41alow/CD42alow cells exhibited twofold to
threefold higher migrating ability in response to SDF-1 than
CD41ahigh/CD42ahigh cells, suggesting that
megakaryocyte migration in response to SDF-1 is downregulated during
maturation. This hypothesis is further supported by the fact that
SDF-1 was able to induce P-selectin (CD62) translocation on
CD41alow but not on CD41ahigh cells and on
platelet surface membranes. Because early observations have shown that
almost all CD41ahigh/CD42ahigh cells failed to
undergo cell division as compared to
CD41alow/CD42alow cells,5 this may
imply that SDF-1 is a chemoattractant for immature proliferating
megakaryocytes in the marrow. In addition, as SDF-1 induces
P-selectin expression on immature megakaryocytes, it may be involved in
modulation of their adhesion properties.
Similar loss of function of CXCR4 has been already reported in B-cell
development, suggesting that SDF-1 may function in a stage-specific
manner.11,37 Moreover, as in this study, a difference in
CXCR4 expression and response patterns was observed in mature B cells
that, while expressing significant levels of CXCR4, are not responsive
to SDF-1.11,37 These data suggest that the CXCR4
receptor can be functionally uncoupled or disengaged. Further work will
be necessary to clarify the mechanisms involved in this uncoupling of
expression and function.
The physiological role of SDF-1 in megakaryocyte/platelet
differentiation remains totally speculative. Megakaryocytes are the
only marrow cells that produce blood cells by cytoplasmic fragmentation. This production may be due to long extensions that pass
through the fenestrated bone marrow endothelium barrier (proplatelet formation) and break into platelets under the forces of blood flow.38 Alternatively, the entire megakaryocyte may migrate into the sinusoid.39 Platelets are subsequently formed in
the circulation, especially in the lung circulation, which may act as a
filter for circulating megakaryocytes.40 Regulation of these late stages of platelet production is poorly understood. In the
present study, we had no evidence that SDF-1 is involved in
proplatelet formation because this process as well as in vitro platelet
production were not modulated by addition of this chemokine. As in the
present report, Wang et al19 reported no marked effect of
SDF-1 on megakaryocytopoiesis in vitro. Thus, SDF-1 may be involved in the trafficking of megakaryocytes by controlling their exit
from the marrow into blood and eventually their migration to the lung.
In support of this hypothesis, it was recently shown that SDF-1 was
able to induce a transendothelial migration of megakaryocytes and to
enhance platelet formation by favoring the interaction between
megakaryocytes and endothelial cells.20 However, extension
of this finding to in vivo megakaryopoiesis must be considered with
caution as SDF-1 is unlikely to be the only chemokine factor acting
on megakaryocytes.
It has already been shown that a fraction of megakaryocytes expresses
CD4.41-44 Our data imply that a fraction of megakaryocytes may be infected by lymphocytotropic strains of HIV-1. This possibility is supported by the observation that megakaryocytes from
thrombocytopenic HIV patients express HIV transcripts or
proteins.21,45 However, it has also been shown that HIV
particles can be taken up by megakaryocyte and platelets without true
internalization because viral particles are trapped in the demarcation
membrane system.46 Knowledge of the expression of CXCR4 on
megakaryocytes may facilitate the understanding of the mechanisms of
HIV-related thrombocytopenia.
| |
ACKNOWLEDGMENT |
We are grateful to J.-L. Nichol (Amgen) for providing the SCF and
PEG-rhuMGDF. We are grateful to surgeons for providing bone marrow
samples and to Dr Van Nifderick from the St Vincent de Paul hospital
for cord blood samples.
| |
FOOTNOTES |
Submitted April 29, 1998; accepted October 21, 1998.
Supported by grants from the Institut National de la Santé et de
la Recherche Médicale, the Institut Gustave Roussy, the Agence
Nationale pour la Recherche sur le SIDA (ANRS), and SIDACTION. C.R. is
a fellowship of the French Ministere de la recherche.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Fawzia Louache, MD, INSERM U
362, Institut Gustave Roussy, PR1, 39 Rue Camille Desmoulins, 94805 Villejuif, France.
| |
REFERENCES |
1.
Tavassoli M:
The marrow-blood barrier.
Br J Haematol
41:297, 1979[Medline]
[Order article via Infotrieve]
2.
Dexter TM, Allen TD, Lajtha LG:
Conditions controlling the proliferation of haematopoietic stem cells in vitro.
J Cell Physiol
91:335, 1977[Medline]
[Order article via Infotrieve]
3.
Hofmann R:
Regulation of megakaryocytopoiesis.
Blood
74:1196, 1989[Free Full Text]
4.
Vainchenker W, Kieffer N:
Human megakaryocytopoiesis: In vitro regulation and characterization of megakaryocyte precursor cells by differentiation markers.
Blood Rev
2:102, 1988[Medline]
[Order article via Infotrieve]
5.
Higuchi T, Koike K, Sawai N, Koike T:
Proliferative and differentiative potential of thrombopoietin-responsive precurseurs: Expression of megakaryocytic and erythroid lineages.
Exp Hematol
25:463, 1997[Medline]
[Order article via Infotrieve]
6.
Rollins BJ:
Chemokines.
Blood
90:909, 1997[Free Full Text]
7.
Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T:
Signal sequence trap: A cloning strategy for secreted proteins and type I membrane proteins.
Science
261:600, 1993[Abstract/Free Full Text]
8.
Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA:
The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry.
Nature
382:829, 1996[Medline]
[Order article via Infotrieve]
9.
Oberlin E, Amara A, Bachelerie F, Bessia C, Virelizier J-L, Arenzana-Seisdedos F, Schwartz O, Heard J-M, Clark-Lewis I, Legler DF, Loetscher M, Baggiolini M, Moser B:
The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1.
Nature
382:833, 1996[Medline]
[Order article via Infotrieve]
10.
Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC:
The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.
J Exp Med
185:111, 1997[Abstract/Free Full Text]
11.
D'Apuzzo M, Rolink A, Loetscher M, Hoxie JA, Lewis IC, Melchers F, Baggiolini M, Moser B:
The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4.
Eur J Immunol
27:1788, 1997[Medline]
[Order article via Infotrieve]
12.
Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S-I, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T:
Defects of B-cell lymphopoiesis and bone marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.
Nature
382:635, 1996[Medline]
[Order article via Infotrieve]
13.
Loetscher M, Geiser T, O'Reilly T, Zwahlen R, Baggiolini M, Moser B:
Cloning of a human seven-transmembrane domain receptor, LESTR, that is higly expressed in leukocytes.
J Biol Chem
269:232, 1994[Abstract/Free Full Text]
14.
Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, Berger EA:
CC CKR-5: A RANTES, MIP-1, MIP-1 receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272:1955, 1996[Abstract]
15.
Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper SC, Schall TJ, Littman DR, Landau NR:
Identification of a major co-receptor for primary isolates of HIV-1.
Nature
381:661, 1996[Medline]
[Order article via Infotrieve]
16.
Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP, Paxton WA:
HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
Nature
381:667, 1996[Medline]
[Order article via Infotrieve]
17.
Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, Parmentier M, Collman RG, Doms RW:
A dual-tropic primary HIV-1 isolate that uses fusin and the -chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.
Cell
85:1149, 1996[Medline]
[Order article via Infotrieve]
18.
Feng Y, Broder CC, Kennedy PE, Berger EA:
HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.
Science
272:872, 1996[Abstract]
19. Wang JF, Zy L, Groopman JE: The alpha-chemokine receptor CXCR4
is expressed on megakaryocytic lineage from progenitor to platelets and
modulates migration and adhesion. Blood 756, 1998
20.
Hamada T, Mohle R, Hesselgesser J, Hoxie J, Nachman RL, Moore MAS, Rafli S:
Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelets formation.
J Exp Med
188:539, 1998[Abstract/Free Full Text]
21.
Louache F, Bettaieb A, Henri A, Oksenhendler E, Farcet J-P, Bierling P, Seligmann M, Vainchenker W:
Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura.
Blood
78:1697, 1991[Abstract/Free Full Text]
22.
Debili N, Wendling N, Katz A, Guichard J, Breton-Gorius J, Hunt P, Vainchenker W:
The Mpl-Ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood
86:2516, 1995[Abstract/Free Full Text]
23.
Debili N, Coulombel L, Croisille L, Katz A, Breton-Gorius J, Vainchenker W:
Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow.
Blood
88:1284, 1996[Abstract/Free Full Text]
24.
Norol F, Vitrat N, Cramer E, Guichard J, Burstein SA, Vainchenker W, Debili N:
Effects of cytokines on platelet production from blood and marrow CD34 positive cells.
Blood
91:830, 1998[Abstract/Free Full Text]
25.
Endres MJ, Clapham PR, Marsh M, Ahuja M, Davis Turner J, McKnight A, Thomas JF, Stoebenau B, Choe S, Vance PJ, Wells TNC, Power CA, Sutterwala SS, Doms RW, Landau NR:
CD4-independent infection by HIV-2 is mediated by fusin/CXCR4.
Cell
87:745, 1996[Medline]
[Order article via Infotrieve]
26.
Mazur EM, Basilico D, Newton JL, Cohen JL, Charland C, Sohl PA, Narandran A:
Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral progenitor cells.
Blood
76:1771, 1990[Abstract/Free Full Text]
27.
Tomer A, Harker LA, Burstein SA:
Flow cytometric analysis of normal human megakaryocytes.
Blood
71:1244, 1988[Abstract/Free Full Text]
28.
Fedderspiel B, Melhado IG, Duncan AMV, Delaney A, Schappert K, Clark-Lewis I, Jirik FR:
Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen.
Genomics
16:707, 1993[Medline]
[Order article via Infotrieve]
29.
Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR:
The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.
Proc Natl Acad Sci USA
94:1925, 1997[Abstract/Free Full Text]
30.
Kitchen SG, Zack JA:
CXCR4 expression during lymphopoiesis: Implications for human immunodeficiency virus type 1 infection of the thymus.
J Virol
71:6928, 1997[Abstract]
31.
Deichmann M, Kronenwett R, Haas R:
Expression of the human immunodeficiency virus type-1 coreceptors CXCR-4 (fusin, LESTR) and CKR-5 in CD34+ hematopoietic progenitor cells.
Blood
89:3522, 1997[Abstract/Free Full Text]
32.
Power CA, Clemetson JM, Clemetson KJ, Wells TNC:
Chemokine and chemokine receptor mRNA expression in human platelets.
Cytokine
7:479, 1995[Medline]
[Order article via Infotrieve]
33.
Gewirtz AM, Zhang J, Ratajczack J, Patajczak M, Park KS, Li C, Yan Z, Poncz M:
Chemokine regulation of human megakaryocytopoiesis.
Blood
86:25598, 1995
34.
Gewirtz AM, Calabretta B, Rucinski B, Niewarowski S, Xu WY:
Inhibition of human megakaryocytopoiesis by platelet factor 4 (PF4) and a synthetic C-terminal PF4 peptide.
J Clin Invest
83:1477, 1989
35.
Han ZC, Sensebe L, Abgrall JF, Briere J:
Platelet factor 4 inhibits human megakacaryocytopoiesis in vitro.
Blood
75:1234, 1990[Abstract/Free Full Text]
36.
Han ZC, Bellucci S, Walz A, Baggiolini M, Caen JP:
Negative regulation of human megakaryocytopoiesis by human platelet factor 4 (PF4) and connective tissue-activating peptide III (CTAP-III).
Int J Cell Cloning
8:253, 1990[Medline]
[Order article via Infotrieve]
37.
Bleul CC, Schultze JL, Springer TA:
B lymphocyte chemotaxis regulated in association with microanatomic localisation, differentiation state, and B cell receptor engagement.
J Exp Med
187:753, 1998[Abstract/Free Full Text]
38.
Radley JM, Scurfield G:
The mechanism of platelet release.
Blood
56:996, 1980[Abstract/Free Full Text]
39.
Tavassoli M, Aoki M:
Migration of entire megakaryocyte through the marrow-barrier.
Br J Hematol
48:25, 1981[Medline]
[Order article via Infotrieve]
40.
Trowbridge EA, Martin JF, Slater DN:
Evidence for a theory of physical fragmentation of megakaryocytes implying that all platelets are produced in the pulmonary circulation.
Thromb Res
28:461, 1982[Medline]
[Order article via Infotrieve]
41.
Basch R, Kouri Y, Karpatkin S:
Expression of CD4 by human megakaryocytes.
Proc Natl Acad Sci USA
87:8085, 1990[Abstract/Free Full Text]
42.
Dolzhanskiy A, Basch RS, Karpatkin S:
Development of human megakaryocytes: I. Hematopoietic progenitors (CD34+ bone marrow cells) are enriched with megakaryocytes expressing CD4.
Blood
87:1353, 1996[Abstract/Free Full Text]
43.
Gewirtz A, Boghosian-Sell L, Catani L, Ratajczak MZ, Shen YM:
Expression of Fc RII and CD4 receptors by normal human megakaryocytes.
Exp Hematol
20:512, 1992[Medline]
[Order article via Infotrieve]
44.
Kouri YH, Borkowsky W, Nardi M, Karpatkin S, Basch R:
Human megakaryocytes have a CD4 molecule capable of binding human immunodeficiency virus-1.
Blood
81:2664, 1993[Abstract/Free Full Text]
45.
Zucker-Franklin D, Cao Y:
Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA.
Proc Natl Acad Sci USA
86:5595, 1989[Abstract/Free Full Text]
46.
Zucker-Franklin D, Seremetis S, Zheng ZY:
Internalization of human immunodeficiency virus type 1 and other retroviruses by megakaryocytes and platelets.
Blood
75:1920, 1990[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
L. Gilles, D. Bluteau, S. Boukour, Y. Chang, Y. Zhang, T. Robert, P. Dessen, N. Debili, O. A. Bernard, W. Vainchenker, et al.
MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9
Blood,
November 5, 2009;
114(19):
4221 - 4232.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Yang, M. Koupenova, D. J. McCrann, K. J. Kopeikina, H. M. Kagan, B. M. Schreiber, and K. Ravid
The A2b adenosine receptor protects against vascular injury
PNAS,
January 15, 2008;
105(2):
792 - 796.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Jin, Y. Tabe, S. Konoplev, Y. Xu, C. E. Leysath, H. Lu, S. Kimura, A. Ohsaka, M.-B. Rios, L. Calvert, et al.
CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells
Mol. Cancer Ther.,
January 1, 2008;
7(1):
48 - 58.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. Dhanjal, C. Pendaries, E. A. Ross, M. K. Larson, M. B. Protty, C. D. Buckley, and S. P. Watson
A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice
Blood,
May 15, 2007;
109(10):
4237 - 4244.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. von Hundelshausen and C. Weber
Platelets as Immune Cells: Bridging Inflammation and Cardiovascular Disease
Circ. Res.,
January 5, 2007;
100(1):
27 - 40.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Berthebaud, C. Riviere, P. Jarrier, A. Foudi, Y. Zhang, D. Compagno, A. Galy, W. Vainchenker, and F. Louache
RGS16 is a negative regulator of SDF-1-CXCR4 signaling in megakaryocytes
Blood,
November 1, 2005;
106(9):
2962 - 2968.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Mondal, C. A. Williams, M. Ali, M. Eilers, and K. C. Agrawal
The HIV-1 Tat Protein Selectively Enhances CXCR4 and Inhibits CCR5 Expression in Megakaryocytic K562 Cells
Experimental Biology and Medicine,
October 1, 2005;
230(9):
631 - 644.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-F. Geay, D. Buet, Y. Zhang, A. Foudi, P. Jarrier, M. Berthebaud, A. G. Turhan, W. Vainchenker, and F. Louache
p210BCR-ABL inhibits SDF-1 Chemotactic Response via Alteration of CXCR4 Signaling and Down-regulation of CXCR4 Expression
Cancer Res.,
April 1, 2005;
65(7):
2676 - 2683.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. S. Li, A. Forslow, and K.-G. Sundqvist
Autocrine Regulation of T Cell Motility by Calreticulin-Thrombospondin-1 Interaction
J. Immunol.,
January 15, 2005;
174(2):
654 - 661.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Schafer, C. Schulz, M. Eigenthaler, D. Fraccarollo, A. Kobsar, M. Gawaz, G. Ertl, U. Walter, and J. Bauersachs
Novel role of the membrane-bound chemokine fractalkine in platelet activation and adhesion
Blood,
January 15, 2004;
103(2):
407 - 412.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. van Buul, C. Voermans, J. van Gelderen, E. C. Anthony, C. E. van der Schoot, and P. L. Hordijk
Leukocyte-Endothelium Interaction Promotes SDF-1-dependent Polarization of CXCR4
J. Biol. Chem.,
August 8, 2003;
278(32):
30302 - 30310.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Youssefian, A. Drouin, J.-M. Masse, J. Guichard, and E. M. Cramer
Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation
Blood,
May 13, 2002;
99(11):
4021 - 4029.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Fruehauf, K. Srbic, R. Seggewiss, J. Topaly, and A. D. Ho
Functional characterization of podia formation in normal and malignant hematopoietic cells
J. Leukoc. Biol.,
March 1, 2002;
71(3):
425 - 432.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Mattia, F. Vulcano, L. Milazzo, A. Barca, G. Macioce, A. Giampaolo, and H. J. Hassan
Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release
Blood,
February 1, 2002;
99(3):
888 - 897.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Hattori, B. Heissig, K. Tashiro, T. Honjo, M. Tateno, J.-H. Shieh, N. R. Hackett, M. S. Quitoriano, R. G. Crystal, S. Rafii, et al.
Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells
Blood,
June 1, 2001;
97(11):
3354 - 3360.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Guerriero, G. Mattia, U. Testa, C. Chelucci, G. Macioce, I. Casella, P. Samoggia, C. Peschle, and H. J. Hassan
Stromal cell-derived factor 1{alpha} increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells
Blood,
May 1, 2001;
97(9):
2587 - 2595.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. R. L. Gear, S. Suttitanamongkol, D. Viisoreanu, R. K. Polanowska-Grabowska, S. Raha, and D. Camerini
Adenosine diphosphate strongly potentiates the ability of the chemokines MDC, TARC, and SDF-1 to stimulate platelet function
Blood,
February 15, 2001;
97(4):
937 - 945.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Majka, A. Janowska-Wieczorek, J. Ratajczak, M. A. Kowalska, G. Vilaire, Z. K. Pan, M. Honczarenko, L. A. Marquez, M. Poncz, and M. Z. Ratajczak
Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis
Blood,
December 15, 2000;
96(13):
4142 - 4151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. J. Lane, S. Dias, K. Hattori, B. Heissig, M. Choy, S. Y. Rabbany, J. Wood, M. A. S. Moore, and S. Rafii
Stromal-derived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases
Blood,
December 15, 2000;
96(13):
4152 - 4159.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Krzysiek, E. A. Lefevre, J. Bernard, A. Foussat, P. Galanaud, F. Louache, and Y. Richard
Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3alpha /CCL20 in human B cells
Blood,
October 1, 2000;
96(7):
2338 - 2345.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Kowalska, M. Z. Ratajczak, M. Majka, J. Jin, S. Kunapuli, L. Brass, and M. Poncz
Stromal cell-derived factor-1 and macrophage-derived chemokine: 2 chemokines that activate platelets
Blood,
July 1, 2000;
96(1):
50 - 57.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Shiraga, A. Ritchie, S. Aidoudi, V. Baron, D. Wilcox, G. White, B. Ybarrondo, G. Murphy, A. Leavitt, and S. Shattil
Primary Megakaryocytes Reveal a Role for Transcription Factor Nf-E2 in Integrin {alpha}iib{beta}3 Signaling
J. Cell Biol.,
December 27, 1999;
147(7):
1419 - 1430.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Chelucci, I. Casella, M. Federico, U. Testa, G. Macioce, E. Pelosi, R. Guerriero, G. Mariani, A. Giampaolo, H.J. Hassan, et al.
Lineage-Specific Expression of Human Immunodeficiency Virus (HIV) Receptor/Coreceptors in Differentiating Hematopoietic Precursors: Correlation With Susceptibility to T- and M-Tropic HIV and Chemokine-Mediated HIV Resistance
Blood,
September 1, 1999;
94(5):
1590 - 1600.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Haddad, E. Cramer, C. Riviere, P. Rameau, F. Louache, J. Guichard, D. L. Nelson, A. Fischer, W. Vainchenker, and N. Debili
The Thrombocytopenia of Wiskott Aldrich Syndrome Is Not Related to a Defect in Proplatelet Formation
Blood,
July 15, 1999;
94(2):
509 - 518.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction