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The expression of CXCR4 is down-regulated on the CD34+ cells of patients
with myelofibrosis with myeloid metaplasia
Vittorio Rosti
a
, Margherita Massa
b
, Alessandro M. Vannucchi
c
, Gaetano Bergamaschi
d
,
Rita Campanelli
a
, Alessandro Pecci
e
, Gianluca Viarengo
f
, Valentina Meli
g
, Monia Marchetti
h
,
Paola Guglielmelli
c
, Edward Bruno
i,j
, Mingjiang Xu
i,j
, Ronald Hoffman
i,j
, Giovanni Barosi
h,j,
⁎
on behalf of the Investigators of the Italian Registry of Myelofibrosis with Myeloid Metaplasia and
the Myeloproliferative Disorders Research Consortium
a
Transplant Research Area, IRCCS Policlinico S. Matteo, Pavia, Italy
b
Laboratory of Biotechnology, IRCCS Policlinico S. Matteo, Pavia, Italy
c
Department of Hematology, Azienda Ospedaliera Universitaria Careggi, Florence, Italy
d
Unit of Internal Medicine I, IRCCS Policlinico S. Matteo, Pavia, Italy
e
Unit of Internal Medicine III, IRCCS Policlinico S. Matteo, Pavia, Italy
f
Unit of Clinical Immunology, Immunohematology, and Transfusion Service, IRCCS Policlinico S. Matteo, Pavia, Italy
g
Department of Pediatrics, IRCCS Policlinico S. Matteo, Pavia, Italy
h
Unit of Clinical Epidemiology, IRCCS Policlinico S. Matteo, Pavia, Italy
i
Section of Hematology/Oncology, Department of Medicine, University of Illinois College of Medicine, Chicago, IL, USA
j
MPD Research Consortium, Chicago, IL, USA
Submitted 8 January 2007
Available online 9 March 2007
(Communicated by R. Hoffman, M.D., 8 January 2007)
Abstract
Purpose: We studied the expression of the chemokine receptor CXCR4 on circulating CD34+ cells of patients with myelofibrosis with myeloid
metaplasia (MMM), and examined its relationship to the severity of disease.
Patients and methods: Surface and intracellular CXCR4 expression were measured flow cytometrically in 84 consecutive MMM patients, 16
patients with polycythemia vera (PV), and 20 healthy subjects. In 23 MMM patients, CXCR4 gene expression level was also quantitated by real
time-RT-PCR in CD34+ cells.
Results: The expression of CXCR4 on circulating CD34+ cells was significantly reduced in patients with MMM (P< 0.001) as compared to
normal controls and patients with PV (P= 0.01). The levels of CXCR4 mRNA in CD34+ cells were lower in patients with MMM as compared
with normal subjects, and were directly correlated with the degree of CXCR4 surface expression, demonstrating that transcriptional defects were
the major cause for receptor down-regulation. No statistical association was found between JAK2V617F mutational status and the extent of
CXCR4 down-regulation. CXCR4 expression on CD34+ cells inversely correlated with the number of circulating CD34+ cells (R=−0.55;
P< 0.001), and was severely down-regulated in high risk patients and patients with a high “myelodepletion severity index”. CXCR4 down-
regulation was associated with advanced patient age, the presence of severe anemia, thrombocytopenia, and degree of bone marrow fibrosis.
Conclusions: Reduced expression of CXCR4 by CD34+ cells is a characteristic of MMM which is associated with the constitutive mobilization of
CD34+ cells and occurs in patients with advanced forms of the disease.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Myelofibrosis; CXCR4; SDF-1; Mobilization
Blood Cells, Molecules, and Diseases 38 (2007) 280 –286
www.elsevier.com/locate/ybcmd
⁎Corresponding author. Laboratorio di Epidemiologia Clinica, IRCCS Policlinico S. Matteo, Viale Golgi 19, 27100 Pavia, Italy. Fax: +39 0382 503917.
E-mail address: barosig@smatteo.pv.it (G. Barosi).
1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcmd.2007.01.003
Introduction
Myelofibrosis with myeloid metaplasia (MMM) is a
Philadelphia chromosome-negative chronic myeloproliferative
disorder (Ph-CMPD). MMM is characterized by the con-
stitutive release of hematopoietic progenitor cells (HPCs) and
their accumulation in the blood and extramedullary sites
[1,2]. This biological feature distinguishes MMM from other
Ph-CMPD.
Mobilization of HPCs in humans is regulated by a network
of hematopoietic cytokines, chemokines and adhesion mole-
cules, among which stromal derived factor-1 (SDF-1,
CXCL12) and its receptor CXCR4 play a major role [3–5].
Increased protease activity in the bone marrow microenviron-
ment with SDF-1 degradation and CXCR4 inactivation has
been reported to be an indirect mechanism by which
chemotherapy or G-CSF mobilizes HPCs [6,7]. Recently, Xu
and coworkers provided evidence that plasma levels of
neutrophil elastase (NE) and matrix metalloproteinases were
increased in MMM patients, and that the absolute number of
CD34+ cells in the peripheral blood was correlated with the
levels of soluble VCAM-1 [8]. In addition, Passamonti et al.
documented that granulocytes were activated in patients with
MMM in a manner similar to that induced by G-CSF
administration [9]. These results support the hypothesis that a
G-CSF-like, protease-driven mechanism underlies HPCs
mobilization in MMM.
Down-regulation of CXCR4 and disruption of the CXCR4/
SDF-1 axis might also affect cell mobilization in MMM.
Blocking of CXCR4 by specific antibodies or peptides has
been reported to result in mobilization of tumor cells in
xenograft mouse models [10,11]. Furthermore down-regula-
tion of CXCR4 expression with a CXCR4 antagonist results
in mobilization of polymorphonuclear neutrophils and HPCs
into the peripheral blood of mice [12]. Moreover, transplanta-
tion of CXCR4-deficient HPCs into lethally irradiated
recipient mice, leads to a reconstituted marrow in which the
more mature myeloid elements are noticeably reduced due to
premature release of CXCR4-negative myeloid cells into the
blood stream [13]. The down regulation of CXCR4 expression
by CD34+ cells from patients in blast crisis of chronic
myeloid leukemia (CML), as compared with patients in the
chronic phase or control individuals has also been observed
[14]. More recently, G-CSF mobilization of HPCs has been
shown to be associated with the down-regulation of CXCR4
expression by bone marrow myeloid cells, probably in
association with the accumulation of neutrophil-derived
proteolytic enzymes [15].
The above mentioned results provide a rationale for studying
CXCR4 expression in MMM. These investigations are even
more compelling due to the recent reports that a Val617Phe
mutation in the exon 12 of the JAK2 gene (JAK2V617F) affects
approximately 50% of patients with MMM [16–18]. The
possibility that the JAK2V617F mutation might lead to down
regulation of CXCR4 is not unprecedented. Down regulation of
CXCR4 in CML has been previously linked to the dose of BCR/
ABL in CML [14].
Materials and methods
Patients and study protocol
Eighty-four consecutive patients with a diagnosis of MMM,
referred between July, 2003 and April, 2006 to RIMM (Italian
Registry for Myelofibrosis) were included in this study. Patients
were included if they had not been previously treated with
cytoreductive drugs, thalidomide, interferon or corticosteroids,
or if these treatments had been stopped for at least 3 months. In
63 patients, the diagnosis of MMM was established according
to the Italian consensus conference criteria [19]. Nineteen cases
had been diagnosed with a “prefibrotic form of myelofibrosis”
according to the WHO classification system [20]. The study
protocol was approved by the Institutional Review Board of the
IRCCS Policlinico S. Matteo in Pavia, Italy, and all patients
provided written informed consent.
Patients were assigned a prognostic score based on the
scoring system developed by Dupriez et al. [21], and a
“severity”score, as previously reported [22].
Sixteen patients with PV were also studied for comparison (8
females and 8 males with a median age of 46 years; range, 22 to
70 years). The diagnosis was made according to the WHO
criteria [20]. PV patients were studied at diagnosis, or after
diagnosis but before any drug therapy was started. Control
samples were obtained from 20 healthy individuals.
Flow cytometric enumeration of CD34+ circulating
hematopoietic progenitor cells
One hundred microliters of ethylenediaminetetraacetic acid
(EDTA) anticoagulated blood was stained with fluorescein
isothiocyanate (FITC)-, phycoerythrin (PE)-, and 7-amino-
actinomycin D (7-AAD)-conjugated monoclonal antibodies
(moAb). The CD45-FITC/CD34-PE/7-ADD antibodies were
obtained from Becton Dickinson (San Jose, CA, USA), and the
analyses were performed utilizing a FACScalibur (Becton
Dickinson) [23].
Surface and intracellular flow cytometric analysis
One hundred microliters of EDTA anticoagulated blood was
stained with anti-CD34-FITC and anti-CXCR4-PE (Becton
Dickinson, San Jose, CA, USA). After red cell lysis the samples
were centrifuged and the pellets resuspended in 300 μL of saline.
Cells (2 × 10
5
) were flow cytometrically analyzed using the
CellQuest software (Becton Dickinson, San Jose, CA, USA).
Results were expressed both as percentage of CD34+ cells co-
expressing CXCR4, or as mean fluorescence intensity (MFI).
MFI was calculated as ratio of geometric mean of CD34+/
CXCR4+ cells and geometric mean of CD34+ co-stained with
the appropriate isotype controls.
The cytofluorimetric surface expression of CXCR4 on
CD34+ cells was measured immediately after blood sampling
or no more than 6 h apart in samples maintained at +4 °C.
Since it has been reported that upregulation of CXCR4
expression on circulating CD34+ cells occurs after incubation
281V. Rosti et al. / Blood Cells, Molecules, and Diseases 38 (2007) 280–286
at 37 °C [25], in 5 samples the intra-assay variance of the
CXCR4 expression was assessed by repeating the measure-
ment immediately after sampling, after 1 h, 2 h, 3 h and 6 h.
Maintaining the sample at +4 °C did not alter the signal
intensity (intra assay CV = 1.5%).
For intracellular analysis prior to fixation of CXCR4, red
cells lysis and surface staining with anti-CD34-FITC was
performed. Cells were then fixed with 4% paraformaldehyde for
30 min and permeabilized with 0.1% saponin in 1% FCS/PBS
for 10 min. CXCR4-PE was then added for 30 min in the dark,
and the samples washed twice with ice-cold 0.1% saponin in 1%
FCS/PBS. The percentage of CD34+/CXCR4+ cells was then
determined.
In order to investigate CXCR4 surface expression on mature
hematopoietic cells, flow cytometry studies on separated
granulocytes was performed using a whole blood lysis
technique (NH4Cl) as previously described [24].
Real time RT-PCR quantification of CXCR4 gene expression
level
For the gene expression analysis in CD34+ HPC, mono-
nuclear cells were separated over a Ficoll gradient (Lymphoprep,
Nycomed Pharma, Oslo, Norway), and processed through two
sequential steps utilizing immunomagnetic CD34
+
cell selection
devices (Miltenyi Biotech, Gladbach, Germany). The purity of
the CD34
+
cell preparation was assessed flow cytometrically
(FACScan, Becton Dickinson, San Jose, CA, USA) after
staining with CD34-PE/CD45-FITC antibodies. Aliquots of
CD34
+
cells were immediately lysed using a guanidine
thiocyanate/phenol solution (Trizol, Gibco BRL, Paisley, UK)
and total RNA purified in the presence of carrier glycogen,
according to the manufacturer's instructions. The level of
CXCR4 gene expression was determined by quantitative real-
time PCR using the Pre-Developed TaqMan Assay Reagent
(#Hs00607978_s1) from Applied Biosystems (Foster City, CA,
USA), concurrently with the housekeeping GAPDH gene. Total
RNA (1 μg) was reverse transcribed with random hexamers and
50 ng cDNA was amplified in a ABI PRISM 7300 Sequence
Detection System (Applied Biosystems). The SDS software was
used to analyze fluorescent signals and to calculate the cycle
threshold (C
T
). Quantitative normalization of cDNA in each
sample was obtained by the ΔC
T
method (CXCR4 C
T
−
GAPDH C
T
). Each sample was assayed in triplicate, and both
negative and positive controls were included in each assay.
For the analysis of CXCR4 gene expression levels in
separated granulocytes, cDNA was reverse transcribed and
processed for real-time PCR as described above, except that
RNAse P was used as the reference gene.
Plasma levels of SDF-1α
Plasma SDF-1αlevels were measured using commercially
available high-sensitivity ELISA kits (R&D Systems, Minnea-
polis, MN, USA) in platelet-depleted samples (centrifuged at
11,000×gfor 10 min) stored at −40 °C. The limit of detection of
the assay was 1560 pg/mL.
JAK2 V617F genotyping
DNA was extracted by standard procedures, after isolation of
total peripheral blood granulocytes. Screening for the JAK2
V617F mutation was initially performed by an allele-specific
PCR in which the mutated allele was specifically amplified
together with a fragment common to the mutated and wild type
genes, with a detection limit for the mutated allele of 5% to
10%. Samples positive for the mutation were subsequently
analysed by PCR amplification and digestion with the
restriction endonuclease BsaXI which allows for an estimation
of the ratio between the mutated and wild type alleles. Samples
were scored as homozygous if the proportion of the mutant
allele was greater than 50%.
Statistical methods
Skewed variables were logarithmically transformed before
entering a parametric analysis. Comparisons between groups
were performed by the Student t-test, the Mann–Whitney U-
test or Chi-squared test when appropriate. Associations between
patient characteristics (covariates) were assessed for pairs of
numerical variables by Spearman's correlation, and for
categorical and continuous variables by Wilcoxon–Mann–
Whitney statistics. Results were considered statistically sig-
nificant when Pvalues were less than 0.05. All calculations
were performed with STATISTICA software (Statsoft, Tulsa,
OK, USA).
Results
Patient characteristics
The clinical characteristics of the 84 MMM patients enrolled
in the study are summarized in Table 1. The JAK2 V617F
mutation was identified in 39 of 56 (69.6%) patients, 10
(25.6%) of whom were homozygous for the mutation. Patients
who were homozygous for the JAK2V617F mutation were
characterized by a larger spleen index (mean, 346.8 cm
2
) than
patients heterozygous for the mutation (mean, 202 cm
2
;
P= 0.010), or non-mutat ed (mean, 163 cm
2
;P= 0.007). JAK2
V617F homozygous patients had a higher number of circulating
CD34+ cells than patients who were heterozygous for
JAK2V617F (199.8 × 10
6
/L vs. 31.8× 10
6
/L; P= 0.0015).
CXCR4 expression in CD34+ circulating cells and
granulocytes
The percentage of CD34+ cells expressing CXCR4 was
significantly lower (P< 0.001) in MMM patients (mean, 41.6%,
range, 0.63–93.2%) than in the normal controls (mean, 70.2%;
range, 37–97%) (Fig. 1). The MFI of CXCR4 expression was
also significantly lower in MMM patients (mean, 1.45; range,
0.02–7.05) than in normal controls (mean, 3.50; range, 0.37–
24.1; P= 0.003). The percentage of CXCR4-expressing CD34+
cells and MFI of CXCR4 staining cells were also statistically
lower in MMM patients than in PV patients (mean, 63.5% for
282 V. Rosti et al. / Blood Cells, Molecules, and Diseases 38 (2007) 280–286
CD34/CXCR4 co-expression and 2.21 for MFI; P= 0.013 and
P= 0.02, respectively). In patients with PV, the percentage of
CD34+ cells co-expressing CXCR4 and the MFI of CXCR4
expression was not different from that observed in normal
controls.
CD34+ cells from MMM patients were also characterized by
a lower intracellular CXCR4 MFI than from the normal
unstimulated controls (6.48 vs. 12.4). A direct correlation was
evidenced between surface and intracellular CXCR4 MFI in
MMM patients (R= 0.48; P= 0.004) (Fig. 2).
In 16 patients with MMM, the surface CXCR4 expression
was also assayed on circulating granulocytes. MFI was 1.86
(mean; range 0.59–3.64) in contrast to 3.43 (mean; range 2.19–
4.40) in normal subjects (N= 10; P< 0.001).
Correlation between plasma levels of SDF-1 and CXCR4
expression
The levels of SDF-1 in the plasma of the 84 MMM patients
(4160 pg/mL, range 1560–14875 pg/mL) was significantly
higher than that found in healthy controls (2830 pg/mL, range
1560–5530 pg/mL) (P< 0.005). The levels of circulating SDF-1
did not correlate however with the number of circulating CD34+
cells. Since it is known that high levels of SDF-1 concentration
result in CXCR4 internalization [25], we examined the relation-
ship between the surface CXCR4 expression (both as percentage
of CXCR4-expressing CD34+ cells and MFI) and the levels of
SDF-1 in plasma. No such correlation between plasma SDF-1
levels and the degree of CXCR4 expression by CD34+ cells was
documented.
CXCR4 mRNA levels in cells purified from MMM patients and
controls
In 23 MMM patients the level of CXCR4 mRNA present in
CD34+cellswasmeasuredbyReal-TimeRT-PCRand
compared to that in normal CD34+ cells. The median (±S.D.)
ΔC
T
value for MMM CD34+ cells was significantly higher
than for normal CD34+ cells, thus documenting signifi-
cantly reduced CXCR4 mRNA content (Table 2). CXCR4
mRNA levels were also significantly decreased in MMM
granulocytes (N= 28) compa red to healt hy controls (N= 15)
(Table 2).
In 5 patients with MMM, we documented an inverse
correlation between the CXCR4 surface expression on CD34+
cells and ΔC
T
measurement (R=−0.93), documenting an
association between decreased mRNA level and decreased
protein expression on the surface of the CD34+ cells. In 16
Table 1
Clinical characteristics of the 84 patients with MMM at the time of enrollment into the study
Characteristics All patients
(N=84)
Patients with a fibrotic
stage of MMM (N= 63)
Patients with a prefibrotic
form of MMM (N= 21)
Age, years, median (range) 51 (20–84) 57 (20–84) 45 (24–74)
Sex, male, no. of patients (%) 42 (50%) 37 (58.7%) 5 (23.8%)
Hemoglobin, g/L, median (range) 113 (59–167) 110 (59–167) 117 (86–165)
White blood cell count, × 10
9
/L, median (range) 8.5 (2.3–59.7) 7.6 (2.3–59.7) 8.6 (4.4–17.8)
Circulating immature myeloid cells + blasts, %,
median (range)
4.5 (0–21) 5 (0–21) 4 (1–10)
Circulating erythroblasts, %, median (range) 1.0 (0–120) 1.0 (0–120) 1.0 (0–2)
Platelet count, ×10
9
/L, median (range) 275 (10–1331) 257 (10–1268) 507 (126–1331)
Spleen volume index, cm
2
, median (range)
a
190 (80–672) 229 (90–672) 120 (80–600)
Time from diagnosis to examination, months,
median (range)
26 (1–311) 22.5 (1–205) 41 (2–311)
Dupriez prognostic score at the time of the study,
number of patients (%)
b
0 (low risk) 52 (61.9%) 34 (54.0%) 18 (85.7%)
1 (intermediate risk) 23 (27.4%) 20 (31.7%) 3 (14.3%)
2 (high risk) 9 (10.7%) 9 (14.3%) 0 (0%)
Severity score, number of patients (%)
c
0–3 52 (61.9%) 34 (54.0%) 18 (85.7%)
4–5 32 (38.1%) 29 (46.0%) 3 (14.3%)
a
Spleen size was measured by ultrasonography by recording the spleen index, i.e. the product of the longitudinal by the transverse spleen axis.
b
A Dupriez score of 0 was assigned to a hemoglobin concentration greater than 100 g/L and a white blood cell count between 4×10
9
/L and 30 × 10
9
/L, a score of 1
to either a hemoglobin concentration less than 100 g/L or a white blood cell count greater than 30× 10
9
/L or less than 4 × 10
9
/L, and a score of 2 was given if both the
hemoglobin concentration and white blood cell count were in the aberrant ranges.
c
The severity score is the sum of myeloproliferation index and myelodepletion index derived from the values of hemoglobin, white blood cell count, platelet count
and spleen volume. Myeloproliferation index scored leukocytosis (corrected for the circulating erythroblasts), thrombocytosis and splenomegaly (or hepatomegaly in
splenectomized patients), while myelodepletion index scored anemia, leukopenia and thrombocytopenia. The following parameters were scored according to 3 grades:
splenomegaly (0=non-palpable or removed, 1 = 10 cm or less below the costal margin, 2=more than 10 cm below the costal margin) and anemia (0 = hemoglobin
concentration greater or equal to 120 g/L; 1 = hemoglobin concentration from 100 to 120 g/L; and 2 =hemoglobin concentration lower than 100 g/L or transfusion-
dependency). Two grades were considered for leukocytosis (0 = leukocyte count lower than 15 × 10
9
/L; 1= leukocyte count higher or equal to 15 × 10
9
/L),
thrombocytosis (0 = platelet count lower than 500 × 10
9
/L; 1 = platelet count higher or equal to 500× 10
9
/L), thrombocytopenia (0 = platelet count higher than
150 × 10
9
/L; 1 = platelet count equal or lower than 150× 10
9
/L) and leukopenia (0 = leukocyte count higher or equal to 4 × 10
9
/L; 1 = leukocyte count equal or lower
than 4× 10
9
/L). Myelodepletion and myeloproliferation indexes ranged from 0 to 4, while the overall severity score ranged from 0 to 6.
283V. Rosti et al. / Blood Cells, Molecules, and Diseases 38 (2007) 280–286
patients with MMM a similar relationship was observed in
peripheral blood granulocytes (R=−0.62; P= 0.01).
Correlation between CXCR4 expression and disease
characteristics
An inverse correlation was observed between the expres-
sion of CXCR4 by CD34+ cells and the number of circulating
CD34+ cells present in the peripheral blood (R=−0.55;
P< 0.001 for percentage of CXCR4 expressing CD34+ cells,
and R=−0.35, P= 0.001 for MFI) (Fig. 3).
The decreased intensity of CXCR4 expression by MMM
CD34+ cells, as measured by MFI, was associated with
lower Hb levels (R= 0.384; P= 0.001), older age (R=−0.31;
P= 0.026), longer disease duration (R=−0.37; P= 0.008), and
lower platelet count (R= 0.42; P= 0.007). Patients with a
“prefibrotic form of myelofibrosis”had higher levels of
CXCR4 expression at the time of the study than those with
fibrotic bone marrows (mean, 56.4% vs. 36.5%, P= 0.001 for
percentage of CXCR4-expressing CD34+ cells; 3.05 vs. 0.98,
P= 0.011 for MFI). Both the patient's Dupriez prognostic
score and “severity”score were correlated with surface CXCR4
down-regulation (CXCR4-expressing CD34+ cell, percentage,
F= 4.04; P= 0.021 for Dupriez score; F= 2.208, P= 0.05 for
“severity score”). The “myelodepletion index”, which includes
leukopenia, thrombocytopenia and anemia, contributed to a
greater extent to the correlation between the “severity score”
and the degree of CXCR4 down-regulation (F= 2.78; P= 0.03)
(Fig. 4).
Correlation between CXCR4 expression and JAK2 V617F
mutational state
The expression of CXCR4 on CD34+ cells did not differ in
MMM patients heterozygous for JAK2V617F mutation as
compared to those with a wild-type genotype. Although a
Fig. 1. CXCR4 expression on circulating CD34+ cells from patients with MMM,
PVor control subjects. Patients with MMM include idiopathic myelofibrosis and
post-ET and post-PV myelofibrosis. CXCR4 expression levels (percent of
CXCR4-expressing CD34+ cells) are shown as a box plot where dots indicate the
mean value, bars indicate the standard deviation, while the lower and upper edges
of the boxes indicate the standard error. The Student t-test showed significant
differences between MMM patients and PV patients or normal subjects
(P<0.001).
Fig. 2. Relationship between circulating CD34+ intracellular CXCR4 mean
flurescence intensity (MFI) and CD34+ cells surface CXCR4 MFI. Linear
regression analysis demonstrated a significant correlation (R= 0.48; P= 0.004).
Table 2
Measurement of CXCR4 gene expression levels in MMM and normal cell
populations
ΔC
T
(mean ± S.D.) Range P
Cell source
MMM CD34+ cells (N= 23) 5.12 ± 2.42 1.15–9.77 0.003
Normal CD34+ cells (N= 7) 1.53 ± 0.79 0.31–2.68
MMM Granulocytes (N= 28) 2.36 ± 0.93 1.04–5.09 0.014
Normal Granulocytes (N= 15) 1.60 ± 0.91 0.3–2.77
CD34+ cells and granulocytes were purified from the peripheral blood (PB) of
MMM patients, or from peripheral blood of normal volunteer donors. CXCR4
mRNA levels were quantified by a real time RT-PCR assay, and expressed as
ΔC
T
using GAPDH or RNAseP (in case of granulocytes) as the housekeeping
reference gene.
Fig. 3. Relationship between the expression of CXCR4 on the surface of CD34+
cells of patients with MMM and the number of CD34+ hematopoietic progenitor
cells (HPCs) in peripheral blood. The CXCR4 expression was measured as
CXCR4-expressing CD34+ cells (percentage). Linear regression analysis
demonstrated a significant correlation (R=−0.55; P< 0.001).
284 V. Rosti et al. / Blood Cells, Molecules, and Diseases 38 (2007) 280–286
reduction of CXCR4 expression on CD34+ cells was evident in
JAK2V617F homozygous patients as compared to heterozy-
gous and wild-type patients (mean, CXCR4-expressing CD34+
cells, 29.6% vs. 41.6%; MFI, 0.56 vs. 1.68, respectively), these
differences did not, however, reach statistical significance.
Discussion
MMM has been shown to be characterized by an unusual
degree of mobilization of HPCs, stem cells as well as
endothelial cells into the peripheral blood [2,26]. The mechan-
ism underlying the constitutive mobilization of CD34+ cells in
MMM patients remains poorly understood. In this study, we
analysed the hypothesis that down-regulation of the chemokine
receptor CXCR4 might contribute to this process. SDF-1 is the
natural ligand for CXCR4. The CXCR4/SDF-1 axis has been
documented to play a role in the homing and retention of
primitive HPCs and stem cells within the bone marrow [27].
Disruption of this ligand/receptor interaction has been impli-
cated in cytokine mediated mobilization of CD34+ cells [28].In
CML cross talk between BCR/ABL and the CXCR4 pathway
has been shown to result in the disruption of chemokine
signalling and chemotaxis [29–32]. Geay et al. have recently
reported that cells expressing high levels of BCR/ABL are
characterized by a significant signalling defect accompanied by
a reduction of CXCR4 expression at the transcriptional level
[14]. Furthermore the tyrosine kinase activity of BCR/ABL was
clearly shown to down regulate the expression and function of
CXCR4. We found that circulating CD34+ cells of MMM
patients, on average, displayed lower levels of CXCR4 than
CD34+ cells from patients with PV or normal individuals. We
documented that the CXCR4 mRNA levels in CD34+ cells and
granulocytes from MMM patients were lower as compared to
controls. Moreover, by correlating CXCR4 gene and receptor
expression, we showed a strict correlation between reduced
mRNA content and cell surface protein expression, demonstrat-
ing that the CXCR4 down-regulation in CD34+ cells and
granulocytes of MMM is the consequence of a transcriptional
event.
The down-regulation of CXCR4 on CD34+ cells was asso-
ciated with increasing numbers of circulating CD34+ cells,
suggesting that the down regulation of CXCR4 by CD34+ cells
may play a role in the constitutive HPC mobilization. Although
CXCR4 down-regulation on CD34+ cells is able to directly
influence cell mobilization via the disruption of the SDF-1 axis
in bone marrow, the constitutive mobilization of CD34+ cells in
MMM is more likely the result of multiple events affecting stem
cell trafficking.
The down-regulation of CXCR4 by CD34+ cells and
granulocytes were shown in this report to be due to reduced
gene expression. Both genetic and epigenetic mechanisms
have been documented to affect transcriptional regulation of
the CXCR4 gene [33,34]. We present evidence here to indicate
that the decreased expression of CXCR4 in MMM occurs
irrespective of the JAK2V617F mutational status of patients
with MMM, suggesting that other intracellular signals are
likely responsible for this event. Further studies are clearly
required to gain greater understanding of the precise molecular
mechanism responsible for this transcriptional silencing of
CXCR4.
In our series of cases, down-regulation of CXCR4 in MMM
peripheral blood CD34+ cells was associated with disease
characteristics that reflected the severity of the disease process,
including older age [35], overt bone marrow fibrosis, anemia,
leukopenia and thrombocytopenia. Since CXCR4 participates
in the regulation of bone marrow myelopoiesis, and in the long
term bone marrow repopulation by pluripotent stem cells [36–
38], the down-modulation of CXCR4 receptor in MMM may
represent a crucial event in the progression of the disease that
may contribute to the aberrant trafficking of HPCs and the
depletion of bone marrow stem cells [39], leading to the
development of a bone marrow failure state that characterizes
the more advanced stages of the disease.
Acknowledgments
Research Grant Support: This work was supported by a grant
(CS30, 2003) from Istituto Superiore di Sanità; a grant from the
Italian Ministry of Health (Ricerca Finalizzata 2002), a grant
from MIUR (2003, #06488803); a grant from Associazione
Italiana per la Ricerca sul Cancro (Milano) and the MPD
Research Consortium funded by the National Cancer Institute
(CA P01 CA 108671-01A2).
References
[1] G. Barosi, R. Hoffman, Idiopathic myelofibrosis, Semin. Hematol. 42
(2005) 248–258.
[2] G. Barosi, G. Viarengo, A. Pecci, et al., Diagnostic and clinical relevance
of the number of circulating CD34+ cells in myelofibrosis with myeloid
metaplasia, Blood 98 (2001) 3249–3255.
[3] A. Peled, I. Petit, O. Kollet, et al., Dependence of human stem cell
Fig. 4. CXCR4 expression on the surface of CD34+ cells of MMM patients
categorized according their “myelodepletion index”. CXCR4 expression levels
(percent of CXCR4-expressing CD34+ cells) are shown as a box plot where dots
indicate the mean value, bars indicate the standard deviation, while the lower
and upper edges of the boxes indicate the standard error. Myelodepletion index,
which scores leukopenia, thrombocytopenia and anemia was correlated with
surface CXCR4-down regulation (F=2.78; P=0.03).
285V. Rosti et al. / Blood Cells, Molecules, and Diseases 38 (2007) 280–286
engraftment and repopulation of NOD/SCID mice on CXCR4, Science
283 (1999) 845–848.
[4] T. Lapidot, I. Petit, Current understanding of stem cell mobilization: the
roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines,
and stromal cells, Exp. Hematol. 30 (2000) 973–981.
[5] M. Kucia, R. Reca, K. Miekus, et al., Trafficking of normal stem cells and
metastasis of cancer stem cells involve similar mechanisms: pivotal role of
the SDF-1-CXCR4 axis, Stem Cells 23 (2005) 879–894.
[6] L.M. Pelus, H. Bian, A.G. King, S. Fukuda, Neutrophil-derived MMP-9
mediates synergistic mobilization of hematopoietic stem and progenitor
cells by the combination of G-CSF and the chemokines GRObeta/CXCL2
and GRObetaT/CXCL2delta4, Blood 103 (2004) 110–119.
[7] J.P. Levesque, J. Hendy, Y. Takamatsu, et al., Mobilization by either
cyclophosphamide or granulocyte colony-stimulating factor transforms the
bone marrow into a highly proteolytic environment, Exp. Hematol. 30
(2002) 440–449.
[8] M. Xu, E. Bruno, J. Chao, et al., Constitutive mobilization of CD34+ cells
into the peripheral blood in idiopathic myelofibrosis may be due to the
action of a number of proteases, Blood 105 (2005) 4508–4515.
[9] F. Passamonti, E. Rumi, D. Pietra, et al., Relation between JAK2 (V617F)
mutation status, granulocyte activation, and constitutive mobilization of
CD34+ cells into peripheral blood in myeloproliferative disorders, Blood
107 (2006) 3676–3682.
[10] F. Bertolini, C. Dell'Agnola, P. Mancuso, et al., CXCR4 neutralization, a
novel therapeutic approach for non-Hodgkin's lymphoma, Cancer Res. 62
(2002) 3106–3112.
[11] A. Larochelle, A. Krouse, M. Metzger, et al., AMD3100 mobilizes
hematopoietic stem cells with long-term repopulating capacity in nonhu-
man primates, Blood 107 (2006) 3772–3778.
[12] L.M. Pelus, H. Bian, S. Fukuda, et al., The CXCR4 agonist peptide,
CTCE-0021, rapidly mobilizes polymorphonuclear neutrophils and
hematopoietic progenitor cells into peripheral blood and synergizes with
granulocyte colony-stimulating factor, Exp. Hematol. 33 (2005) 295–307.
[13] Q. Ma, D. Jones, P.R. Bonghesan, et al., Impaired B-lymphopoiesis,
myelopoiesis, and derailed cerebellar neuron migration in CXCR4-
and SDF-1 deficient mice, Proc. Natl. Acad. Sci. U. S. A. 95 (1998)
9448–9453.
[14] J.F. Geay, D. Buet, Y. Zhang, et al., p210BCR-ABL inhibits SDF-1
chemotactic response via alteration of CXCR4 signaling and down-
regulation of CXCR4 expression, Cancer Res. 65 (2005) 2676–2683.
[15] H.K. Kim, M.D. Sierra, C.K. Williams, et al., G. G-CSF downregulation of
CXCR4 expression identified as a mechanism for mobilization of myeloid
cells, Blood prepublished online March 14, 2006.
[16] R.A. Mesa, H. Powell, T. Lasho, et al., A longitudinal study of the JAK2
(V617F) mutation in myelofibrosis with myeloid metaplasia: analysis at
two time points, Haematologica/THJ 91 (2006) 415–416.
[17] P.J. Campbell, M. Griesshammer, K. Dohner, et al., V617F mutation in
JAK2 is associated with poorer survival in idiopathic myelofibrosis, Blood
107 (2006) 2098–2100.
[18] A. Tefferi, T.L. Lasho, S.M. Schwager, et al., The JAK2(V617F) tyrosine
kinase mutation in myelofibrosis with myeloid metaplasia: lineage
specificity and clinical correlates, Br. J. Haematol. 131 (2005) 320–328.
[19] G. Barosi, A. Ambrosetti, C. Finelli, et al., The Italian Consensus
Conference on diagnostic criteria for myelofibrosis with myeloid
metaplasia, Br. J. Haematol. 104 (1999) 730–737.
[20] J. Thiele, R. Pierre, M. Imbert, et al., in: E.S. Jaffe, N. Harris, N. Stan, J.W.
Vardiman (Eds.), Chronic Idiopathic Myelofibrosis in World Health
Organization of Tumors of Hematopoietic and Lymphoid Tissues, IARC
Press, Washington, DC, 2001, pp. 35–38.
[21] B. Dupriez, P. Morel, J.L. Demory, et al., Prognostic factors in agnogenic
myeloid metaplasia: a report on 195 cases with a new scoring system,
Blood 88 (1996) 1013–1018.
[22] M. Marchetti, G. Barosi, F. Balestri, et al., Low-dose thalidomide
ameliorates cytopenias and splenomegaly in myelofibrosis with myeloid
metaplasia: a phase II trial, J. Clin. Oncol. 22 (2004) 424–431.
[23] M. Keeney, I. Chin-Yee, K. Weir, et al., Single platform flow cytometric
absolute CD34+ cell counts based on the ISHAGE guidelines. Interna-
tional Society of Hematotherapy and Graft Engineering, Cytometry 34
(1998) 61–70.
[24] G.T. Stelzer, G. Marti, A. Hurlly, et al., U.S.–Canadian Consensus
recommendations on the immunophenotypic analysis of hematologic
neoplasia by flow cytometry: standardization and validation of laboratory
procedures, Cytometry 30 (1997) 214–230.
[25] J.-J. Lataillade, D. Clay, C. Dupuy, et al., Chemokine SDF-1 enhances
circulating CD34+ cells proliferation in synergy with cytokines: possible
role in progenitor survival, Blood 95 (2000) 756–768.
[26] M. Massa, V. Rosti, I. Ramajoli, R. Campanelli, A. Pecci, G. Viarengo, V.
Meli, M. Marchetti, R. Hoffman, G. Barosi, Circulating CD34+, CD133+,
and vascular endothelial growth factor receptor 2-positive endothelial
progenitor cells in myelofibrosis with myeloid metaplasia, J. Clin. Oncol.
23 (2005) 5688–5695.
[27] A. Aiuti, I.J. Webb, C. Bleul, T. Springer, J.C. Gutierrez-Ramos, 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 (1997)
111–120.
[28] T. Lapidot, I. Petit, Current understanding of stem cell mobilization: the
roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines,
and stromal cells, Exp. Hematol. 30 (2002) 973–981.
[29] R. Salgia, E. Quackenbush, J. Lin, et al., The BCR/ABL oncogene alters
the chemotactic response to stromal-derived factor-1alpha, Blood 94
(1999) 4233–4246.
[30] A. Ptasznik, E. Urbanowska, S. Chinta, et al., Crosstalk between BCR/
ABL oncoprotein and CXCR4 signaling through a Src family kinase in
human leukemia cells, J. Exp. Med. 196 (2002) 667–678.
[31] A. Peled, I. Hardan, L. Trakhtenbrot, et al., Immature leukemic
CD34+CXCR4+ cells from CML patients have lower integrin-depen-
dent migration and adhesion in response to the chemokine SDF-1,
Stem Cells 20 (2002) 259–266.
[32] J. Durig, C. Rosenthal, A. Elmaagacli, et al., Biological effects of stroma-
derived factor-1 alpha on normal and CML CD34+ haemopoietic cells,
Leukemia 14 (2000) 1652–1660.
[33] T. Mori, J. Kim, T. Yamano, et al., Epigenetic up-regulation of C–C
chemokine receptor 7 and C–X–C chemokine receptor 4 expression in
melanoma cells, Cancer Res. 65 (2005) 1800–1807.
[34] N. Sato, H. Matsubayashi, N. Fukushima, et al., The chemokine receptor
CXCR4 is regulated by DNA methylation in pancreatic cancer, Biol. Ther.
4 (2005) 70–76.
[35] K. Strasser-Weippl, M. Steurer, M. Kees, et al., Age and hemoglobin level
emerge as most important clinical prognostic parameters in patients with
osteomyelofibrosis: introduction of a simplified prognostic score, Leuk.
Lymphoma 47 (2006) 441–450.
[36] J. Kahn, T. Byk, L. Jansson-Sjostrand, et al., Overexpression of CXCR4 on
human CD34+ progenitors increases their proliferation, migration, and
NOD/SCID repopulation, Blood 103 (2004) 2942–2949.
[37] T. Nagasawa, S. Hirota, K. Tachibana, et al., Defects of B-cell
lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC
chemokine PBSF/SDF-1, Nature 382 (1996) 638–653.
[38] Q. Ma, D. Jones, T.A. Springer, The chemokine receptor CXCR4 is
required for the retention of B lineage and granulocytic precursors within
the bone marrow microenvironment, Immunity 10 (1999) 463–471.
[39] H. Ni, G. Barosi, D. Rondelli, R. Hoffman, Studies of the site and
distribution of CD34+ cells in idiopathic myelofibrosis, Am. J. Clin.
Pathol. 123 (2005) 833–839.
286 V. Rosti et al. / Blood Cells, Molecules, and Diseases 38 (2007) 280–286
