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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3658-3667
Chronic Lymphocytic Leukemia B Cells Express Functional
CXCR4 Chemokine Receptors That Mediate Spontaneous Migration
Beneath Bone Marrow Stromal Cells
By
Jan A. Burger,
Meike Burger, and
Thomas J. Kipps
From the Department of Medicine, the Division of Hematology/Oncology,
University of California, San Diego, La Jolla, CA; and the Department
of Immunology, The Scripps Research Institute, La Jolla, CA.
 |
ABSTRACT |
Chemokines play a central role for lymphocyte trafficking and
homing. The mechanisms that direct the tissue localization of B cells
from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We
found that CLL B cells express functional CXCR4 receptors for the
chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by
receptor endocytosis, calcium mobilization, and actin polymerization
assays. Moreover, CLL B cells displayed chemotaxis to this chemokine
that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4,
pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase
inhibitor. That this chemotaxis may be involved in the homing of CLL
cells is argued by studies in which CLL B cells were cocultured with a
murine marrow stromal cell line that secretes SDF-1. Within 2 hours,
CLL B cells spontaneously migrated beneath such stromal cells in vitro
(pseudoemperipolesis). This migration could be inhibited by
pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1 , or
pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4
on CLL B cells that migrated into the stromal cell layer. These
findings demonstrate that the chemokine receptor CXCR4 on CLL B cells
plays a critical role for heterotypic adherence to marrow stromal cells
and provide a new mechanism to account for the marrow infiltration by
neoplastic B cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
B-CHRONIC LYMPHOCYTIC leukemia (B-CLL)
represents the most common type of adult leukemia in western societies
and is characterized by the relentless accumulation of anergic,
self-reactive, mature CD5+ B cells in the blood, secondary
lymphoid tissues, and the marrow.1 The marrow invariably is
infiltrated with leukemia cells. Furthermore, the extent of marrow
infiltration correlates with clinical stage and
prognosis.2,3 Some studies suggest that CLL B cells can respond to regulatory signals in the marrow microenvironment. In
particular, close contact with marrow stromal cells can provide factors
favorable for the accumulation and survival of CLL B
cells.4,5 At this time, it is not known whether CLL cells
originate or home to the marrow.
The trafficking and homing of normal lymphocytes between the blood and
lymphoid tissues is a multistep process that requires the sequential
engagement of adhesion molecules and the activation through chemokine
receptors.6,7 These steps are thought to be critical for
extravasation and homing to distinct lymphoid-tissue microenvironments
that provide supportive growth and regulatory factors.
Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that is
constitutively expressed at high levels by bone marrow stromal cells.8-10 It exists in 2 forms derived from alternative
RNA splicing, SDF-1 or SDF-1 . The SDF-1 gene was highly conserved
during evolution, and there exists only 1 amino acid difference between
murine and human SDF-1, allowing for the action of this chemokine
across species.8,11 SDF-1 signals through a G
protein-coupled receptor termed CXCR4.12,13 In
lymphocytes, SDF-1 triggers rapid integrin-dependent arrest under
physiological flow conditions, indicating that SDF-1 can induce
lymphocyte recruitment in vivo.14
There is substantial evidence from in vitro and in vivo experi-
ments that SDF-1 plays an important role in B-cell development and
trafficking. Mice lacking the gene encoding SDF-1 or CXCR4 have
severely reduced B lymphopoiesis, but normal T
lymphopoiesis.15-18 In vitro, SDF-1 is chemotactic for pro-
and pre-B cells.19 These results suggest that SDF-1 may
direct progenitor B cells into the appropriate bone marrow
microenvironments, where regulatory factors are released.20
This interpretation is supported by the recent finding that the marrow
of CXCR4-deficient mice contains reduced numbers of pro-B and pre-B
cells, whereas abnormally high numbers are found in the blood due to a
premature release from the marrow.21
CLL B cells can be described as "incompetent" resting B cells.
More than 99% are in the G0 phase of the cell cycle, they
respond poorly to mitogenic signals, and are inefficient
antigen-presenting cells. Therefore, mechanisms that control the
trafficking of normal B cells may not be functional in CLL B cells, and
it has not yet been established that CLL B cells can migrate.
Several studies, however, described the expression of adhesion
molecules and soluble factors related to the trafficking of lymphocytes
on CLL B cells or in the serum of CLL patients and related expression
pattern to disease subsets and prognosis.22-24 Because of
the importance of SDF-1 for B-lymphocyte development and trafficking,
we examined for expression and function of the chemokine receptor CXCR4
on CLL B cells.
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MATERIALS AND METHODS |
Chemokine, antibodies, flow cytometry.
Synthetic, human SDF-1 (1-67) was provided by Dr I. Clark-Lewis
(University of British Columbia, Vancouver, Canada) and purchased from
Upstate Biotechnology (Lake Placid, NY). The following monoclonal antibodies (MoAbs) specific for human surface antigens were used: anti-CXCR4-phycoerythrin (PE) (12G5),
anti-CD3-fluorescein isothiocyanate (FITC),
anti-CD19-allophycocyanine (APC), anti-CD49d,
anti-CD49e, anti-CD54, and the appropriate isotype controls from
Pharmingen (San Diego, CA). The National Institutes of Health (NIH)
AIDS research and reference reagent program (Rockville, MD) provided the nonconjugated CXCR4 MoAb 12G5 and the CCR3-specific MoAb 7B11. For
flow cytometry, the cells were adjusted to a concentration of 5 × 106 cells/mL in RPMI 1640 with 0.5% bovine serum albumin
(BSA). A total of 5 × 105 cells were stained with
saturating antibody concentrations for 30 minutes at 4°C, washed 2 times, and then analyzed on a FACSCalibur (Becton Dickinson, Mountain
View, CA). Flow cytometry data were analyzed by using the FlowJo 2.7.4 software (Tree Star, Inc, San Carlos, CA).
Cell purification, cell lines.
After informed consent, blood samples were obtained from patients
fulfilling diagnostic and immunophenotypic criteria for common B-cell
CLL at the University of California, San Diego (UCSD) Medical Center.
Blood mononuclear cells (PBMC) were isolated via density gradient
centrifugation over Ficoll Paque (Pharmacia, Uppsala, Sweden). Cells
were used fresh or viably frozen in fetal calf serum (FCS) plus 10%
dimethyl sulfoxide (DMSO) for storage in liquid nitrogen. Frozen cells
were cultured overnight at 37°C in 5%CO2/air in
RPMI-1640 supplemented with 10% FCS and
penicillin-streptomycin-glutamine (GIBCO-BRL, Grand Island, NY).
Fluorescence-activated cell sorting (FACS) analysis of the CLL cells
showed an average of 93.1% ± 4.4% (mean ± standard deviation
[SD]) CD19-positive cells, representing the CLL B cells. The T cells
are the second most predominant population in the blood lymphocyte
population of CLL patients. These cells constituted an average of 3.9% ± 2.1% (mean ± SD, n = 16) of the total lymphocytes
of all patient samples examined. The viability of the CLL cells was
always greater than 85%, as determined by staining with propidium
iodine (PI). The human pro-B cell line, Reh, and the pre-B cell line,
Nalm-6, were provided by Dr J. Scheele (Department of Biochemistry and
Chemistry, UCSD). The murine stromal cell line M2-10B4 was purchased
from the American Type Culture Collection (ATCC; Rockville, MD). Cell
lines were cultured at 37°C in RPMI-1640 supplemented with 10% FCS
and penicillin-streptomycin-glutamine (GIBCO-BRL).
CXCR4 reverse transcriptase-polymerase chain reaction (RT-PCR)
analysis.
RNA was isolated from nonpurified (n = 12) or CD19-selected CLL PBMC (n = 3), using the Quiagen RNeasy kit (Quiagen, Santa Clarita, CA). RNA
then was used for first strand cDNA synthesis with the SuperScript
preamplification system (GIBCO-BRL, Rockville, MD), according to the
manufacturer's instructions. The following CXCR4-specific primers were
used: 5' primer: GGA GAA TTC TTA CCA TGG AGG GGA TCA; 3'
primer: GGA GAA TTC AGC TGG AGT GAA AAC TTG. The annealing temperature
was 58°C and the reaction proceeded for 35 cycles. To normalize for
the amount of RNA, we performed RT-PCR for human
glyceraldehyde-3-phosphate dehydrogenase (GA3PD), as
described.25
CXCR4 receptor endocytosis assay.
Receptor downmodulation of CXCR4 by SDF-1 was performed as
described.26,27 Briefly, CLL cells, the pro-B cell line,
Reh, or the pre-B cell line, Nalm-6, each were adjusted to 5 × 106/mL in RPMI-1640 with 0.5% BSA. The cells were cultured
with SDF-1 at various concentrations for 1 hour at
37°C in 5%CO2 in air. Cells were
washed with a 20-fold volume of ice-cold buffer without FCS, stained at
4°C with saturating concentrations of PE-conjugated anti-CXCR4
MoAb, and then analyzed by flow cytometry.
Ca2+ mobilization.
Ca2+ mobilization in response to SDF-1 was performed as
described.28 Briefly, the cells were loaded with Indo-1AM
(Molecular Probes, Eugene, OR), washed 2 times with
Ca2+-free modified Gey's buffer (MGB), suspended in MGB
containing 1.5 mmol/L Ca2+, and then warmed to 37°C for
2 minutes in a stirred cuvette. The emission ratio at 400/480 nm was
followed kinetically after addition of the chemokine on an SLM 8000 fluorometer (Spectronic Instruments, Inc, Rochester, NY). To induce
maximal Ca2+ release, cells were subsequently stimulated
with 2.5 µg/mL ionomycin (Sigma Chemicals Co, St Louis, MO).
Actin polymerization assay.
Actin polymerization was tested as described.8,29 Briefly,
cells (1.25 × 106/mL) were suspended in RPMI-1640
medium with 0.5% BSA at 37°C and incubated with 100 ng/mL SDF-1
for varying amounts of time. To determine actin polymerization in CLL B
cells, CLL cells were prelabeled with anti-CD19 MoAbs. At the indicated
time points, 400 µL of the cell suspension were added to 100 µL of
a solution containing 4 × 10 7 mol/L
FITC-labeled phalloidin, 0.5 mg/mL 1--lysophosphatidylcholine (both
from Sigma), and 18% formaldehyde in phosphate-buffered saline (PBS).
The fixed cells were analyzed by flow cytometry on a FACSCalibur and
all time points are plotted relative to the mean relative fluorescence
of the sample before addition of the chemokine.
Chemotaxis assay.
The chemotaxis assay across bare polycarbonate was preformed as
described.8 Briefly, CLL cells or B-cell lines were
suspended in RPMI-1640 with 0.5% BSA. A total of 100 µL, containing
5 × 105 cells, was added to the top chamber of a
6.5-mm diameter Transwell culture inserts (Costar, Cambridge, MA) with
a pore size of 5 µm. Filters then were transferred to wells
containing medium with or without SDF-1. The chambers were incubated
for 2 hours at 37°C in 5% CO2. After this incubation,
the cells in the lower chamber were suspended and divided into aliquots
for counting with a FACSCalibur for 20 seconds at 60 µL/min in
triplicates or for immunophenotyping.
A 1:20 dilution of input cells was counted under the same conditions.
Antibody inhibition was performed by preincubating the cells with
different concentrations of anti-CXCR4 MoAb 12G5 or anti-CCR3 MoAb 7B11
for 30 minutes at 4°C before use in the chemotaxis assay. For
pertussis toxin treatment, cells were preincubated with 200 ng/mL
pertussis toxin (List Biological Laboratories, Inc, Campbell, CA) at
37°C for 2 hours, washed twice, and subsequently applied to the top
chamber of the chemotaxis assay. For inhibition of phosphatidylinositol
3-kinase (PI-3 kinase), B-CLL cells were incubated with different
concentrations of Wortmannin (Calbiochem, San Diego, CA) at
37°C for 30 minutes and then examined for
chemotaxis in response to 100 ng/mL SDF-1, as described above.
SDF-1 expression by the murine M2-10B4 marrow stromal cell line.
For SDF-1 mRNA detection, RNA was extracted from the M2-10B4 stromal
cell line and used for cDNA synthesis as described above. The sequences
of the murine SDF-1-specific primers we used were: 5' primer:
CCT AAG TCG ACA CGC CAT GGA CGC CAA; 3' primer: CCT ATC TCG AGT
CAC ACC TCT CAC ATC. The conditions of the PCR reaction were the same
as described above. A sequenced plasmid containing the murine SDF-1
cDNA was used as a positive control. Conditioned medium from this cell
line was used to assay for secretion of bioactive SDF-1. For this
purpose, the culture medium was replaced with serum-free medium (X-VIVO
15, Bio Whittaker, Walkersville, MD) when cells had reached 70%
confluency. After 3 days, the conditioned medium was removed and used
for chemotaxis and receptor-endocytosis assays with the pro-B cell
line, Reh, as described above. For blocking of the CXCR4 receptor, Reh
cells were preincubated with 30 µg/mL anti-CXCR4 MoAb before being
applied to the chemotaxis chambers.
In vitro migration assay of CLL cells beneath stromal cells
(Pseudoemperipolesis).
To determine the role of SDF-1 in the interaction of CLL cells with
stromal cells in vitro, we developed an assay that allows us to count
and phenotype the cells that migrate into a stromal cell layer. The
murine stromal cell line M2-10B4 was seeded the day before the assay
onto collagen-coated 24-well plates at a concentration of 1.5 × 105 cell per well in RPMI-1640 supplemented with 10% FCS
and penicillin-streptomycin-glutamine. CLL cells were suspended in
RPMI-1640/10% FCS and added to the stromal cell layer. The plates were
incubated at 37°C in 5%CO2. After incubation for 2 hours or at the indicated time points in the time course experiment,
cells that had not migrated into the stromal cell layer were removed by
vigorously washing the wells with RPMI medium 3 times. The complete
removal of nonmigrated cells and the integrity of the stromal cell
layer containing transmigrated cells was assessed by phase contrast
microscopy and documented photographically. The stromal cell layer
containing transmigrated cells was detached by incubation for 1 minute
with trypsin/EDTA solution prewarmed to 37°C (ATV solution;
GIBCO-BRL). Cells were then immediately suspended by adding 1 mL
ice-cold RPMI/10% FCS, washed, and suspended in 0.5 mL cold medium for
counting by flow cytometry or staining of aliquots for flow cytometry.
A lymphocyte gate was set using the different relative size and
granularity (forward scatter and side scatter) characteristics to
exclude stromal cells from the counts. Duplicate samples were counted at high flow for 20 seconds to determine the relative number of migrated cells. Control samples, in which 1 × 107 CLL
B cells were added to the wells immediately before the washing step, or
samples that only contained stromal cells consistently had counts
<200 events/20 seconds (background). To calculate the percentage of
CLL cells that had migrated into the marrow stromal cells
(MSC) layer, a 1/10 diluted sample of the input cell
suspension was counted under the same conditions. For the analysis of
the phenotype of the transmigrated CLL B cells, cells were costained with anti-human CD19 MoAb along with anti-CXCR4, anti-CD49d,
anti-CD49e, anti-CD54, or anti-CD3 MoAbs. For the antibody inhibition
studies, CLL cells were preincubated with 30 µg/mL anti-CXCR4 MoAb
(12G5) for 30 minutes, washed twice, and applied to the assay. SDF-1 pretreatment was performed by preincubating CLL cells (1 × 107 cells/mL) with synthetic SDF-1 at a concentration of
200 ng/mL for 1 hour at 37°C in 5% CO2, before adding
the SDF-1 containing CLL cell suspension to the assay. Pertussis
toxin pretreatment was performed as described above.
Data analysis, statistics.
Results are shown as mean ± standard deviation (SD) or standard
error about the mean (SEM) of at least 3 experiments each. For
statistical comparison between groups, the Student paired t-test or Bonferroni t-test were used. Analyses were
performed using the Biostatistics software developed by Stanton A. Glantz (UC San Francisco). Flow cytometry data were analyzed using the FlowJo software.
| |
RESULTS |
Expression of CXCR4 mRNA and surface protein in B-CLL.
We detected CXCR4 mRNA in 12 of 12 CLL blood samples tested
(Fig 1A). The amount of CXCR4 mRNA detected
did not vary if the CLL B cells were purified to greater than 98%
purity before RNA extraction. RT-PCR for the GA3PD gene indicated that
the samples contained similar amounts of RNA (data not shown).
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| Fig 1.
(A) RT-PCR analysis for CXCR4 mRNA in CLL samples from 12 different patients. The specific 1,058-bp PCR fragment is visible in 12 representative B-CLL samples (lanes 1 through 12). The arrow points to
the 1,018-bp marker of the 1-Kb control DNA ladder shown in the lanes
flanking the test samples. (B) Logarithmic fluorescence histograms
depicting the expression of CXCR4 on mononuclear blood cells of a
representative patient with CLL (left panel) and Nalm-6 (center) or Reh
cells (right panel). The left panel depicts the logarithmic red
fluorescence of electronically-gated CD3+ T cells (bold
line) or the CD19+ CLL cells (shaded) stained with the
anti-CXCR4-PE MoAb or with a PE-labeled isotype control antibody (thin
lined histogram). The center and right histograms depict the Nalm-6 or
Reh cells, respectively, stained with the anti-CXCR4-PE MoAb (shaded)
or isotype control antibody (open histogram).
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We stained CLL blood cells with anti-CD3-FITC, anti-CXCR4-PE, and
anti-CD19-APC, and examined for B- and T-cell expression of CXCR4 by
flow cytometry (Fig 1B). As expected, the pro-B cell line, Reh, and the
pre-B cell line, Nalm-6, also expressed high levels of CXCR4 (Fig 1B).
We found that CLL B cells from each of 12 patients expressed high
levels of CXCR4. The T cells in the samples expressed lower levels of
CXCR4 than CLL B cells (Fig 2A). The mean
fluorescence intensity ratio of CLL cells from 12 different patients
was 274 ± 68 (mean ± SD, n = 12), whereas it was 122 ± 34 for the T cells of the same patients (Fig 2A).
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| Fig 2.
(A) Leukemic CLL B cells express higher levels of CXCR4
than the T cells from CLL B patients. The average MFIR of CLL B cells
(n = 12; shaded) was significantly higher than the mean CXCR4-MFIR of
T cells (n = 12; hatched). The dots represent CXCR4-MFIR values for
CLL B and T cells from individual patients, with lines connecting the
values from the same patient sample. (B) SDF-1 induces CXCR4
receptor downmodulation on CLL B cells and T cells. Using anti-CXCR4
along with anti-CD19 and anti-CD3 MoAbs, we determined the CXCR4 MFIR
values for CLL B cells (shaded boxes) and T cells from the same CLL
patients (hatched boxes) after preincubation with 1, 10, 100, 1,000, or
2,500 ng/mL SDF-1 or medium alone. The data represent the MFIR
values (±SD) from 3 different patient samples.
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SDF-1 induces dose-dependent CXCR4 receptor endocytosis.
Receptors internalization by endocytosis is characteristic for
chemokine receptors and may allow for continuous sampling of
chemoattractants, permitting the cells to follow a chemotactic gradient. Figure 2B shows the mean fluorescence of B-CLL cells stained
with anti-CXCR4 MoAbs after incubation with varying concentrations of
SDF-1 or medium alone. We found that the amount of SDF-1 required
to induce maximum downmodulation on CLL B cells was equal to 1,000 ng/mL, or 125 nmol/L. This is similar to the
amount required for optimal downmodulation of CXCR4 on T cells of the
same patient (Fig 2B) or Nalm-6 (data not shown). In addition, we
observed a partial CXCR4 downmodulation on CLL B cells (Fig 2B) or
Nalm-6 in response to SDF-1 at concentrations as low as 1 ng/mL.
This was lower than the concentration of SDF-1 that was required to induce detectable CXCR4 downmodulation on T cells of the same CLL
patients and might indicate high sensitivity of CXCR4 on CLL B cells to
SDF-1.
SDF-1 induces calcium mobilization in CLL B cells.
Binding of chemokines to their receptors causes a characteristic
increase in cytosolic calcium. This is one of the earliest biochemical
events that occur in response to chemokines. To examine intracellular
calcium flux, we labeled CLL cells or the B-cell lines, Nalm-6 and Reh,
with indo-1-AM before adding SDF-1 to 100 ng/mL. Evaluation of the
fluorescence of stimulated cells showed that both CLL B cells and
normal B cells mobilize Ca2+ in response to SDF-1.
Compared with the immature B-cell lines, Nalm-6 or Reh
(Fig 3A and B), CLL cells (n = 6) had a
prolonged elevation in intracellular free Ca2+ after
stimulation with SDF-1.
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| Fig 3.
(A) SDF-1 induces mobilization of intracellular
calcium in CLL B cells (bold line). (B) The immature B-cell lines, Reh
(bold line) and Nalm-6 (thin line). Increases of intracellular
Ca2+ were recorded on a fluorometer after addition of 100 ng/mL SDF-1 to cells loaded with Indo-1. Adding ionomycin induced
maximum release of intracellular Ca2+. A representative
experiment of at least 3 is shown. (C) SDF-1 induces actin
polymerization in CLL B cells, which can be inhibited by pertussis
toxin. Intracellular F-actin was measured using FITC-labeled phalloidin
in CD19-prelabeled CLL B cells (boxes) after the addition of 100 ng/mL
SDF-1 at time 0. Results are shown as percent of intracellular
F-actin relative to the value before the addition of SDF-1 and are
the mean and SD of 3 independent experiments. Pertussis toxin inhibits
actin polymerization in CLL B cells (diamonds).
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SDF-1 induces actin polymerization in CLL B cells.
Reorganization of the actin cytoskeleton is an early event in
the migratory response to chemokines.20 To evaluate the
ability of SDF-1 to induce changes in the actin cytoskeleton of CLL
B cells, we examined for changes in filamentous actin (F-actin) of CLL
cells in response to 100 ng/mL SDF-1. We detected a significant, transient increase in F-actin within 15 seconds after exposure of the
cells to the chemokine, followed by a subsequent depolymerization, as
shown in Fig 3C. Actin polymerization after SDF-1 stimulation was
inhibited by preincubation of CLL B cells with pertussis toxin (Fig
3C), indicating that this response is mediated through pertussis toxin-sensitive Gi proteins.
Chemotaxis of CLL cells in response to SDF-1.
We performed a chemotaxis assay in which B-CLL cells were evaluated for
their ability to migrate through 5-µm pores of bare polycarbonate
filters. This assay allowed us to determine the absolute number, as
well as the phenotype of the transmigrated cells.
Figure 4A shows the chemotaxis response of
CLL B cells to various concentrations of synthetic SDF-1, as
determined by immunophenotyping of input and transmigrated cells. The
average proportion of CLL B cells that migrated to chambers with the
optimal concentration of SDF-1 (100 ng/mL) was 16% ± 9% of
input cells (mean ± SD, n = 16 patients). In contrast, the
proportion of input cells that migrated to control chambers without
SDF-1 was 1% ± 2% (Fig 4A). The response to SDF-1 had a
biphasic curve that is characteristic for chemoattractant-induced
chemotaxis (Fig 4C). The pro-B cell line, Reh, and the pre-B cell line,
Nalm-6, also migrated to chambers containing SDF-1, as described
earlier.19 The maximum migration response also was noted to
chambers containing 100 ng/mL SDF-1.
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| Fig 4.
SDF-1 induces chemotaxis in CLL B cells. (A) Blood
lymphocytes from 16 CLL patients were assayed in the bare filter
chemotaxis assay for migration to buffer (control) or different
concentrations of SDF-1. Input and transmigrated cells were stained
with anti-CD19 and anti-CD3 MoAbs to determine the percentages of the
input CLL B cells that migrated into the chambers. The bars represent
the mean values (±SD) for the migration of CLL B cells from 16 different patients. (B) For antibody inhibition, CLL PBMC were
preincubated with different concentrations of MoAbs against the
chemokine receptors CXCR4 (12G5) or a control MoAb directed against the
chemokine receptor CCR3 (7B11) before addition to the chemotaxis assay.
The controls were preincubated in buffer alone. Results indicate the
relative migration compared with control samples migrating to 100 ng/mL
SDF-1 (100%) and represent the mean values ± SD of 2 experiments
with CLL B cells from 4 different patients. The stars indicate
statistically different values, compared with the controls with
P values < .05. (C) SDF-1 attracts CLL B cells by a
pertussis toxin-sensitive mechanism. The migration of CLL B cells is
completely blocked by pretreatment with 200 ng/mL pertussis-toxin (PT,
diamonds). Data represent the mean values ± SEM of CLL B from 16 CLL
B patients for the chemotaxis assays (boxes) and 4 different CLL
samples for pertussis-toxin treatment (diamonds). (D) Migration of
B-CLL cells is partially inhibited by Wortmannin, as selective
inhibitor of PI-3 kinase. CLL cells were pretreated with 10, 100, 500, and 2,500 nmol/L concentrations of Wortmannin and subject to the
chemotaxis assay in the presence of 100 ng/mL SDF-1. The bars
represent the mean values (±SD) for migration of Wortmannin-treated
B-CLL cells (n = 6), relative to the migration without the inhibitor.
The stars indicate statistically different values, compared with the
controls with P values < .05.
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The chemotaxis of CLL cells was significantly inhibited by
preincubation of the input cells with the anti-CXCR4 MoAb 12G5, indicating that a direct interaction between SDF-1 and CXCR4 was necessary for chemotaxis (Fig 4B). In contrast, preincubation with a
control MoAb against the chemokine receptor, CCR3, had no significant effect.
Chemotaxis of CLL B cells to SDF-1 was completely blocked by
pertussis toxin (Fig 4C), indicating that this activity was dependent
on signaling through a Gi protein(s). Furthermore,
pretreatment with Wortmannin, a selective inhibitor of PI-3 kinase,
partially inhibited SDF-1-induced migration of CLL B cells (Fig
4D). The phenotype of the migrated cells indicated that the proportion of CLL B cells and T cells did not differ between samples pretreated with Wortmannin or controls, indicating that Wortmannin inhibited the
migration of both cell types.
M2-10B4 stromal cells express SDF-1 mRNA and secrete bioactive SDF-1.
Using SDF-1-specific PCR primers, we amplified a PCR product of the
expected size (296 bp) from M2-10B4 cDNA and a sequenced plasmid
containing the murine SDF-1 cDNA served as a positive control,
demonstrating SDF-1 expression by the stromal cell line (Fig 5A). Conditioned medium from this cell
line induced chemotaxis of Reh cells in a dose-dependent fashion, and
this migration was blocked by preincubation of the cells with
anti-CXCR4 MoAbs (Fig 5B). Moreover, preincubation with
M2-10B4-conditioned medium induced a dose-dependent downmodulation of
CXCR4 receptors on Reh cells, compared with cells preincubated in
medium alone (data not shown). These observations indicated that the
M2-10B4 line expresses and secretes SDF-1.
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| Fig 5.
(A) RT-PCR analysis for murine SDF-1 mRNA. Using cDNA
from M2-10B4 cells (lane 3) and plasmid DNA encoding murine SDF-1 as
a positive control (lane 2), PCR fragments of the expected size of 296 bp were amplified in both test samples (100-bp marker in lane 1). (B)
Chemotaxis of the Reh B-cell line in response to conditioned medium
(CM) from M2-10B4 cells. Compared with medium (Control), M2-10B4 CM at
different concentrations (100, 50, and 20 vol%) induced chemotaxis of
Reh cells. This migration was inhibited by preincubation of Reh cells
with 30 µg/mL CXCR4 MoAb. Error bars indicate the range of
duplicate samples.
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CLL B cells migrate beneath heterologous MSC (Pseudoemperipolesis).
Coculture of CLL cells with the murine-marrow-stroma cell-line,
M2-10B4, results in spontaneous migration of CLL cells into the stromal
cell layer. This in vitro phenomenon termed pseudoemperipolesis is
characterized by the dark appearance of cells that have migrated into
the same focal plane as the stromal cells, whereas the more superficial, nonmigrated cells remain refractile
(Fig 6).30 Time-course
experiments showed that pseudoemperipolesis of CLL cells mostly
occurred within the first 2 hours of coculture
(Fig 7B). Titration of the
input CLL cell numbers showed that concentrations above 1 × 107 cells per 24 well plate did not significantly increase
the number of migrated cells (Fig 7C). A 2-hour assay with 1 × 107 input cells was found to be the optimal condition for
this assay and hence was used in subsequent inhibition studies. Under
these conditions, an average of 7.4% ± 3.7% (mean ± SD) of
input CLL cells from 6 different patients migrated into the stromal
layer. For comparison, we assessed the migration of Nalm-6 cells and found that 6.9% ± 0.5% (mean ± SE of duplicate tests) of the
input cells migrated into the stromal layer under the same experimental conditions.
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| Fig 6.
(A) Representative phase contrast photomicrograph of
pseudoemperipolesis of CLL B cells after 2-hour culture on the
heterologous murine stromal cell line, M2-10B4. Cells that had not
migrated beneath the stromal cells washed off, and the stromal cell
layer containing the migrated CLL cells was photographed (200x
magnification). Pseudoemperipolesis is characterized by the dark
appearance of lymphocytes that have migrated into the same focal plane
as the stromal cells. (B) For comparison, this photomicrograph shows
reduced pseudoemperipolesis after pretreatment of CLL cells with
pertussis toxin.
|
|
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| Fig 7.
Measurement, time course, and titration of CLL
B-cell pseudoemperipolesis. (A) To measure pseudoemperipolesis, CLL
cells that had migrated into the stromal cell layer were harvested by
treating the washed stromal cell layer with trypsin. The removed cells
were analyzed via flow cytometry. We collected the data on the cells
that had characteristic forward- and side-light scatter characteristics
of lymphocytes, allowing us to exclude the marrow stromal cells from
the analyses (for demonstrative purposes, the stromal cell population
was centered for the acquisition of this sample). (B) The time course
of pseudoemperipolesis of CLL B cells from 3 different patients. A
continuous increase of CLL B cells, as determined by counting and
anti-CD19 staining of cells that had migrated into the stromal cell
layer, was detected over the first 2 hours. (C) Titration of
pseudoemperipolesis of CLL cells using increasing numbers of input CLL
cells. Lymphocytes from 3 different CLL patients that migrated into the
stromal cell layer within 2 hours were counted for 20 seconds at high
flow using a lymphocyte gate. Displayed are the mean (±SD) relative
numbers of duplicate samples.
|
|
Pseudoemperipolesis of CLL B cells is associated with strong CXCR4
downmodulation and increased expression of CD49d.
CLL B cells that had migrated into the stromal cell layer and input CLL
cells were analyzed for surface marker expression by costaining with
FITC-labeled anti-CD19 MoAb and PE-conjugated MoAbs to the epitopes of
interest. First, we examined for CD19 and CD3 expression on the
migrated lymphocytes. Similar to the input CLL cells, the CD19-positive
CLL B cells were the predominant population of transmigrated cells
(82% ± 14.7% of the migrated lymphocytes, n = 6). Comparing the
mean fluorescence intensity ratios (MFIR ± SD), we noted a
significantly lower CXCR4 expression (CXCR4 MFIR, 50 ± 21 v
423 ± 184, n = 4; P < .05) and higher CD49d expression
(CD49d MFIR, 13 ± 2 v 7 ± 2; n = 4, P < .05)
on the transmigrated CLL B cells than on the input CLL B cells
(Fig 8A), whereas no difference was
observed for the expression of CD49e and CD54 (CD49e MFIR, 6 ± 5 v 6 ± 5; n = 4; CD54 MFIR, 24 ± 11 v 25 ± 9; n = 4).
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| Fig 8.
(A) CLL B cells that migrated into the stromal cell layer
have lower CXCR4- and higher CD49d (VLA-4)-surface expression than
input CLL B cells. CLL cells from 3 representative patients that
migrated into the MSC layer were stained with anti-CD19 and anti-CXCR4
or anti-CD49d MoAbs. An aliquot of the input CLL cells before addition
to the assay was stained for comparison. The bold lines represent the
staining for the specific MoAb, while the thin lines show the isotype
control staining of the respective sample. The numbers indicate the
mean fluorescence intensity for CXCR4 or CD49d, respectively. (B)
Pseudoemperipolesis of CLL cells was significantly inhibited by
anti-CXCR4 MoAb, pretreatment with 200 ng/mL SDF-1, and pertussis
toxin, while preincubation with an isotype-matched MoAb to an
irrelevant antigen had no inhibitory effect. The bars represent the
number of CLL B cells that had migrated into the stromal cell layer
after 2 hours (1 × 107 input cells), relative to the
nontreated controls (100%) and are the means (±SD) of 6 different
CLL patients tested in 3 independent experiments. The stars indicate
significant differences with P < .05, using Bonferroni's
t-test.
|
|
Role of SDF-1 in pseudoemperipolesis of CLL B cells.
To establish the role of SDF-1 in the migration of CLL cells into the
stromal layer, we used inhibitors that specifically or nonspecifically
interfered with the interaction of SDF-1 or CXCR4 on CLL cells.
Pertussis toxin was the strongest inhibitor of CLL cell
pseudoemperipolesis: only 14% ± 11% (mean ± SD; n = 6) cells
compared with untreated control samples (100%) had migrated after 2 hours. Significant inhibition was also observed after SDF-1
pretreatment and addition to the coculture (35% ± 19%; n = 6),
and anti-CXCR4 MoAb preincubation of CLL cells (58% ± 16%; n = 6;
Fig 8B).
| |
DISCUSSION |
In this study, we investigated the expression and function of the
chemokine receptor, CXCR4, on B cells from patients with CLL and
characterized its role in heterotypic adherence to marrow stromal
cells. First, we stimulated CLL B cells with synthetic SDF-1 and
detected responses that are characteristic for the activation of
leukocytes by chemoattractants (chemokine receptor endocytosis, calcium
mobilization, actin polymerization). Moreover, CLL B cells migrated in
response to SDF-1, showing a biphasic dose response curve that is
characteristic for chemokine-induced migration. The chemotaxis of CLL B
cells toward SDF-1 was mediated by signaling through the CXCR4
receptor, as demonstrated by our ability to inhibit such migration with
anti-CXCR4 MoAb 12G5. Preincubation with this MoAb significantly
reduced chemotaxis of CLL B cells in response to SDF-1 (72%
inhibition with 30 µg/mL anti-CXCR4 MoAb; Fig 4B). The failure to
achieve 100% inhibition under these conditions has been noted
earlier,31,32 and may reflect the competition of relatively
high concentrations of chemokine (eg, 100 ng/mL) with anti-CXCR4 MoAbs
during a 2-hour assay. Internalization and recycling of CXCR4 receptors
after MoAb binding26 or partial dissociation of this MoAb
at the physiologic temperatures of the chemotaxis assay may play a role
in this context.
We also found that the migration of CLL B cells could be inhibited by
pretreatment of the CLL cells with pertussis toxin or Wortmannin,
indicating that SDF-1 signaling for migration of CLL B cells is
linked to a CXCR4 coupled Gi subunit of a
heterotrimeric guanosine triphosphate (GTP)-binding
protein and activation of PI3-kinase, respectively.
In hematopoietic cells, SDF-1 treatment also can activate p44/42
mitogen-activated protein kinase (Erk1 and Erk2) through CXCR4.33,34 This downstream signaling pathway may play a
role in SDF-1-induced transcriptional activation.34 In
addition, there are reports that the specific p44/42 mitogen activated
protein kinase (MAPK) inhibitor, PD98059, could
inhibit chemotaxis of eosinophils induced by eotaxin35 or
of neutrophils induced by N-formyl peptide, C5a, or
interleukin-8,36 suggesting that the Erk-pathway also may
be involved in signaling for chemotaxis. However, this remains
controversial, as other reports indicate that PD98059 could not inhibit
neutrophil-chemotaxis induced by f-Met-Leu-Phe (fMLP)37 or
interleukin-838 or the platelet-derived growth
factor-induced chemotaxis of fibroblasts.39 Consistent with
such reports are our preliminary studies in which we did not observe
significant inhibition of SDF-1-induced chemotaxis with 50 µmol/L
PD98059 for CLL cells from any 1 of 5 different donors (data not
shown). As such, it appears that the p44/42 MAPK pathway may not be necessary for SDF-1-induced CLL cell migration.
Having established that CLL B cells express functional CXCR4 chemokine
receptors, we went on to investigate the importance of this receptor
for interactions with marrow stromal cells, which are a major source of
the chemokine SDF-1 in vivo. We found that CLL B cells can
spontaneously migrate beneath marrow stromal cells within 2 hours. This
striking in vitro phenomenon termed pseudoemperipolesis was
demonstrated by phase contrast microscopy by the dark appearance of
lymphocytes that migrated into the same focal plane as the stromal
cells. The term pseudoemperipolesis is used to describe symbiotic
complexes of leukemia cells with their stromal cell component in
vitro.30,40 In this context, stromal cells also are
referred to as "nurse-like cells." During this cell interaction, leukemia cells migrate beneath the adherent cells or are trapped by
membrane projections, but do not become internalized.40 It is used to distinguish this type of cell interaction from "true" emperipolesis, the internalization of lymphocytes by sessile cells, first described in situ for thymocytes internalized by thymic stromal
cells (thymic nurse cells).41
Earlier studies on the molecular mechanism of heterotypic adherence
between marrow stromal cells and B cells demonstrated that this is a
biphasic process, characterized by an early phase of adherence ( 15
minutes). This process partly depends on the interaction between very
late antigen-4 (VLA-4; or CD49d) on B lymphocytes with vascular cell
adhesion molecule-1 (VCAM-1; or CD106), expressed on stromal
cells.40,42
The higher expression of CD49d (VLA-4) integrins on migrated CLL B
cells compared with input CLL B cells can either be interpreted as an
enrichment of CLL B cells expressing higher levels of CD49d, allowing
those to enter the stromal cell layer, or could be explained as an
induction of CD49d expression by signals delivered during the migration
process. In either case, this observation suggests that CLL B cells
interact with VCAM-1 or fibronectin on marrow stromal cells.
The late phase of heterotypic adherence ( 30 minutes), associated with
pseudoemperipolesis, does not depend on CD49d/VLA-4 engagement.42 While the early phase of adherence may allow
for the initial homing to the marrow, the late phase, where leukemia cells come into closer contact with stromal cells, may confine malignant B cells within the bone marrow, as suggested by Patrick et
al.42
In this study, we demonstrated that the chemokine SDF-1 plays a
critical role for pseudoemperipolesis, the late phase of heterotypic adherence of CLL B cells to marrow stromal cells according to the
above-mentioned model. The almost complete inhibition of migration into
the stromal cell layer by pertussis toxin demonstrates that signaling
through a Gi protein coupled receptor is required for this
migration. The pretreatment of CLL B cells with SDF-1 and the
addition of this chemokine to the coculture significantly reduced the
pseudoemperipolesis by about 65% compared with nontreated controls.
This condition uses 2 antagonizing mechanisms that can interfere with
SDF-1-induced migration of CLL B cells into the stromal layer. First,
the pretreatment of the CLL cells with this chemokine induces a
downmodulation of the CXCR4 receptor on CLL B cells to levels that are
 10% of nontreated cells, as demonstrated in the receptor
endocytosis assay (Fig 2B). Second, by adding these cells in SDF-1
containing medium to the coculture, the exogenous SDF-1 can interfere
with a SDF-1 gradient established by SDF-1 secreting stromal cells that
otherwise may allow for the directional migration into the stromal cell
layer. Direct evidence for the importance on SDF-1 for this migration
is provided by significantly blocking pseudoemperipolesis with an
anti-CXCR4 MoAb, whereas a control antibody was without any effect (Fig
8B). Finally, the strong downmodulation of CXCR4 on CLL B cells that
had migrated into the stromal cell layer provides further evidence that
SDF-1 contributes to this migration process. From these observations, we propose that heterotypic adhesion between CLL B cells and marrow stromal cells is a multistep process comparable to transendothelial migration, involving the sequential engagement of adhesion molecules and activation through chemokine receptors, in particular CD49d/VLA-4 and CXCR4, respectively.
Earlier studies by Lagneaux et al and others4,43
demonstrated that CLL B cells could adhere to human marrow stromal
cells. They noted that this adhesion was partly mediated through 1
and 2 integrins, and direct contact with stromal cells prevented spontaneous or steroid-induced apoptosis of CLL B cells in
vitro.4,5 In contrast, only very few normal
CD5+ B cells adhered to marrow stromal cells, and those
were not protected from spontaneous apoptosis. It has therefore been
proposed, that the marrow microenvironment, in particular marrow
stromal cells, provide factors that allow for the accumulation of CLL B
cells and make them more resistant to chemotherapy.
Because SDF-1 induces chemotaxis of CLL B cells and is critical for the
spontaneous migration of leukemic CLL B cells beneath marrow stromal
cells, we propose that this chemokine allows for the infiltration of
the marrow by leukemic CLL cells. In addition, SDF-1 may not only
direct, but also confine CLL B cells within the medullary cavity,
similar to the recently demonstrated mechanism of progenitor B-cell
retention in the marrow by SDF-1.21 Attraction through the
CXCR4 chemokine receptor therefore provides a new mechanism to explain
how neoplastic B cells can access limited supportive microenvironmental
niches in the marrow, which usually are restricted to progenitor cells.
Further studies will have to define whether inhibiting the heterotypic
adhesion of B-CLL cells to marrow stromal cells, for example by
interfering with CXCR4-receptor signaling in CLL B cells, could modify
the growth or survival of CLL B cells. Therefore, future studies on the
role of SDF-1 in the interaction of CLL B cells with marrow stromal
cells may lead to new therapeutic strategies for patients with this disease.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs L.Z. Rassenti and K. Kato for providing
B-CLL cDNA samples and to T.A. Johnson for excellent technical
assistance. We thank Dr I. Clark-Lewis for the generous gift of
SDF-1, Dr I.U. Schraufstatter for providing the p44/42 MAPK
inhibitor PD98059, and Dr N.J. Zvaifler for suggestions and the
critical evaluation of the manuscript.
| |
FOOTNOTES |
Submitted June 1, 1999; accepted August 4, 1999.
Supported in part by Grant No. D-96-17136 (to J.A.B.) from the Deutsche
Krebshilfe, Bonn, Germany, Grant No. SA 623/2-1 (to M.B.) from the
Deutsche Forschungsgemeinschaft, Bonn, Germany, and Grant No. 5 R37CA49870-11 from the National Institutes of Health
(to T.J.K.).
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 Thomas J. Kipps, MD, PhD, Department of
Medicine, Division of Hematology/Oncology, University of California San
Diego, School of Medicine, 9500 Gilman Dr, La Jolla, CA 92093-0663;
e-mail: tkipps{at}ucsd.edu.
| |
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TOP
ABSTRACT
INTRODUCTION