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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2387-2396
Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are
Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells
By
L. Lagneaux,
A. Delforge,
D. Bron,
C. De Bruyn, and
P. Stryckmans
From the Service de Médecine Interne et Laboratoire
d'Investigation Clinique Henri Tagnon, Institut J. Bordet, Brussels,
Belgium.
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ABSTRACT |
The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL)
patients have a long survival in vivo, although ex vivo they rapidly
die by apoptosis. To further investigate the mechanism of this, we have
studied the influence of bone marrow stromal cells from normal subjects
on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B
lymphocytes. After 48 hours of incubation in medium alone, leukemic and
normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the
percentage of leukemic cells undergoing apoptosis (8 ± 2%, P < .0005) and prevented the loss of bcl-2 protein expression. In
contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was
found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were
obtained with stromal cell conditioned medium. The difference in
behavior observed between normal and leukemic B cells plated on stromal
cells can be explained by the fact that only a few normal B cells
adhere to stromal cells in comparison with B-CLL cells. B-CLL cell
adhesion to stromal cells is mediated by  1 and 2
integrins acting simultaneously. Contact between B-CLL cells and bone
marrow stromal cells seems to play a major role in the accumulation and
survival of B-CLL cells in the bone marrow.
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INTRODUCTION |
B-CHRONIC LYMPHOCYTIC leukemia (B-CLL) is
a malignant disease characterized by the accumulation of mature
monoclonal CD5+ B cells.1 The majority of
circulating cells appear to be nondividing, and it has been suggested
that a prolonged life span could be responsible for the accumulation of
the malignant B cells in vivo.2
The longevity of B cells is controlled by programmed cell death or
apoptosis, an active process involving activation of an internally
coded suicidal program and characterized by specific morphologic and
biochemical changes leading to endonucleolytic degradation of the DNA
at nucleosomal intervals.3,4 Apoptosis can be influenced by
various factors that may be the appearance or disappearance of a
stimulus, such as the withdrawal of growth factors for growth
factor-dependant cells.5
Despite an apparent long half-life in vivo, B-CLL cells die in vitro
during short-term culture in media supplemented by either autologous or
fetal bovine serum.6,7 This fact suggest that the prolonged
survival of B-CLL cells may be attributed to humoral or cellular
factors capable of protecting these cells from apoptotic cell death. A
variety of cytokines, such as interleukin-4 (IL-4), interferon  (IFN-), IFN- , and IL-10, have been shown to be involved with the
process of apoptosis in B-CLL cells, many exerting their effects by way
of bcl-2 expression.8-11 In addition to cytokines, cell-cell and cell-matrix interactions have been shown to be critical in preventing apoptosis of malignant lymphocytes.12
Recently, Long et al13 reported that apoptosis of B-CLL
cells can be prevented by contact with endothelial cell hybrids by way
of integrins expressed on the malignant B lymphocytes. Given that
B-cell proliferation and death involve interactions with stromal cells
and their products,14,15 we have evaluated whether
interactions with bone marrow (BM) stromal cells could play a major
role in maintaining the malignant clone in this disease.
Examination of the factors and mechanisms involved in the process of
apoptosis could be of value in the development of new therapeutic
strategies for B-CLL.
This study provides evidence that BM stromal cells produce soluble
factors inducing apoptosis of B-CLL cells, but that adhesion of these
B-CLL cells to stromal cells rescues them from apoptosis and extends
their life span in vitro.
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MATERIAL AND METHODS |
Patients.
Twenty B-CLL patients, 6 women and 14 men, with a median age of 61 (47 to 84) years were included in this study. Diagnosis was based on
clinical examination, peripheral blood count, and immunophenotyping
(Table 1). These patients were either
untreated or had not received treatment for the previous 6 months.
Purification of CLL B-lymphocytes.
Heparinized venous blood from all patients was centrifugated over
Ficoll-Hypaque (Lymphocyte Separation Medium, International Medical
Products, Brussels, Belgium), and mononuclear cells were depleted from
monocytes and T cells as previously described.16 After
purification, the samples were stained with selected monoclonal antibodies and contained greater than 98% of CD19+ cells
of which 67 ± 6% coexpressed CD5.
Purification of normal B lymphocytes.
CD19+ cells were purified from umbilical cord blood (UCB)
on Ceprate LC CD19 affinity columns (CellPro, Bothell, WA) according to
the manufacturer's instructions. Mononuclear cells were incubated 25 minutes at 4°C with a biotinylated mouse monoclonal Ig antihuman CD19. The cells were then washed and loaded onto an
avidin-immunoaffinity column. Nonadsorbed cells were removed by washing
the column with phosphate-buffered saline (PBS), and adsorbed cells
were gathered by squeezing and eluting off the column. Cell numbers
before and after separation were measured using Trypan blue staining.
The ratio of CD19+ cells in the mononuclear cell population
obtained from 11 UCB samples varied from sample to sample (range 4% to
52%, mean 23 ± 5%), and 14 ± 5% of CD19+
coexpressed the CD5 antigen. The Ceprate LC separation of UCB cells
resulted in a population containing 89 ± 2% CD19+
cells of which 57 ± 6% coexpressed CD5.
Phenotypic analysis.
Peripheral blood mononuclear cells or purified B lymphocytes were
resuspended in PBS and washed and stained by direct immunofluorescence. The cells were incubated at room temperature for 30 minutes with specific monoclonal antibodies.
Double-labeling fluorescence staining was performed using fluorescein
isothiocyanate (FITC)-conjugated CD5 and phycoerythrin (PE)-conjugated CD19 antibodies (Dakopatts, Glostrup, Denmark). Anti-CD25 (IL-2R1), CD3, CD14, and antibodies against surface Ig
anti- , anti- , anti-, anti-µ, and anti- were also
purchased from Dakopatts.
The cells were analyzed with an EPICS-XL flowcytometer (Coulter,
Hialeah, FL). The percentage of positive cells was determined by
reference to nonspecific staining with antibodies of the same isotype.
BM stromal cell cultures.
After informed consent, BM from normal volunteer donors and B-CLL
patients were collected by sternal aspiration and BM-mononuclear cells
(BM-MNC) were isolated by layering on a Ficoll-Hypaque density gradient
(International Medical Product). BM stromal layers were established as
previously described.16
In brief, 5 × 105 BM-MNC were plated in 35-mm petri
dishes containing 1 mL -minimal essential medium (-MEM; GIBCO,
Grand Island, NY) supplemented with 15% fetal calf serum (FCS;
Seralab, Sussex, UK) and 2 × 10 6 M methyl
prednisolone (Pharmacia and Upjohn, Kalamazoo, MI) at 37°C, 7.5%
CO2 in humidified air.
The adhering cells were fed at weekly intervals by complete replacement
of the medium until a confluent layer of fibroblasts, macrophages, and
fat cells had formed in each dish. Confluence was usually achieved
after 4 to 6 weeks of culture.
Preparation of stromal cell-conditioned medium (stroma CM).
Conditioned media were prepared from 6-week-old stromal cell layers
established from normal subjects when these cells had grown to form a
confluent layer. Stromal cells were washed three times to remove methyl
prednisolone, fed with fresh medium, and cultured for 48 hours.
The stromal cell supernatants were then collected after centrifugation
and stored at  80°C until use.
Measurement of cytokines.
Cytokines were measured in conditioned media using specific immunologic
procedures (enzyme-linked immunosorbent assay [ELISA]): IL-6
(Eurogenetics, Tessenderlo, Belgium; sensitivity 5 pg/mL), leukemia-inhibiting factor (LIF; Eurogenetics; sensitivity 10 pg/mL),
IL-7 (Eurogenetics; sensitivity 10 pg/mL), granulocyte colony-stimulating factor (G-CSF; Quantikine, R&D Systems, Minneapolis, MN; sensitivity 11 pg/mL), stem cell factor (SCF; Quantikine; sensitivity 3 pg/mL), granulocyte-macrophage colony-stimulating factor
(GM-CSF; Medgenix, Fleurus, Belgium; sensitivity 3 pg/mL), IL-10 (DNAX,
Palo Alto, CA; sensitivity 40 pg/mL), macrophage inhibitory
protein-1 (MIP-1; ImmunoSource, Halle, Belgium; sensitivity 195 pg/mL), and transforming growth factor (TGF-; Promega Corp, Madison, WI; sensitivity 25 pg/mL).
Source of neutralizing antibodies.
Neutralizing antibodies to IL-6 were a murine IgG antibody
(Eurogenetics), antibody to IL-10 was a goat IgG (R&D Systems), and
chicken anti-human TGF- was purchased from R&D systems. These antibodies were used at 10 µg/mL.
Cell culture studies.
Before each experiment, the media from stromal cell cultures was
removed and these cells were washed three times with -MEM.
Purified leukemic and normal B lymphocytes were resuspended in -MEM + 15% FCS at a final concentration of 106/mL. Then, 2 mL
of this cell suspension was placed in the wells of a 6-well plate or
seeded on BM stromal cells in identical plates. In parallel
experiments, cells were seeded onto 24 mm transwell diffusion chambers
(0.4 µm microporous filter; Costar, Catalog No. 3408) and placed into
stroma-coated 6-well plates.
Leukemic and normal B lymphocytes were also cultured in -MEM
supplemented with 50% stroma CM. All cell cultures were incubated for
48 hours at 37°C in 5% CO2 with 100% humidity.
Determination of cell viability.
Viability was determined at various time intervals by Trypan blue
exclusion in a Neubauer counting chamber (Vel, Leuven,
Belgium). Stromal cells were identifiable by their large size and
different morphology. In some cases, nonviable cells were stained with
propidium iodide (20 µg/mL) in PBS and counted by flow cytometry.
Determination of DNA fragmentation by flow cytometry.
Quantification of cells with degraded DNA was performed using a method
described by Nicoletti et al.17
Cells to be analyzed were collected, washed, permeabilized, and
incubated with solution containing propidium iodide (PI) and RNAse
(Coulter DNA-Prep Reagent). The tubes were placed at 4°C in the
dark overnight before analysis by flow cytometry.
Data collection was gated using forward light scatter and side light
scatter to exclude cell debris and aggregates.
The PI fluorescence of individual nuclei was measured using a Coulter
EPIC XL. At least 5 × 103 cells of each sample were
analyzed. Apoptotic cells are represented by a broad hypodiploid peak
of cells that is easily discriminable from the peak of cells with
diploid DNA content in the red fluorescence channel. All data were
analysed using EPICS Elite 4.0 software.
When B-lymphocytes were cultured in the presence of stromal cells, cell
cycle analysis was performed by labeling cells prestained with
biotinylated anti-CD19 and streptavidin-FITC (Pharmingen, San Diego,
CA) with PI and determining their DNA content by flow cytometry as
previously described.
Tunel assay.
DNA fragmentation in apoptotic cells was detected according to the
method described by Gavrieli.18 B cells were fixed in 4%
buffered formaldehyde (pH 7.4) for 30 minutes at room temperature. Then, after washing in PBS, B cells were resuspended in
permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate)
for 2 minutes on ice. After washing, B cells were resuspended in TUNEL
reaction mixture (dUTP-FITC) at 37°C for 1 hour according to the
manufacturer's instruction (Boehringer Mannheim Biochemical,
Indianapolis, IN). Cells were washed and analyzed by flow cytometry.
Control staining was performed on aliquots of the same cells treated
with the staining mixture without terminal deoxynucleotidyl transferase
(TdT). As previously described, TUNEL assay was performed in cocultures with B cells prestained with biotinylated anti-CD19 and streptavidin-PE (Pharmingen).
Adhesion assay.
Normal and leukemic B cells were radiolabeled by incubating 1 to 5 × 106 cells with 100 µCi
Na251CrO4 for 1 hour at 37°C.
51Cr-labeled B cells were washed and plated into wells
containing confluent BM stromal cells and incubated at 37°C for 2 hours. After two washes to remove nonadherent cells, the stromal
cell-adherent B cells were quantitated on a -counter. All the
experiments were performed in triplicate, and the percentage of
adhesion was calculated as follows:
For
inhibition studies, B-CLL cells were incubated for 1 hour with the
following blocking antibodies at 10 µg/mL: anti-CD11a, anti-CD18,
anti-CD29, and anti-CD49d (Immunotech, Marseille, France) before
addition to wells. Stromal cells were incubated with anti-CD54 and
anti-CD106 1 hour before the addition of labeled B-CLL cells. Results
were compared with those of wells containing isotype-matched irrelevant
antibody.
Bcl-2 expression.
Cells were permeabilized by treatment with Permeafix (Ortho Diagnostics
Systems, Beerse, Belgium) for 40 minutes and then washed in cold PBS.
The washed cells were then incubated with FITC-labeled mouse antihuman
bcl-2 monoclonal antibody (MoAb; Dako, Glostrup, Denmark) for 30 minutes.
The cells were then washed in PBS, and cell-associated fluorescence
analysis was performed on the flowcytometer. A mouse IgG1 MoAb was used
as a negative control. In cell cocultures, bcl-2 expression was
evaluated in B cells prestained with biotinylated anti-CD19 and
streptavidin-PE.
To quantify bcl-2 expression, the cytometer was calibrated using
FITC-labeled micro-beads (Immuno-Brite, Coulter). The set of four beads
used titred, respectively, 31,500, 115,000, 460,000, and 1,115,000 mean
equivalent of soluble fluorochrome (MESF) units.
A linear regression model was used to correlate mean values of the
fluorescent bead peaks to the log10 of corresponding MESF units. This method allows quantification of mean bcl-2 expression of
the tested population and direct comparison of samples analyzed at
different times.
Statistical analysis.
The Wilcoxon's nonparametric test was used to analyze the statistical
significance of the experimental results.
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RESULTS |
Spontaneous cell death of B lymphocytes in vitro.
B cells from 20 patients and 11 UCB samples were cultured in medium
alone, and apoptotic cell death was evaluated by light microscopy and
flow cytometry.
Purified normal and leukemic B lymphocytes rapidly initiated apoptosis
when placed in culture as previously described.6,19
After 48 hours of culture, B cells showed morphologic evidence of
apoptosis, such as nuclear fragmentation and loss of cell volume. As
measured by flow cytometry, the mean percentage of apoptotic cells was
31 ± 5% and 22 ± 3%, respectively, for normal and leukemic B
cells (Fig 1). These B cells were 80% to
100% dead after 7 days of culture, and the appearance of cells with
degraded DNA was associated with the loss of viability through the
culture period.
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| Fig 1.
Percentage of apoptotic B cells after 48 hours of culture
in medium alone (control MEM) or in the presence of stromal cells (with
or without contact). DNA fragmentation was revealed by flow cytometry.
Results are expressed as mean ± SEM of 11 experiments for
normal B cells and 20 for B-CLL cells. ( ), Control MEM; ( ), stromal cell contact; ( ), no stromal cell contact.
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BM stromal cells prevent apoptosis of B-CLL cells.
To determine whether the presence of normal stromal cells could
influence cell viability, B cells were cultured for 48 hours on BM
stromal cells. Quantification of the percentage of cells with degraded
DNA was performed by PI staining of permeabilized cells prestained with
CD19. As shown in Fig 1, apoptosis of leukemic B cells was
significantly reduced by plating these cells on normal allogeneic
stromal cells (8 ± 2 v 22 ± 3%, P < .0005).
In contrast, when normal B cells were cocultured with stromal cells,
the percentage of B cells undergoing apoptosis was slightly but not
significantly increased (37 ± 6% v 31 ± 5%).
When a microporous membrane was inserted between stroma and leukemic B
cells, the protective effect of stromal cells was lost and strikingly,
the percentage of apoptotic cells was increased (38 ± 7% v
22 ± 3%, P < .001; Fig 1).
These observations suggest that stromal cells secrete factors that
diffuse through microporous membrane and are able to induce apoptosis
of leukemic B cells. Moreover, intimate B-cell-stroma contact is
essential to rescue leukemic cells from spontaneous apoptosis and
apoptosis induced by soluble factors produced by stromal cells.
Stroma CM induces apoptosis of normal and leukemic B cells.
To confirm that stromal cells secrete factors inducing B-cell
apoptosis, we have cultured normal and leukemic B cells in -MEM supplemented with 50% stroma CM.
As shown in Fig 2, increased apoptosis of normal and
leukemic B cells was obtained in the presence of stroma CM, confirming that stromal cells secrete factors responsible for B-cell apoptosis.
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| Fig 2.
Effect of stroma CM on B cell apoptosis. Stroma CM was
prepared as described in Materials and Methods. Results are expressed as the mean ± SEM of nine experiments and represent the percentage of
apoptotic B cells after 48 hours of culture in the presence of -MEM
supplemented with stroma CM at 50% compared with cultures in -MEM
alone or in the presence of stromal cells but without contact. (),
Control MEM; (), stroma CM; (), no stromal cell contact.
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After 48 hours, in all CLL patients tested (n = 9), spontaneous and
stroma CM-induced apoptosis mean percentages were, respectively, 21 ± 6% and 42 ± 5% (P < .012), and similar results
were obtained when B cells were separated from stroma by filter (43 ± 7%, P < .008). Stroma CM also induced apoptosis of
normal B cells (51 ±14% v 43 ± 13%, in medium alone,
n = 4) but this difference was not significant.
Profile of DNA fluorescence in CLL and UCB B cells cultured in these
various conditions are shown in Fig 3. Some samples were labeled with biotinylated dUTP using the TUNEL assay as described previously and showed comparable results (data not shown).
To establish the nature of soluble factors produced by stromal cells
and inducing apoptosis, the cytokines present in conditioned medium and
the effect of neutralizing antibodies to specific cytokines have been
evaluated. BM stromal cells produce constitutively a wide variety of
cytokines but at various levels (Fig 4A).
Using neutralizing antibodies, we have determined the potential role of
IL-6, IL-10, and TGF- as inducers of apoptosis. Indeed, IL-6 represents the major cytokine constitutively released by stromal cells
(>5 ng/mL), and IL-10 or TGF- have been described as modulators of
apoptosis in several cell types including B lymphocytes. Our observations, detailed in Fig 4B, showed that these cytokines are not
involved in the induction of apoptosis by stroma CM.
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| Fig 4.
(A) Cytokine production by stromal cells. Supernatant
from stromal cells were collected 2 days after media change and assayed by ELISA for the above cytokines. Values are the mean ± SEM of 10 experiments. (B) Induction of B-cell apoptosis by stroma CM and
influence of neutralizing antibodies to cytokines. Results are
expressed as percentage of apoptotic cells and represent the mean of
five experiments.
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Survival of leukemic B cells on BM stromal cells.
As shown in Fig
5, the culture of B cells in medium alone led to a significant decrease
in cell recovery. The number of B cells recovered after 48 hours and 7 days of culture was, respectively, 49 ± 4% (mean ± SEM) and
28 ± 6% of those originally seeded. After 14 days of culture, only
9 ± 4% of B cells remained alive. However, in the contact with
stromal cells, the recovery of viable cells increased significantly,
with a mean cell recovery of 61± 9% and 45 ± 10%,
respectively, after 7 and 14 days. In contrast, after 7 days of culture
with or without stromal cells, the majority of normal B cells were
dead. These results confirmed our previous observations and showed a
prolonged survival of leukemic B cells seeded on stromal cells compared
with that of their normal counterpart in the same culture conditions.
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| Fig 5.
Percentage of leukemic B cells recovered from culture in
the presence or absence of stromal cells. Values are the mean of four
experiments and refer to viable B cells after various periods of
culture, determined by Typan blue exclusion and compared with those
originally seeded.
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Viability was also assessed using flow cytometry to quantify the
percentage of cells, prestained with CD19, capable of excluding propidium iodide. After 7 days of culture, in medium alone, the mean
proportion of viable cells declined to 56 ± 2% (n = 3). In contrast with stromal cells, the viability of B-CLL cells was increased
(88 ± 1%; data not shown).
Adhesion of B cells to stromal cells.
After having observed that contact between leukemic B cells and stromal
cells is essential for their protection from apoptosis, we examined the
adhesion of B-CLL cells and normal B lymphocytes to stromal cells.
Figure 6 shows experiments comparing the
level of adhesion of 51Cr-labeled normal (n = 5) and
leukemic B cells (n = 15). After 2 hours, 25 ± 4% (range 16% to
66%) of B-CLL cells adhered to stromal cells, whereas adhesion of UCB
B cells was 6 ± 2%.
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| Fig 6.
Adhesion of normal (n = 5) and leukemic (n
= 15) B cells to stromal cells. Percentage of adhesion was calculated
as described in Materials and Methods.
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The differences in behavior observed between normal and leukemic B
cells plated on stromal cells could be related to the level of cell
adhesion.
To investigate the mechanism of adhesion, the binding of leukemic
B-lymphocytes to stromal cells was evaluated in the presence of several
blocking antibodies to antagonize 1- and
2-integrin-dependent pathways. As shown in
Fig 7A, blocking of CD18- or CD29-dependant adhesion mechanisms did not reduce the binding of B-CLL cells to
stromal cells (<10% inhibition). There was also no inhibition of
B-CLL binding after treatment with anti-CD11a. In six out of eight
cases examined, anti-CD49d induced moderate inhibition of B-CLL
adhesion (14 ± 6%). However, when anti-CD11a was associated to
anti-CD18, more than 20% inhibition was observed (P < .04). Addition of anti-CD49d to the combination of anti-CD18/anti-CD11a increased significantly the inhibition of binding in all cases tested
(45 ± 10%, P < .04). Adhesion of B-CLL cells to stromal cells is thus partly mediated by 1- and
2-integrins acting simultaneously.
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| Fig 7.
(A) Blocking of adhesion of B-CLL cells to stromal cells.
Leukemic cells were preincubated for 1 hour with blocking antibodies before plating on stromal cells. Each bar represents the mean of
adhesion inhibition of eight experiments. (B) Blocking of B-CLL cell
adhesion to stromal cells untreated or treated with anti-CD54 and/or anti-CD106. B-CLL cells were simultaneously preincubated with antibodies to CD11a/CD18 or CD49d.
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To evaluate the potential role of ICAM-1 (CD54) and VCAM-1 (CD106) in
the adhesion of B-CLL cells, BM stromal cells were incubated with
antibodies directed to these two ligands. B-CLL cells were simultaneously preincubated with antibodies to 1- and
2-integrins (VLA-4/VCAM-1 and LFA-1/CD54). Blocking
antibodies against ICAM-1 and VCAM-1 inhibited adhesion by 22 ± 7%
and 34 ± 5% (Fig 7B). However, the combination of these two
antibodies impaired significantly B-CLL cell adhesion to stromal cells
(inhibition of 48 ± 9, P < .04) compared with cells
treated with anti-CD54 or anti-CD106 alone. These results show that
both 1- and 2-integrins VLA-4 and LFA-1
and their ligands VCAM-1 and ICAM-1 are involved in the adhesion of
B-CLL cells to stromal cells. However, the fact that the combination of
1- and 2- integrins, associated with their ligands CD106 and CD54, was unable to block completely adhesion process suggests that other mechanisms are involved in the adhesion of
B-CLL cells to stroma.
Bcl-2 analysis by flow-cytometry.
Bcl-2 was studied at the protein level by flow-cytometry on
permeabilized cells in eight cases of B-CLL
(Fig 8). Before culture, more than 90% of
cells from all cases tested constitutively expressed bcl-2 at a high
level (39 ± 10 × 103 MESF). After 7 days of
culture in medium alone, the level of bcl-2 expression was
downregulated (26 ± 4 × 103 MESF),
although the percentage of bcl-2 positive cells remained constant.
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| Fig 8.
Bcl-2 expression of B-CLL cells cultured in the presence
or absence of stromal cells. The results are expressed as the mean ± SEM of eight experiments and represent the level of bcl-2 expressed as
MESF.
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However, after 7 days of culture in the presence of stromal cells, the
expression of bcl-2 in B cells was 44 ± 9 × 103
MESF and comparable with the level of bcl-2 seen at day 0. The loss of
bcl-2 protein was thus inhibited in the presence of stromal cells.
For nine B-CLL cases tested, the reduction in bcl-2 expression was
found to correlate with the rate of B-CLL apoptosis
(Fig 9).
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| Fig 9.
Relationship between the level of bcl-2 protein in B-CLL
cells and their degree of apoptosis during in vitro culture. These two
parameters were determined in nine cases.
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DISCUSSION |
B-CLL cells in vivo display enhanced survival, and their level of
apoptosis remains low compared with that of normal B lymphocytes. In
contrast, B-CLL cells in vitro die spontaneously and are difficult to
keep alive. We have shown in the present study that leukemic B cells
become able to survive in vitro when BM stromal cells are added in
vitro, as they do in vivo and as long as a cell contact is maintained
between these two cell types.
BM stromal cells synthesize several cytokines, including CSFs, IL-6,
IL-7, IL-10, TGF-, and SCF, and exert a complex regulatory function
mediated by these soluble growth factors and extracellular matrix
proteins.20 Self-renewal and differentiation of B-cell precursors depend on interactions with BM stromal cells and
extracellular matrix.21 B-lymphocyte production is also
regulated by cytokines produced in the microenvironment.22
A number of previous studies have shown that contact with BM stroma
induces proliferation and survival of acute lymphoblastic
leukemia (ALL) cells in cultures.23,24 The
adhesion of B-CLL cells to stromal cells is crucial for their survival
because these cells die when separated from the stromal cells by a
microporous membrane. Moreover, the protective effect from apoptosis,
mediated by contact, cannot be substituted by addition of stroma CM.
Our results obtained with stroma CM indicate that soluble factors or
cytokines produced by stromal cells are unable to protect B-CLL cells
from apoptosis but on the contrary significantly increase their
apoptosis. Indeed, it has been described that B-CLL cell apoptosis can
be influenced by various cytokines such as IL-4, IFN-, IFN-, and
IL-10.8-11 Soluble factors produced constitutively by
stromal cells are responsible for the induction of apoptosis in
B-CLL cells. So far, only two cytokines, IL-5 and IL-10, have been described as inducers of apoptosis in CLL.11, 25
However, IL-5 is not produced by stromal cells, and our results obtained with neutralizing antibodies to IL-6, IL-10, and TGF- show
that these cytokines are not involved in the increase of apoptosis by
stroma CM. It seems thus interesting to isolate factor(s) inducing
apoptosis by methods such as gel filtration and reverse-phase chromatography.
Signals other than soluble factors present in conditioned medium are
clearly required for the survival of these B-CLL cells, such as matrix-
or membrane-bound cytokines and/or messages transduced to the
nucleus by way of other cell surface receptors.
The survival of B-CLL cells in coculture with stroma has been reported
recently. Panayiotidis and coworkers26 described the
survival of leukemic B cells up to 6 weeks in an in vitro BM
environment. Other in vitro studies revealed that stromal cell cultures
supplemented with conditioned media from the human bladder carcinoma
line 5637 were able to support the growth of B-CLL cells for up to 9 to
13 weeks.27
In our study, CD5+ B cells, which have been reported to be
abundant in UCB and have been suggested to represent a normal
counterpart of B-CLL cells, were also examined.28 In
contrast to B-CLL cells, normal CD5+ B cells were unable to
survive in coculture with stromal cells. The difference in behavior
observed between normal and leukemic B cells plated on stromal cells
can be explained by our observation that only a few UCB B cells adhered
to stromal cells in comparison with B-CLL cells. It has been suggested
that specific binding to marrow fibroblasts is part of the
differentiation program of B-lineage precursors and that this binding
activity decreases during B-lineage differentiation.29 This
could therefore explain a different adhesion between normal and
leukemic CD5+ B lymphocytes. However, another hypothesis
could be that normal B cells are insensitive to the "survival
signal" mediated by contact between B cells and stromal cells.
Indeed, two cytokines so far, IL-5 and IL-13, can induce the apoptosis
in B-CLL but not in normal peripheral B cells.25,30 However, although the intimate mechanism of stroma-mediated cell survival remains unknown, our data at least show that the survival of
leukemic cells depends on direct cell-cell contact but not on soluble
cytokines produced constitutively by stromal cells.
Survival signals may be provided by interactions between adhesion
proteins expressed on B-CLL cells and their ligands in the microenvironment. Such adhesion interactions have been shown to modulate apoptosis in others systems.31 Long et
al13 have shown that, in B-CLL, apoptosis and IL-7 gene
expression can be mediated by cell-cell contact with endothelial cells.
It has been described that B-CLL cells express integrins, such as
VLA-4, and various adhesion molecules, such as LFA-1 and ICAM-1, at a
variable level.32,33 The fact that VLA-4 and its ligand
VCAM-1 are obligatory proteins in the adhesion of B precursors to
stromal cells led us to evaluate the role of 1- and
2-integrins in the adhesion of B-CLL cells to stromal
cells.
As reported by other groups, we were unable to inhibit significantly
B-CLL adhesion using individual blocking antibodies directed to CD11a,
CD18, CD29 or CD49d. Approximately 30% to 50% inhibition of B-CLL
adhesion could be obtained with the combination of
anti-CD11a/CD18/CD29/CD49d suggesting that binding of leukemic cells to
stromal cells requires the simultaneous action of 1- and
2-integrins. CD54 and CD106 represent the ligands on
stromal cells for B-CLL cell binding and interact with CD11a/CD18 and
CD49d expressed by leukemic cells. However, concomitant blockade of
1- and 2-integrins was essential to
obtain significant inhibition of B-CLL adhesion. This simultaneous intervention of 1- and 2-integrins has
been previously reported by other groups evaluating the adhesion of
acute myeloid leukemia (AML) cells or normal hematopoietic
progenitors to stromal cells.34,35 It has been recently
shown that binding through both the CD11a/CD18-CD54 and CD49d-VCAM-1
pathways can prevent apoptosis of germinal center B cells and may thus
contribute to the process of B-cell selection.36
Presently, we can only speculate on the nature of signals involved in
the survival of B-CLL cells observed in this study; further studies are
required to answer this question. For instance, the contact between B
cells and stromal cells could induce the release of specific factors
able to protect B cells from apoptosis. The factors crucial for the
survival of B-CLL cells could be either membrane-bound or concentrated
in the extracellular matrix. In this context, Verfaillie et
al37 have suggested that, although not necessary for
proliferation, the close association of progenitor cells with the
stroma may be crucial for the regulation and ordered progression of
normal hematopoiesis. This effect may be mediated by high levels of
cytokines concentrated by glycoaminoglycans in the stromal
extracellular matrix.37
Because apoptosis seems to be controlled, at least in part, by the
proto-oncogene bcl-2, we investigated the possibility that stromal
cells maintain CLL B-cell viability by regulating bcl-2 expression.
Using flow cytometry, we observed a reduction in bcl-2 expression in
B-CLL cells during in vitro culture, and this reduction was found to
correlate with the level of apoptosis as reported recently.38,39
Contact between B cells and stromal cells could trigger delivered
"signals" that prevent the loss of bcl-2 and consequently inhibit
cell death. In a recent study, BM stromal cells have also been shown to
regulate not only bcl-2 but also bax expression in pro-B cells,
confirming the importance of stromal cell function in the maintenance
of cell viability of B-lineage cells.40 Bcl-2 is only one
factor among several genes regulating apoptosis. Indeed, several groups
have reported the existence of bcl-2-related genes that produce a
range of heterodimers that function as dominant regulators of
apoptosis.41 Bcl-2, bax, bcl-xL, and bcl-xS have an
interrelated role in the control of apoptosis, and their expression in
CLL is skewed toward prevention of apoptosis .42,43
Additional experiments will address the influence of stromal cells in
the regulation of bcl-2 gene family.
In summary, we have shown that contact with BM stromal cells protects
leukemic B cells from spontaneous apoptosis and apoptosis induced by
soluble factors produced constitutively by stromal cells. However,
further investigations will be necessary to identify other molecules
involved in the interaction between stromal cells and leukemic cells
and the signals able to rescue leukemic cells from apoptosis.
Understanding of the role of adhesion proteins in B-CLL cell adhesion
to stromal cells and the mechanisms responsible for the prolonged
survival of B-CLL cells in vivo could lead to the elucidation of the
pathogenesis of B-CLL and to the development of new therapeutic
strategies.
| |
FOOTNOTES |
Submitted April 16, 1997;
accepted November 7, 1997.
Supported by Télévie-FNRS Grant No. 7.4506.94 and by a
grant from the Bekales Foundation.
Address reprint requests to L. Lagneaux, Institut J. Bordet,
Laboratoire d'hématologie, 1 Rue Héger-Bordet, 1000 Brussels, Belgium.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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M. Burger, T. Hartmann, M. Krome, J. Rawluk, H. Tamamura, N. Fujii, T. J. Kipps, and J. A. Burger
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Blood,
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F. K. Stevenson and F. Caligaris-Cappio
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I. M. Ghobrial, N. D. Bone, M. J. Stenson, A. Novak, K. E. Hedin, N. E. Kay, and S. M. Ansell
Expression of the Chemokine Receptors CXCR4 and CCR7 and Disease Progression in B-Cell Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma
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M. J. Terol, M. Tormo, J. A. Martinez-Climent, I. Marugan, I. Benet, A. Ferrandez, A. Teruel, R. Ferrer, and J. Garcia-Conde
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L. Granziero, P. Circosta, C. Scielzo, E. Frisaldi, S. Stella, M. Geuna, S. Giordano, P. Ghia, and F. Caligaris-Cappio
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I. M. Pedersen, S. Kitada, L. M. Leoni, J. M. Zapata, J. G. Karras, N. Tsukada, T. J. Kipps, Y. S. Choi, F. Bennett, and J. C. Reed
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Blood,
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M. T. de la Fuente, B. Casanova, J. V. Moyano, M. Garcia-Gila, L. Sanz, J. Garcia-Marco, A. Silva, and A. Garcia-Pardo
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N. Tsukada, J. A. Burger, N. J. Zvaifler, and T. J. Kipps
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J. A. Burger, N. Tsukada, M. Burger, N. J. Zvaifler, M. Dell'Aquila, and T. J. Kipps
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Abstract
Introduction
Abstract
