From the Departamento de Inmunología, Centro de Investigaciones Biológicas, Velázquez 144, 28006 Madrid, Spain
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ABSTRACT |
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The very late antigen (VLA)-4 and VLA-5 integrins
mediate hematopoietic progenitor cell attachment to bone marrow (BM)
stroma. Transforming growth factor-1 (TGF-
1) is a cytokine
present in the BM microenvironment that has been shown to regulate the
synthesis of adhesion elements in several cell types. We have
investigated whether TGF-
1 action on human BM stromal cells affected
the adhesion of progenitor cells involving integrins VLA-4 and VLA-5.
Two precursor cell lines, pre-B Nalm-6 and the multipotential UT-7,
attached to untreated primary stroma and to the human BM stromal cell
line Str-5 preferentially using VLA-4. However, treatment of the stroma with TGF-
1 resulted in a significant reduction in the participation of VLA-4 in mediating precursor cell adhesion to stroma and a concomitant increase in the utilization of VLA-5. This effect was not
exclusive of normal BM stroma. Treatment with TGF-
1 of stroma from
multiple myeloma BM samples produced a substantial increase in VLA-5
use by the myeloma cell line NCI-H929 to adhere to this stroma. The
differential use of VLA-4 and VLA-5 correlated with an increase in
fibronectin surface expression by stromal cells in response to
TGF-
1. Adhesion assays to purified fibronectin using Nalm-6 cells
showed a predominant utilization of VLA-4 at low concentrations of this
ligand, whereas higher concentrations resulted in a preferential use of
VLA-5. These results indicate that regulation of fibronectin expression
on BM stromal cells by TGF-
1 results in a modulation of the pattern
of integrins used by the precursor and myeloma cells to adhere to BM
stroma, which could have important consequences on the proliferation
and differentiation of hematopoietic precursor cells as well as on the
localization and growth of myeloma cells.
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INTRODUCTION |
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The bone marrow stromal cells (BMSC)1 constitute a population of different cell types that provide the bone marrow (BM) microenvironment with a wealth of cytokines necessary for sustained hematopoiesis and that express membrane ligands for adhesion receptors on hematopoietic progenitor cells (1). The integrins VLA-4 and VLA-5 are among the main adhesion receptors used by CD34high, precursor, and leukemic cells to attach to stroma, interacting with different regions on fibronectin (FN), whereas VLA-4 can additionally bind to VCAM-1, which is constitutively expressed by BMSC (2-5).
TGF- is a multifunctional cytokine present in the BM
microenvironment that is mainly produced by megakaryocytes and by BMSC (6, 7) and that regulates stem cell proliferation and B lymphopoiesis
(8-10). The levels of TGF-
have been found to be higher in several
leukemias, such as multiple myeloma and B cell chronic lymphocytic
leukemia, compared with normal bone marrow (11, 12). We have recently
characterized the TGF-
1 receptor system on primary human BMSC,
showing the expression of the serine/threonine kinase transducing type
I and II receptors as well as endoglin (13). Little is known about the
effects of TGF-
on stromal cell function. Potential targets for its
action include the control of cell proliferation and the regulation of
the synthesis of adhesion elements, such as extracellular matrix
proteins, and extracellular matrix protein-degrading proteases (14).
Changes in the expression of adhesion elements on BMSC at different
niches or during different stages of differentiation could result in
variations in the adhesion of maturing precursor cells and could also
influence the localization of tumor cells in the BM. In this work, we
have studied the involvement of integrins VLA-4 and VLA-5 in progenitor
cell adhesion to TGF-
1-treated stroma.
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MATERIALS AND METHODS |
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Stromal Cell Cultures--
Normal BM was obtained after informed
consent from donors for allogeneic bone marrow transplants. Multiple
myeloma BM samples were obtained from untreated patients with active
disease. The stromal cultures were generated as described previously
(13) and maintained in Iscove's modified Dulbecco's medium (IMDM)
supplemented with 10% fetal calf serum (complete medium) and
105 M hydrocortisone sodium succinate.
Non-adherent cells were removed after 1 week, and upon confluency, the
stromal cells were passaged by trypsin/EDTA. After four passages, they
were seeded 1 week before use in 96-well plates for adhesion and cell
ELISAs. Our BM stromal monolayers consisted mainly of fibroblasts and
macrophages, with a low percentage of endothelial cells (13). The human
bone marrow stromal cell line Str-5 was maintained in complete medium (13). For TGF-
1 treatments, stromal cultures were first incubated in
IMDM supplemented with 1% fetal calf serum and serum replacement medium (2× TCM, ICN Biomedicals) for 24 h, followed by a 24-h incubation in the same medium in the presence of recombinant human TGF-
1 (R&D Systems, Abingdon, United Kingdom), and this medium was
removed by aspiration before assays.
Cells and Antibodies--
The pre-B Nalm-6, myeloma NCI-H929,
and Burkitt lymphoma Ramos cell lines were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum (and 50 µM
2-mercaptoethanol for the NCI-H929 cells). The multipotential cell line
UT-7 (15), which expresses CD34 and CD33, was grown in -minimum
Eagle's medium in the presence of 10% fetal calf serum and 2 ng/ml
granulocyte-macrophage colony-stimulating factor. Antibodies used in
this study included monoclonal P3X63 (16), anti-
4 HP1/2 (17),
anti-
5 SAM-1 (18), and P1D6 (Life Technologies, Inc.), Bear-1
anti-CD11b (18), anti-VCAM-1 4B9 (19), and polyclonal anti-FN central
cell-binding domain Rb110 and anti-FN carboxyl-terminal CS-1-containing
cell-binding domain Rb113.
Measurement of Bioactive TGF---
TGF-
levels from
stromal supernatants were determined in a bioassay by analyzing the
growth inhibition of mink lung epithelial Mv1Lu cells (American Type
Culture Collection CCL 64). Mv1Lu cells were subcultured in IMDM
supplemented with 10% fetal calf serum in 24-well tissue culture
plates at 3 × 104 cells/well. TGF-
1 (0.1-10
ng/ml) or 24-h stromal supernatants in IMDM supplemented with 1% fetal
calf serum and 2× TCM were added without previous acidification to
Mv1Lu cells in triplicates, which were incubated for 24 h at
37 °C, the last 4 h in the presence of 1 µCi/well
[3H]thymidine (Amersham International, Buckinghamshire,
UK). At the end of incubation, cells were washed with
phosphate-buffered saline and extracted with 0.2 N NaOH,
and extracts were neutralized with 1 M HCl before adding
scintillation liquid and analyzed in a
-counter.
Adhesion Assays--
Cell lines were labeled in their respective
growth medium with the fluorescent dye BCECF-AM (Molecular Probes,
Leiden, The Netherlands) and finally resuspended in RPMI 1640 medium
containing 0.4% bovine serum albumin (adhesion medium). For cell
adhesion to purified plasma fibronectin (Sigma), increasing
concentrations of this ligand were coated overnight at 4 °C in 100 mM NaHCO3 (pH 8.8), followed by 2-h incubation
at 37 °C and then blocking with 0.4% bovine serum albumin in the
same solution for 2 h at 37 °C. Preincubations with antibodies
were carried out at 37 °C for 15 min, and the antibodies were
removed before adhesion. To untreated or TGF-1-treated stroma or
fibronectin in 96-well dishes (Falcon) was added 50 µl of adhesion
medium 20 min before adhesion, and after removing it, labeled cells
(5 × 104 cells/well) were incubated with the stroma
or with fibronectin for 20 min at 37 °C. Unbound cells were removed
by three washes with RPMI 1640 medium, and adherent cells were
quantified using a fluorescence analyzer (CytoFluor 2300, Millipore
Corp.).
Cellular ELISA--
Untreated or TGF-1-treated stromal
monolayers were fixed with 3.7% formaldehyde in phosphate-buffered
saline, washed three times with Tris-buffered saline, and blocked with
3% bovine serum albumin in Tris-buffered saline. After removing the
blocking solution, cells were incubated for 1 h at 22 °C with
the primary antibodies, washed with Tris-buffered saline in the
presence of 0.05% Tween 20, and further incubated for 1 h at
22 °C with peroxidase-conjugated goat anti-rabbit Ig (Dako,
Glostrup, Denmark). Monolayers were finally washed and incubated with
ABTS (Boehringer Mannheim) in the dark before measuring in an ELISA
reader (Multiskan Bichromatic, Labsystems, Helsinki, Finland) at 405 nm.
Flow Cytometry-- Preparation of the stromal cells for flow cytometry analysis was performed as described previously (13). Approximately 105 stromal cells were incubated with saturating concentrations of primary antibodies at 4 °C for 45 min. After washing, fluorescein isothiocyanate-conjugated rabbit anti-mouse Ig (Dako) was added and further incubated at 4 °C for 30 min. Samples were analyzed using a Coulter Epics XL flow cytometer.
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RESULTS |
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To investigate whether TGF-1 action on BMSC could influence
hematopoietic precursor and myeloma cell adhesion mediated by integrins
VLA-4 and VLA-5, we first measured the levels of bioactive TGF-
present in primary human BMSC (HBM-Str) cultures, in the stromal cell
line Str-5, and in multiple myeloma BMSC (MM-Str) cultures. Bioassays
for TGF-
activity using the TGF-
responder cell line Mv1Lu showed
TGF-
-dependent activity corresponding to 0.3-0.6 ng/ml
in HBM-Str cells cultured for 24 h, whereas Str-5 and MM-Str
cells secreted bioactive TGF-
in the range of 2-2.5 ng/ml
(Fig. 1).
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Incubation of primary stroma with 1 ng/ml TGF-1 resulted in a
decrease in the adhesion of the pre-B cell line Nalm-6 compared with
the adhesion to untreated stroma (Fig.
2A). The anti-
4 HP1/2 mAb
significantly inhibited (30-40%; p < 0.05) the
adhesion of Nalm-6 cells to untreated stroma, but inhibition by HP1/2
was consistently decreased when the adhesion was performed on
TGF-
1-incubated stroma. In contrast, the anti-
5 SAM-1 mAb
minimally inhibited Nalm-6 cell adhesion to untreated stroma; however,
incubation of stroma with TGF-
1 resulted in a blockade of cell
adhesion by SAM-1, as well as by another anti-
5 mAb called P1D6
(data not shown), of ~50% (p < 0.05) (Fig.
2A). The results of inhibition of Nalm-6 cell adhesion using
a mixture of anti-
4 and anti-
5 mAbs mimicked those of inhibition
with single mAbs. Thus, HP1/2 and SAM-1 together inhibited slightly
better than HP1/2 alone the adhesion of Nalm-6 cells to untreated
stroma (Fig. 2A), reflecting a major VLA-4 use. After
incubation of the stroma with TGF-
1, the combination of both
antibodies inhibited similarly to SAM-1 alone, indicating a predominant
VLA-5 utilization. The anti-VCAM-1 4B9 mAb inhibited by 15-20% the
adhesion of Nalm-6 cells to either untreated or TGF-
1-treated stroma
(Fig. 2A). The effect of TGF-
1 action on Nalm-6 cell
adhesion to HBM-Str was also evident when we preincubated the Str-5
cells with increasing concentrations of TGF-
1, although in this
case, higher concentrations of the cytokine were needed to obtain a
substantial blocking of Nalm-6 cell adhesion by the anti-
5 mAb (Fig.
2B). In parallel experiments, the SAM-1 and P1D6 mAbs did
not inhibit the adhesion of the VLA-5-negative B cell line Ramos either
to untreated or TGF-
1-treated HBM-Str cells (data not shown).
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As for the case of Nalm-6 cells, preincubation of HBM-Str cells with
TGF-1 resulted in a decrease in the adhesion of the multipotential
cell line UT-7 (Fig. 2C). Interestingly, both HP1/2 and, to
a lesser extent, SAM-1 inhibited the adhesion of UT-7 cells to
untreated stroma. However, TGF-
1 treatment of the stroma resulted in
a notable reduction in HP1/2 inhibition of cell adhesion, whereas
inhibition by SAM-1 remained unchanged, indicating a predominant VLA-5
use. We could not observe any clear effect of the anti-VCAM-1 mAb on
UT-7 cell adhesion to stroma, suggesting that VLA-4 on these cells
mainly interacts with fibronectin.
To study if the differential use of VLA-4 and VLA-5 could also be
observed using leukemic BM stroma, we generated stromal cell cultures
from multiple myeloma BM samples and analyzed the effect of their
incubation with TGF-1 on the adhesion of the myeloma-derived cell
line NCI-H929. These monolayers expressed abundant fibronectin and
substantial levels of VCAM-1 (data not shown).2 Similarly to Nalm-6
and UT-7 cells, NCI-H929 cells adhered significantly less to
TGF-
1-treated compared with untreated multiple myeloma stroma (Fig.
3). The anti-
4 HP1/2 mAb inhibited
NCI-H929 cell adhesion to untreated stroma by ~40%
(p < 0.05) and to a lower extent after treatment with
TGF-
1. As observed for Nalm-6 cells, a low degree of inhibition of
NCI-H929 cell adhesion to untreated stroma was detected with the
anti-
5 mAb P1D6, but after TGF-
1 incubation of the multiple
myeloma stroma, a high induction of blocking by this antibody was
obtained (Fig. 3). The anti-VCAM-1 4B9 mAb inhibited normally 15-25%
of NCI-H929 cell adhesion to both untreated and TGF-
1-treated
multiple myeloma stroma.
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One of the targets of TGF- action is the control of extracellular
matrix protein synthesis (14, 20). As fibronectin is expressed by
stromal cells and is a ligand for both VLA-4 and VLA-5, we analyzed
whether FN levels on the surface of stromal cells were affected by
TGF-
1. Cellular ELISAs consistently showed an augmentation in FN
deposition by HBM-Str cells in response to TGF-
1, as measured by
antibodies against the FN central cell-binding domain and the
CS-1-containing region (Fig. 4). These
results were confirmed by [35S]Met/Cys metabolic labeling
of stroma, where a 2.5-fold increase in gelatin-bound extracellular FN
was observed upon TGF-
1 treatment (Fig. 4). When the expression of
VCAM-1 on stromal cells was analyzed by flow cytometry, a significant
reduction was obtained in response to TGF-
1 (data not shown),
similar to previous results (21). The same experiments showed no effect
of TGF-
1 on VLA-
1 or CD11b expression on these cells.
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To investigate possible mechanisms underlying the potential
preferential receptor usage, we carried out cell adhesion assays with
increasing concentrations of human plasma fibronectin using Nalm-6
cells. The cells adhered to fibronectin in a
concentration-dependent manner in the presence of control P3
antibodies, reaching saturation at ~30 µg/ml (Fig.
5). The inhibition of Nalm-6 cell
adhesion to fibronectin by anti-4 and anti-
5 antibodies displayed
three different patterns, depending on the concentrations of
fibronectin used. At the lowest concentrations of FN (2-4 µg/ml),
the anti-
4 HP1/2 mAb notably inhibited Nalm-6 cell adhesion, whereas
the anti-
5 P1D6 mAb did not block, suggesting a predominant VLA-4 use (Fig. 5). When the concentration of FN was increased (8-12 µg/ml), HP1/2 showed no or modest inhibition of Nalm-6 cell adhesion, whereas P1D6 significantly inhibited this adhesion, indicating a
preferential VLA-5 utilization. At the highest concentrations of
fibronectin (>16 µg/ml), both P1D6 and HP1/2 inhibited Nalm-6 cell
adhesion, although the former inhibited always to a higher extent (Fig.
5). In the same experiments, a mixture of antibodies against the FN
central cell-binding domain and the CS-1-containing region fully
inhibited Nalm-6 cell adhesion when they were preincubated with the
fibronectin-coated wells (data not shown), demonstrating the
specificity of the adhesion.
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DISCUSSION |
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This study describes novel modulatory roles for TGF-1 in the
adhesion of hematopoietic precursor and myeloma cells to BM stroma. The
important finding is that TGF-
1 action on human BMSC resulted in a
significant reduction in the participation of VLA-4 in mediating
precursor and myeloma cell adhesion to the stroma, linked to an
increase in VLA-5 involvement in such adhesion. As shown in Table
I, the ratio of VLA-5 versus
VLA-4 utilization by Nalm-6 and UT-7 cells increased 10-fold after
treatment of the stroma with TGF-
1, whereas this ratio was close to
5-fold in the case of NCI-H929 cells, as determined from values of
inhibition of cell adhesion. The results were obtained using TGF-
1
concentrations similar to those found in the supernatants from normal
and multiple myeloma stromata, although in UT-7 cells, slightly higher
amounts of TGF-
1 were required. We detected lower levels of TGF-
in multiple myeloma stromal cultures compared with a previous report (11), which could be due to the different methods of measurement, as we
analyzed bioactive TGF-
, whereas the other study used an ELISA. The
differential effects on VLA-4- and VLA-5-mediated cell adhesion are
likely at the attachment and possibly spreading levels, as we performed
short adhesion assays (20 min), and thus, little, if any, cell
migration took place in our system. In addition, it has been reported
that most of VLA-4-mediated B cell precursor adhesion to stroma occurs
in the first 15 min of cell attachment (22), and therefore, our results
were obtained under conditions favoring VLA-4 activity. The modulation
of VLA-4 and VLA-5 use by TGF-
1 treatment of stroma takes place in
the absence of any exogenously added TGF-
1 in the precursor cell
lines, and thus, it is independent of a possible increase in VLA-5
expression due to this cytokine, as has been reported for several cell
types (23, 24).
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Associated with the decreased implication of VLA-4 in mediating
precursor and myeloma cell adhesion to TGF-1-treated stroma, there
was a reduction in adhesion to stroma. A diminished B cell precursor
cell adhesion to stroma preincubated with TGF-
1 was also previously
reported, which was linked to a reduced expression of VCAM-1 (21). In
the present work, we observed a moderate participation of the
VLA-4/VCAM-1 adhesion pathway in Nalm-6 and NCI-H929 cells, as detected
by antibody inhibition of the adhesion, but this interaction was not
reduced upon stroma treatment with TGF-
1, suggesting that other
adhesion pathways might be affected. This suggests that most of the
VLA-4-dependent precursor and myeloma cell adhesion to
untreated stroma that we observed takes place by attachment to
fibronectin. The data also indicate that the increase in VLA-5
involvement in cell adhesion to TGF-
1-treated stroma does not
restore the reduced adhesion.
The binding of TGF-1 to the stroma resulted in an increase in the
levels of fibronectin expressed on the surface of stromal cells.
Although both the CS-1 and central cell-binding domains of FN were
augmented by TGF-
1, the precursor and myeloma cells used
predominantly VLA-5 to interact with FN on TGF-
1-treated stroma. To
gain some insight into the mechanisms leading to the preferential use
of VLA-5 after increased fibronectin deposition on BMSC by TGF-
1, we
tested whether changes in the concentration of purified fibronectin
could influence the integrins utilized to mediate cell adhesion. The
results obtained in the lower and likely more physiological range of FN
concentrations mimicked those from adhesion to TGF-
1-treated stroma.
Thus, VLA-4 was the predominant receptor used at the lowest
concentrations of FN inside this range, whereas VLA-5 was the
preferential integrin utilized when the concentration of FN was
increased. At high concentrations of fibronectin, VLA-5 use was still
higher than VLA-4 use, although the latter retained considerable
participation in adhesion to fibronectin. These results suggest that
the concentrations of fibronectin modulate the receptor used to mediate
cell adhesion, and therefore, TGF-
1 regulation of the expression
levels of fibronectin on BMSC finely tunes the pattern of the integrins
that will be used by the precursor and myeloma cells to interact with
the stroma.
The 4 integrins are required for normal development of both B and T
precursor cells in bone marrow (25), and long-term BM cultures have
demonstrated the involvement of VLA-4 in lymphopoiesis (26), evidencing
the importance of VLA-4-mediated adhesion during hematopoiesis. It is
well established that VLA-4 and VLA-5 on stem and precursor cells as
well as on several leukemic cells mediate attachment to their ligands
on BMSC (2-5). This interaction is important for the differentiation
and proliferation of hematopoietic progenitor cells. For instance,
VLA-4/FN interaction is required for terminal maturation of
Ig-secreting BM cells (27). In addition, VLA-4- and VLA-5-mediated cell
adhesion to FN decreases the proliferation of both normal hematopoietic
and chronic myelogenous leukemia progenitors (28, 29), and it has been
reported that VLA-5/FN interaction induces apoptosis of hematopoietic
progenitor cells (30). TGF-
is present in the BM microenvironment,
and it is conceivable that changing amounts of this cytokine in
different niches of BM could influence the regulation of hematopoiesis. BMSC secrete TGF-
1 and have the TGF-
1 receptor system necessary for signaling by this cytokine (13). The differential use of VLA-4 and
VLA-5 by progenitor and myeloma cells to adhere to stroma after binding
of TGF-
1 to their receptors on the stromal cells suggests a
mechanism by which changing adhesive interactions might lead to a
modulation of hematopoietic progenitor cell proliferation and
differentiation. Additionally, the differential contribution of VLA-4
and VLA-5 to the mediation of myeloma cell attachment to multiple
myeloma stroma could influence the localization and proliferation of
the malignant plasma cells in bone marrow.
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ACKNOWLEDGEMENTS |
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We thank Drs. Angeles García-Pardo and Francisco Sánchez-Madrid for critical reading of the manuscript and for providing antibodies. We also thank Dr. Carl G. Figdor for SAM-1 and Dr. John M. Harlan for the 4B9 mAb. We are grateful to the Blood Bank Services of Hospital de la Princesa and Hospital 12 de Octubre (Madrid) for bone marrow samples.
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FOOTNOTES |
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* This work was supported by Grant PM95-0017 from the PGC-MEC (Spain) and by a grant from the Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 34-1-5611800;
Fax: 34-1-5627518; E-mail: cibjt9b{at}fresno.csic.es.
1
The abbreviations used are: BMSC, bone marrow
stromal cells; BM, bone marrow; VLA, very late antigen; FN,
fibronectin; VCAM-1, vascular cell adhesion molecule-1; TGF-,
transforming growth factor-
; IMDM, Iscove's modified Dulbecco's
medium; ELISA, enzyme-linked immunosorbent assay; BCECF-AM,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester;
mAb, monoclonal antibody.
2 M. M. Robledo, F. Sanz-Rodriguez, A. Hidalgo, and J. Teixidó, manuscript in preparation.
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REFERENCES |
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