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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 754-764
High Frequency of Adhesion Defects in B-Lineage Acute Lymphoblastic
Leukemia
By
Teunis B.H. Geijtenbeek,
Yvette van Kooyk,
Sandra J. van Vliet,
Maurits H. Renes,
Reinier A.P. Raymakers, and
Carl G. Figdor
From the Departments of Tumor Immunology and Hematology, University
Hospital Nijmegen, Nijmegen, The Netherlands.
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ABSTRACT |
Aberrant proliferation, differentiation, and/or migration of
progenitors observed in various hematological malignancies may be
caused by defects in expression and/or function of integrins. In this
study, we have developed a new fluorescent beads adhesion assay that
facilitates flow cytometric investigation of lymphocyte function-associated antigen 1 (LFA-1)- and very late activation antigen-4 (VLA-4)-mediated functional adhesion in B-lineage acute lymphoblastic leukemia (ALL) of both the CD10 and
CD10+ (leukemic) cell population within one blood or bone
marrow sample. Surprisingly, of the 20 B-lineage ALL patients
investigated, 17 contained a leukemic cell population with LFA-1-
and/or VLA-4-mediated adhesion defects. Five patients contained
CD10+ cells that did not exhibit any LFA-1-mediated
adhesion due to the lack of LFA-1 surface expression. The
CD10+ cells from 10 ALL patients expressed LFA-1 that
could not be activated by the phorbol ester phorbol 12-myristate
13-acetate (PMA), whereas the CD10 cells
expressed a functional LFA-1. Seven patients contained CD10+ cells that expressed a PMA-unresponsive form of
VLA-4. The PMA unresponsiveness of the integrins LFA-1 and VLA-4
expressed by the CD10+ cells may be due to mutations in
the integrins itself, in protein kinases, or in other intracellular
molecules involved in integrin adhesion. These data clearly demonstrate
the importance of investigating integrin function in addition to
integrin surface expression. The strikingly high frequency (85%) of
adhesion defects in ALL could suggest a causal relationship between
integrin-mediated adhesion and B-lineage ALL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
STEADY-STATE HEMATOPOIESIS occurs in the
bone marrow (BM) microenvironment in which hematopoietic progenitor
cells adhere selectively to stromal cells and extracellular matrix
(ECM).1-3 In these BM niches, the proliferation,
differentiation, and maturation of the progenitors is tightly regulated
by growth factors, chemokines,4,5 and cell adhesion
molecules that are expressed by progenitors as well as stromal
cells.6-8 Cell adhesion molecules not only regulate the
physical association of the progenitors with the BM microenvironment,
but the binding of their counterreceptors also generate intracellular
signals that could directly affect growth and maturation of the
hematopoietic progenitors.9-12 Integrins, particularly
those that belong to the  1-integrin (CD29)13,14 and
2-integrin families (CD18),15,16 represent an important group of cell adhesion molecules expressed by hematopoietic progenitors.
Among the 1-integrins expressed by hematopoietic cells, the very
late activation antigen-4 (VLA-4; CD49d/CD29) and VLA-5 (CD49e/CD29)
have been extensively studied.7,17,18 Both integrins bind
to fibronectin, a major component of the ECM.19,20 VLA-4 is
also able to bind to the vascular cell adhesion molecule 1 (VCAM-1),
which is expressed by stromal cells.21 An important 2-integrin expressed by progenitor cells is the lymphocyte
function-associated antigen 1 (LFA-1; CD11a/CD18).7,18,22
LFA-1 mediates adhesion through binding of the intercellular adhesion
molecule-1 (ICAM-1),23 ICAM-2,24
and ICAM-3.25-27 Both ICAM-1 and ICAM-3 have been found on
early hematopoietic cells,28 whereas stromal cells express ICAM-1 as well as ICAM-2 (R. Torensma, personal communications, January 1998).
The adhesive properties of hematopoietic cells are not only regulated
by the expression levels of integrins and their ligands, but are also
dependent on the activation of integrins by intracellular signaling,
so-called inside-out signaling.29-31 Only activated 1-
or 2-integrin molecules can interact with their ligands, demonstrating that integrin-mediated adhesion is a strictly regulated event. LFA-1 can be activated through intracellular signaling events
triggered by a number of leukocyte surface receptors, such as the
T-cell receptor, CD3, and CD19,30,32,33 or by the phorbol ester phorbol 12-myristate 13-acetate (PMA), which
activates the protein kinase C cascade. Furthermore, integrin-mediated
adhesion can also be stimulated by certain anti-1-integrin or
anti-2-integrin antibodies that induce and stabilize active
conformations of these integrins.34-38
The involvement of integrins in adhesion of hematopoietic progenitor
cells to BM stroma has been assessed in several functional studies.
Infusion of blocking anti-VLA-4 antibodies in primates causes
mobilization of hematopoietic progenitors into the
bloodstream.39 The interaction of VLA-4 with VCAM-1
mediates the binding of both primitive and committed progenitors to
stromal cells,18,40 and antibodies to VLA-4 inhibit
lymphopoiesis, myelopoiesis, and erythropoiesis in
vitro.40-42 These studies implicate a critical role for
VLA-4 in regulating the in vivo migration and trafficking of
hematopoietic progenitors by providing interaction with the BM stromal cells.
In contrast to the role of the 1-integrins, little is understood
about the role of the 2-integrin LFA-1 in hematopoiesis. Infusion of
primates with blocking anti-LFA-1 antibody did not result in any
mobilization of the progenitors into the bloodstream.39 However, interleukin-8 (IL-8) and IL-1 mobilization of hematopoietic progenitor cells in mice could be inhibited by a single injection of
anti-LFA-1 blocking antibodies,43 suggesting a role for
LFA-1 in the localization of early hematopoietic cells in the BM
microenvironment. LFA-1 may be a distinct regulator for growth and
maturation of progenitor cells, because 2-integrins are involved in
the formation of progenitor cell aggregates and can be activated by
CD34-induced intracellular signals.44,45 Furthermore, the
expression of LFA-1 on progenitor cells is highly variable and appears
to be related to the maturation state of the
progenitors.22,46
Interactions between hematopoietic progenitors and components of the BM
microenvironment play a pivotal role in progenitor proliferation,
differentiation, and migration, and adhesion molecules such as VLA-4,
VLA-5, and LFA-1 are critical regulators of these interactions. Changes
in expression and activation states of these adhesion molecules are
likely to reflect diverse stages of hematopoiesis. Therefore, aberrant
progenitor proliferation, maturation, and/or homing in various
hematological malignancies may be correlated to defects in expression
and/or function of cell adhesion molecules. The expression of various
adhesion molecules in hematological malignancies has been extensively
investigated, whereas functional studies on these molecules are very
limited.47-50 In this study, we have analyzed both LFA-1-
and VLA-4-mediated adhesion in B-lymphocyte acute lymphoblastic
leukemia (ALL). ALL is a clonal hematopoietic disorder characterized by
cell maturation arrest and accumulation of malignant lymphoblasts in
marrow, lymphatic, and nonlymphatic tissues, and in most cases
lymphoblasts migrate from the marrow into the peripheral blood.
Aberrant ALL progenitor proliferation, differentiation, and homing
could be correlated to altered adhesion properties of the ALL blasts.
Even though the exact mechanisms of adhesion of ALL cells to BM stroma
are not clear, integrins have been recognized to mediate those cellular
interactions that are important in ALL biology.48,50-52
CD10 (common ALL antigen [CALLA]) is routinely used in the
immunophenotyping of acute lymphatic leukemias as a marker for both
cALL and pre-B-ALL. The CD10 antigen is also expressed on the cell
surface of pre-pre-B, pre-B, and early-B cells during normal B-cell
differentiation. Because healthy BM contains only a low percentage of
these CD10+ cells, CD10 is an excellent marker to identify
the leukemic (CD10+) cell population within cALL or
pre-B-ALL. To study the integrin-mediated adhesion in ALL, we have
developed a novel adhesion assay that allows rapid analysis of LFA-1-
and VLA-4-mediated adhesion of large numbers of samples by flow
cytometry. By using dual-color fluorescence analysis with CD10 as a
marker, we were able to measure the adhesion of both the
CD10 and CD10+ cell population within
one BM or blood sample. The results demonstrate that, even though the
leukemic blasts from most ALL patients express normal levels of LFA-1
and VLA-4, these integrins can not be activated by intracellular
signalling and thus are defective on ALL blasts.
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MATERIALS AND METHODS |
Cells.
Samples were obtained from 20 untreated B-lineage ALL patients 16 to 64 years of age at the time of initial diagnosis. Diagnosis of B-lineage
ALL was based on routine morphological/cytochemical evaluation
according to the standard French-American-British criteria as well as
by immunophenotyping using a panel of well-characterized monoclonal
antibodies (MoAbs). Mononuclear cell fractions were isolated from BM or
peripheral blood samples by Ficoll-Hypaque density gradient
centrifugation. All patients were from the same hospital center
(University Hospital Nijmegen, Nijmegen, The Netherlands). The
B-lineage ALL subclassification for common ALL and pre-B-ALL used in
this study has been described elsewhere.53 Briefly, the
leukemic lymphoblast population in common ALL and pre-B-ALL are
positive for both CD10 and CD19, whereas the more differentiated pre-B-ALL also express cytoplasmic Igµ. The cALL and pre-B-ALL patients chosen here are also positive for CD34, except for patients no. 13, 15, 18, and 19. Patients no. 1 through 15 have been diagnosed as cALL, and patients no. 16 through 20 have been diagnosed as pre-B-ALL. All samples were obtained from BM, except for those from
patients no. 14 and 20, which were obtained from peripheral blood.
Isolation of CD34+ cells from healthy donors
(donors d1 through d4).
CD34+ cells from 4 healthy BM donors were isolated as
follows. CD34+ cells were rosetted with anti-CD34
MoAb-coated magnetic beads (Dynal, Oslo, Norway) for 60 minutes at
4°C with gentle rotation. CD34+ cells were collected
magnetically and subsequently released from the beads with DETACHaBEAD
(Dynal, Oslo, Norway). Isolated cells were free from beads and their
purity exceeded 95% as determined with flow cytometry.54
MoAbs.
The anti-2 chain MoAb KIM185 and the anti-1 chain MoAb TS2/16
were used to activate LFA-155 and VLA-4,37
respectively. The anti- L MoAb NKI-L15 and the anti-4 MoAb HP2/1
were used to specifically block the adhesion of LFA-116 and
VLA-4,56 respectively. CD10, the cALL antigen (CALLA)-MoAb
clone B-E3, was obtained from Immuno Quality Products (Groningen, The Netherlands).
Plate adhesion assay.
Cell adhesion to both ICAM-1 and VCAM-1 was performed as follows. A
96-well flat-bottom plate (MaxiSorp; Nunc, Roskilde, Denmark) was
precoated with 50 µL goat-antihuman Fc-specific
F(ab')2 (4 µg/mL; Jackson Immuno Research
Laboratories, Inc, West Grove, PA) for 1 hour at 37°C and blocked
with 1% bovine serum albumin (BSA) in Tris-sodium buffer (20 mmol/L
Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1 mmol/L CaCl2, 2 mmol/L
MgCl2) for 30 minutes at 37°C. The plate was coated
with 500 ng/mL ICAM-1 Fc or VCAM-1 Fc protein overnight at 4°C.
ICAM-1-Fc or VCAM-1-Fc consist of the extracellular part of both
proteins fused to a human IgG1 Fc fragment. ICAM-1-Fc was produced in
Chinese Hamster Ovary K1 cells cotransfected with the
ICAM-1-IgG1Fc26 (20 µg) and pEE14 (5 µg) vector similar to how it is described for soluble CD4.57 The
ICAM-1-Fc concentration in the supernatant was determined by an IgG1
enzyme-linked immunosorbent assay (ELISA), and the supernatant was used
without further purification. Purified VCAM-1-Fc was kindly provided by
Dr Roy Lobb (Biogen, Cambridge, MA).58 Cells
(20,000 to 40,000/well) were labeled in phosphate-buffered
saline (PBS) with Calcein-AM (25 µg/107 cells/mL;
Molecular Probes, Eugene, OR) for 30 minutes at 37°C. Labeled cells
were washed and preincubated for 15 minutes at room temperature
(RT) with different stimuli (100 nmol/L PMA [Calbiochem, La Jolla, CA], 5 µg/mL activating MoAbs, and/or 10 µg/mL blocking MoAbs). Cells were allowed to adhere for 30 minutes at
37°C. Nonadherent cells were removed by three washes with warm
Tris-sodium-BSA buffer (20 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1 mmol/L CaCl2, 2 mmol/L MgCl2, 0.5% BSA
[wt/vol]). The adherent cells were lysed with 100 µL lysis buffer
(50 mmol/L Tris, 0.1% Triton X-100), and the fluorescence was
quantified using the Cytofluor II (Perseptive Biosystems, Framingham,
MA). Results are expressed as the mean percentage of cells binding from
triplicate wells. Values are depicted as integrin-specific adhesion,
ie, cell adhesion percentage minus cell adhesion percentage in the
presence of an integrin-blocking MoAb.
Ligand coating of fluorescent microspheres.
Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0 µm; Molecular
Probes) were coated with adhesion ligands as follows. Streptavidin was
covalently coupled to the TransFluorSpheres as described by the
manufacturer. Briefly, 20 µL streptavidin (5 mg/mL in 50 mmol/L
MES-buffer) was added to 50 µL TransFluorSpheres. Thirty microliters
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC; 1.33 mg/mL)
was added and the mixture was incubated at RT for 2 hours. The reaction
was stopped by the addition of glycin to a final concentration of 100 mmol/L. The streptavidin-coated beads were washed three times with PBS
(50 mmol/L phosphate, 0.9% NaCl, pH 7.4) and resuspended in 150 µL
PBS, 0.5% BSA (wt/vol). This suspension remains stable for 2 months if
stored at 4°C. The streptavidin-coated beads (15 µL) were
incubated with biotinylated goat-antihuman anti-Fc Fab2
fragments (6 µg/mL) in 0.5 mL PBS, 0.5% BSA for 2 hours at 37°C. The beads were washed once with PBS, 0.5% BSA and
incubated with human IgG1 Fc fused ligands (ICAM-1 Fc, VCAM-1 Fc; 250 ng/mL) in 0.5 mL overnight at 4°C. The ligand-coated beads were
washed, resuspended in 100 µL PBS, 0.5% BSA, and stored at 4°C.
Fluorescent beads adhesion assay.
For cell adhesion to ICAM-1 and VCAM-1, cells were resuspended in
Tris-sodium-BSA (20 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1 mmol/L
CaCl2, 2 mmol/L MgCl2, 0.5% BSA [wt/vol]; 5 × 106 cells/mL). Fifty thousand cells were
preincubated with or without LFA-1- or VLA-4-blocking MoAb (20 µg/mL) for 10 minutes at RT in a 96-well V-shaped-bottom plate. The
ligand-coated beads (20 beads/cell) and different integrin stimuli (100 nmol/L PMA; LFA-1- or VLA-4-activating MoAbs: KIM185 and TS2/16 [10
µg/mL]) were added and the suspension was incubated for 30 minutes
at 37°C. The cells were washed with the Tris-sodium-BSA buffer and
incubated for 10 minutes at RT with fluorescein isothiocyanate
(FITC)-conjugated anti-CD10 antibody. After washing, the cells were
resuspended in 100 µL Tris-sodium-BSA buffer. LFA-1- or
VLA-4-mediated adhesion of the CD10+ and
CD10 cells was measured by flow cytometry using the
FACScan (Becton Dickinson & Co, Oxnard, CA). Values are depicted as
integrin-specific adhesion, ie, cell adhesion percentage minus cell
adhesion percentage in the presence of a specific anti-integrin
blocking MoAb.
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RESULTS |
Fluorescent beads adhesion assay.
To measure the LFA-1- and VLA-4-mediated adhesion of the leukemic
cell population of a large number of B-lineage ALL patients, we
developed a new adhesion assay using fluorescent beads indirectly coated with integrin ligands. This new assay was compared with the
standard plate adhesion assay by measuring the LFA-1- and VLA-4-mediated adhesion of resting peripheral blood lymphocytes (PBL)
in both assays (Fig 1). LFA-1-mediated
adhesion, as measured with the novel fluorescent beads adhesion assay,
is shown in Fig 1A. Thirty-one percent of the PBL have bound ICAM-1
beads after stimulation of LFA-1 with PMA (frame 1). The defined peaks
in frame 2 represent cells that have bound 1 bead, 2 beads, and more beads, respectively. Binding was LFA-1 specific, because it could be
blocked by the blocking anti-LFA-1 MoAb NKI-L15 (Fig 1B, frames 1 and
2). Similar results were obtained with the plate adhesion assay (Fig
1C). VLA-4-mediated adhesion, as measured with both assays, is shown
in Fig 1D. Comparison of both assays demonstrates that the results
obtained with the fluorescent beads adhesion assay are similar to those
obtained with the standard plate adhesion assay, except that the
adhesion measured with the fluorescent beads adhesion assay is
substantially higher and more sensitive. Because the fluorescent beads
adhesion assay is less time-consuming and directly measurable on the
flow cytometer, it is very well suited for screening large numbers of
samples. Furthermore, only the fluorescent beads adhesion assay allows
us to screen the adhesive properties of different subpopulations of
cells within one sample by performing double fluorescent labeling
techniques with distinct FITC-labeled markers.
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| Fig 1.
LFA-1- and VLA-4-mediated adhesion of PBL from a
healthy donor both measured with the fluorescent beads and the plate
adhesion assay. Binding of ICAM-1-coated fluorescent beads by PBL
after stimulation with PMA (A) and in the presence of anti-LFA-1
blocking antibody NKI-L15 (B). (C and D) Comparison of the adhesion as
measured with both assays. Adhesion was measured without any stimulus
and after stimulation with PMA or an activating anti-2 (KIM185) or
anti-1 (TS2/16) MoAb. The specificity was determined by measuring
adhesion in the presence of blocking anti-LFA-1 (NKI-L15) or
anti-VLA-4 (HP2/1) MoAb.
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LFA-1-mediated adhesion of CD10+ ALL cells.
BM or peripheral blood samples were collected from patients that either
suffer from cALL or pre-B-ALL. LFA-1-mediated adhesion of both the
CD10 and leukemic (CD10+) cells in these
B-lineage ALL samples was determined using the fluorescent beads
adhesion assay. A representative set of data (patient no. 11) is shown
in Fig 2. BM aspirate from patient no. 11 contains 3 cell populations with different CD10 antigen expression levels, ie, CD10 (19%), CD10dim (28%),
and CD10bright (53%; Fig 2A, frame 1). Both the
CD10dim and CD10bright cell population
represent the leukemic cell population. As shown, 25% of the
CD10 cells have bound the ICAM-1-coated beads after
PMA activation (frame 2), whereas only 9% of the CD10dim
(frame 3) and 4% of the CD10bright cells (frame 4) have
bound ICAM-1-coated beads despite a high expression of LFA-1 (Fig 2C).
Thus, LFA-1 expressed by the leukemic populations is not responsive to
PMA, whereas LFA-1 expressed by the CD10 cell
population is activated by PMA. However, adhesion of the CD10+ and CD10 cell populations could be
stimulated with the activating anti-2 antibody KIM185 (Fig 2B).
These findings clearly demonstrate that functional adhesion defects can
be observed in B-lineage ALL by measuring adhesion in defined cell
populations (CD10 staining).
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| Fig 2.
LFA-1-mediated adhesion of patient no. 11 and control
PBL measured with the fluorescent beads adhesion assay. Double staining
with CD10-FITC MoAb was used to distinguish between the leukemic
(CD10+) cell population and the CD10 cell
population within one sample. (A) Adhesion of the different CD10 cell
populations after stimulation with PMA. (B) LFA-1-mediated adhesion of
the leukemic (CD10dim and CD10bright) and the
CD10 cell population, in comparison with the adhesion of
PBL from a healthy donor. Standard deviations are less than 5%. (C)
LFA-1 expression of the various CD10 cell populations.
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LFA-1-mediated adhesion of B-lineage CD10+ ALL
cells.
Table 1 summarizes the data of the LFA-1
mediated adhesion and LFA-1 expression of the CD10+
leukemic cell population from 20 B-lineage ALL patients (patients no. 1 through 20) and of the
CD19+/CD34+/CD10+ cell population
from 4 healthy donors (donors d1 through d4). The integrin-mediated
adhesion of the CD19+/CD34+/CD10+
cells from healthy donors was determined by isolating the
CD34+ cells from BM with magnetic beads and subsequently by
selecting for the CD10+ cell population in the fluorescent
beads adhesion assay. These CD34+/CD10+ cells
are appropriate controls for the B-lineage ALL cells, because the
majority are also CD19+59-61 as was determined by triple
CD19/CD10/CD34 fluorescence analysis (>96%; results not shown). The
LFA-1 expression on the CD10+ populations (Table 1) was
determined with a CD10/LFA-1 dual-color fluorescence analysis.
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Table 1.
LFA-1-Mediated Adhesion and Expression of the Leukemic
(CD10+) Cell Populations in B-Lineage ALL Patients
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The leukemic cell populations of 6 patients show a normal
LFA-1-mediated adhesion pattern similar to that of the
CD19+/CD34+/CD10+ cell population
from healthy donors. LFA-1 present on these cells is slightly active
and can be further activated both with the phorbol ester PMA and with
the activating antibody (KIM185). Ten patients show a KIM185-inducible
activation of LFA-1 adhesion; however, the LFA-1 of these ALL patients
fails to respond to intracellular signals (PMA), despite the fact that
LFA-1 is expressed on the cell surface. Finally, 5 ALL patients do not
show any LFA-1-mediated adhesion (neither after PMA nor KIM185
stimulation) due to the absence of LFA-1 on the surface of the leukemic
cells (Table 1). Because 4 of these patients suffer from pre-B-ALL, we
have analyzed the LFA-1 expression on pre-B cells from healthy donors
by cytµ/LFA-1 dual fluorescence analysis on CD34+ cells
(results not shown). This analysis showed that normal pre-B cells do
express LFA-1.
It is of note that the CD10dim population of patient no. 1 does not express LFA-1, whereas the CD10bright cell
population expresses functional LFA-1. In contrast, the CD10dim and CD10bright populations of both
patients no. 8 and 11 have similar LFA-1-mediated adhesive properties.
We could exclude that unresponsiveness of LFA-1 towards PMA activation
was due to low viability of the leukocytes, because the LFA-1-mediated
adhesion of the CD10 cell population of these
patients (no. 5, 9, 12, 14, and 15) was responsive to PMA similar to
the results obtained in patient no. 11 (Fig 2; for other patients, data
not shown).
VLA-4-mediated adhesion of B-lineage CD10+ ALL
cells.
Similar to 2 integrins, we studied the capacity of ALL leukemic
cells to bind VCAM-1-coated fluorescent beads to the 1 integrin VLA-4. The results are summarized in Table
2. The VLA-4 expression on the CD10+ populations was
determined using a CD10/VLA-4 dual-color fluorescence analysis.
In 7 ALL patients, VLA-4 is constitutively active on CD10+
cells. The adhesion is high without any stimulation and cannot be further activated by PMA or the activating anti-1 MoAb TS2/16, indicating that VLA-4 is already maximally active. Similar results were
obtained with CD19+/CD34+/CD10+
cells from a healthy donor (donors d1 through d4), indicating that
VLA-4 is constitutively active on normal
CD19+/CD34+/CD10+ cells. In 7 other
ALL patients, VLA-4 adhesion increases after stimulation with either
PMA or TS2/16. In these patients, VLA-4 activity is very high but not
maximal before stimulation. In 7 other patients, VLA-4 on the leukemic
cells is minimally active without any activation. Furthermore, in these
patients, VLA-4 cannot be activated by treating the cells with PMA,
whereas VLA-4 is readily activated with the activating anti-1
antibody TS2/16. Analysis of the expression pattern of VLA-4 in
B-lineage ALL demonstrates that the defects are not due to lack of
VLA-4 surface expression in these patients. As shown in Table 2, the
leukemic cells of the ALL patients (no. 2, 3, 11, 10, 13, 18, and 19)
express comparable levels of VLA-4 as other ALL patients, suggesting
that VLA-4 is functionally defective in these 7 ALL. This is also shown
in Fig 3, in which identical VLA-4
expression levels on the CD10+ cells from donor d1 and ALL
patients no. 11 and 14 are shown, whereas the VLA-4-mediated adhesion
of these patients is very different (Table 2).
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| Fig 3.
VLA-4 expression of the CD10 cell populations from a
healthy donor and 2 representative B-lineage ALL patients (no. 11 and
14). VLA-4 expression was measured with the anti-VLA-4 antibody HP2/1.
The mean fluorescence of the VLA-4 expression of the
CD10+ cell population is depicted in the top right corner
of the figures.
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The LFA-1- and VLA-4-mediated adhesion defects found in the leukemic
cells from the different patients have been summarized in
Table 3. The leukemic cells
(CD10+) from 17 of 20 patients contain either LFA-1-
and/or VLA-4-mediated adhesion defects, of which the LFA-1-mediated
adhesion defects are most predominant. Furthermore, 5 of these patients
contain leukemic blasts with both a LFA-1 and a VLA-4 adhesion defect.
Comparison of the adhesion of CD10+ ALL cells
present in BM and in peripheral blood.
The egress of specific leukemic cells from the BM into the periphery
could be a result from different adhesive properties. Therefore, we
compared the capacity of both BM- and peripheral blood-derived leukemic
cells of 6 patients to mediate LFA-1- and VLA-4-mediated adhesion
(Table 4). Patients no. 3, 5, 12, and 17 have a similar LFA-1- and VLA-4-mediated adhesion pattern independent of the source, ie, BM or peripheral blood. The leukemic cells of
patients no. 2 and 18 have different adhesion characteristics depending
on the source of cells. Furthermore, the CD10 expression of the
circulating leukemic cells from both patients is higher than that of
the leukemic cells derived from the BM.
| |
DISCUSSION |
In this study, we have investigated the LFA-1- and VLA-4-mediated
adhesion in B-lymphocyte lineage CD10+ ALL using a new
fluorescent beads adhesion assay that enabled us to specifically
measure adhesion of the leukemic cell population within a heterogeneous
BM or peripheral blood sample. We show that the leukemic cells from
85% of the ALL patients investigated have a LFA-1- and/or
VLA-4-mediated adhesion defect. The LFA-1-mediated adhesion defects
observed in the ALL patients are most predominant. The LFA-1-mediated
adhesion defects are either due to the lack of LFA-1 expression on the
surface or due to the presence of nonfunctional LFA-1. The
VLA-4-mediated adhesion defects are due to the cell surface expression
of nonfunctional VLA-4. This study clearly demonstrates the importance
of investigating integrin functionality in addition to integrin
expression on specific leukemic cell populations.
In B-lineage ALL we have focused on both common ALL (cALL) and
precursor B-ALL (pre-B-ALL) using CD10 antigen expression as a
discriminating marker for the leukemic populations. Because samples
from ALL patients contain CD10 and leukemic
(CD10+) cell populations in variable ratios, it is
necessary to distinguish between both populations when measuring
adhesion. The standard plate adhesion method, involving integrin
ligands coated onto plastic, is not suitable to measure the adhesion of
different subpopulations within one BM sample simultaneously.
Furthermore, due to the low amount of material, it is difficult to
obtain enough CD10-sorted cells to perform plate adhesion assays.
Therefore, we have developed a new adhesion assay involving fluorescent
beads indirectly coated with integrin ligands, which can be measured in
a flow cytometer, and by double staining the cells with an FITC-labeled
marker it is possible to distinguish between different cell
populations. We have shown that the LFA-1- and VLA-4-mediated adhesion of PBL from a healthy donor measured with the fluorescent beads adhesion assay is substantially higher than that measured with
the standard plate adhesion assay (Fig 1). In a plate adhesion assay,
the cells have to adhere to and subsequently spread on the
ligand-coated plastic to resist being washed away. In the beads
adhesion assay, the cells do not need to spread; therefore, the
adhesion in the plate adhesion assay is lower. Furthermore, in a
standard adhesion assay, the force of the multiple washing steps is
very important in defining the amount of adhesion measured, and it is
difficult to standardize these washing steps, whereas the single
washing step in the beads adhesion assay does not influence the amount
of beads bound and therefore is more reproducible between researchers
(data not shown).
With this novel fluorescent beads adhesion assay, we could for the
first time discriminate between adhesion of leukemic CD10+
and CD10 cell populations within one BM or blood
sample. Of the 20 ALL patients measured, 6 showed a normal LFA-1
function on the leukemic cells comparable with
CD19+/CD34+/CD10+ cells from
healthy donors (Table 1). The CD10+ cells from 5 ALL
patients (Table 1; patients no. 1, 17, 18, 19, and 20) did not exhibit
any LFA-1-mediated adhesion, due to the lack of LFA-1 expression.
Interestingly, from the 5 pre-B-ALL patients investigated, 4 contained
leukemic cells that did not express LFA-1 on the cell surface. LFA-1 is
expressed on immature CD10+ B cells62 and, as
we showed, on pre-B-cells from healthy donors, which indicates that the
LFA-1 expression on pre-B-ALL cells from these patients is defective. A
greater number of pre-B-ALL patients should be tested to determine
whether the lack of LFA-1 expression is more often observed in
pre-B-ALL patients than cALL. Surprisingly, the CD10+ cells
from 10 B-lineage ALL patients expressed LFA-1 that could not be
activated by PMA through inside-out signalling, whereas the
CD10 cell population responded normally. The phorbol
ester PMA activates protein kinase C and thereby triggers an
intracellular signaling pathway resulting in the activation of both
1- and 2-integrins.29,30 Recently, we and others
observed that certain leukemic T-cell lines (CEM and Jurkat) express
LFA-1 that could not be activated by several stimuli known to induce
ligand binding through intracellular signaling
pathways.63,64 The unresponsiveness of LFA-1 to PMA in
these cell lines could be caused by mutations in the intracellular part
of either the L or 2 chain of LFA-1 or by the absence of crucial
cytoplasmic signaling elements that are involved in the inside-out
activation of 2 integrins. Recently, Mobley et al65 showed that a specific Jurkat mutant contains a mutation associated with an altered form of the mitogen-activated protein kinase ERK1 that
explains the lack of PMA responsiveness of the integrins. The existence
of essential elements necessary for intracellular activation of
integrins is also shown by a defect in LFA-1-mediated adhesion after
PMA triggering observed in the erythroid leukemic CML cell line K562
when transfected with wild-type LFA-1.66 This indicates
that the leukemic cell line K562 lacks crucial signaling elements
necessary to activate LFA-1 through the 2 cytoplasmic tail. It is
tempting to speculate that CD10+ leukemic cells that
express LFA-1 that cannot be activated by PMA might also contain
mutations in protein kinases involved in integrin activation or in
other intracellular signaling molecules such as cytohesin, which has
recently been shown to be involved in integrin signaling.67
The leukemic cells of these patients will be investigated in more
detail to elucidate the signaling defect.
Examining VLA-4-mediated adhesion in B-lineage ALL shows that, in 7 patients, VLA-4 is constitutively active on the leukemic cells, similar
to the CD19+/CD34+/CD10+ cells from
healthy donors (Table 2). In the CD10+ cells from 7 other
ALL patients, unstimulated VLA-4-mediated adhesion is high but can be
further enhanced by PMA or activating antibodies. The leukemic cells
from the remaining 7 patients express VLA-4 that is not functional.
Because the defects are not due to the cell surface expression levels
of VLA-4 (Fig 3 and Table 2), the aberrant adhesion could be caused by
a signaling defect that maintains VLA-4 in a low-affinity state. The
leukemic cells from 3 of these patients also express a nonfunctional
form of LFA-1. This indicates that these leukemic cells have defects
both in the 1- and 2-integrin activation pathways and that
regulatory proteins involved in the adhesion of both 1- and
2-integrins could be defective, such as protein kinase C or certain
cytoskeletal regulatory components.
The differences found in the activation of VLA-4 could be extremely
important to survival, retention, and proliferation of the ALL blast
cells in the BM. VLA-4 and VLA-5 have been shown to be important in the
binding of ALL blasts to the BM stroma.49,51,52,68,69 Manabe et al51 showed that leukemic cell contact with
stromal cells can prevent apoptosis in ALL blasts. It has been
suggested that VLA-4/VCAM-1 interactions could affect ALL blast
survival and proliferation by transmitting signals (outside-in
signaling) to the cells. Blocking studies demonstrated that adhesion of
B-cell precursors to BM stroma is also mediated by VLA-4-VCAM-1 and
could be regulated by cytokines that specifically increase or decrease cell-surface VCAM-1 expression.70 Furthermore, Arroyo et
al71 demonstrated the importance of VLA-4 function in
retaining progenitor cells in the BM for proper maturation. Hence, ALL
blast cell survival may have major effects on leukemic cell growth and
survival. The recent observation that binding to fibronectin may
inhibit progenitor cell proliferation further supports this
notion.72 Thus, differences in VLA-4 activities between
different patients with ALL could reflect different proliferating and
migratory properties, as has been demonstrated for chronic myeloid
leukemia (CML). CML is a leukemia characterized by an abnormal,
premature release of primitive progenitors and precursors in the blood
and by the continuous proliferation of the malignant progenitor
population. In vitro, CML progenitors fail to adhere to or be regulated
by marrow stroma, and Verfaillie et al73 showed that this
is due to aberrant VLA-4- and VLA-5-mediated adhesion. Treatment of
the CML progenitors with interferon- (IFN-) restored the adhesive
properties of these progenitors.74 Because IFN- is known
to induce remission in CML patients, it is feasible that the rescued
integrin-mediated adhesion restores adhesion-mediated proliferation of
the CML progenitors.
Comparison of the clinical characteristics observed in the B-lineage
ALL patients, such as egress of the leukemic cells into the blood,
homing, and cell turn-over, with the aberrant adhesion of the leukemic
cells indicates a trend for a correlation between a high blood cell
count and a defective VLA-4 function (P = .05). From the 20 patients investigated, 6 had high peripheral blood cell counts (>50 × 109/L; data not shown); interestingly, the leukemic
cells from 4 of these patients show a VLA-4 defect. Furthermore, we
demonstrated that, within 1 patient, leukemic cells derived from either
the BM or the periphery have similar adhesive properties (Table 4; patients no. 3, 5, 12, and 17). In 2 patients (Table 4; patients no. 2 and 18), the CD10 antigen expression of the leukemic cells derived from
the BM was different from those derived from the periphery. The
integrin-mediated adhesion of the leukemic cells from both sources was
also different, indicating that, within 1 patient, there may be
different leukemic cell populations with different adhesive properties.
Because the adhesive properties of the leukemic cells present in the
periphery of both patients are different, it is likely that more
factors regulate the egress of malignant lymphoblasts into the periphery.
In summary, we have demonstrated that not only the expression of
integrins in relation to hematopoietic malignancies should be measured,
but more importantly also the function of the integrin receptors. Using
a newly developed fluorescent beads adhesion assay, we were able to
analyze both the LFA-1- and VLA-4-mediated adhesion of the leukemic
cells within B-lineage ALL patients, even though the samples were
heterogeneous. We found that most ALL patients contained leukemic cell
populations exhibiting adhesion defects either in LFA-1- and/or
VLA-4-mediated adhesion, even though the leukemic cells had normal
expression levels of these integrins. Studies to analyze the molecular
basis of these defects in the different patients are in progress.
| |
ACKNOWLEDGMENT |
The authors thank Dr R. Lobb, Dr D. Simmons, Dr E. Martz, and Dr M. Robinson for kindly providing recombinant VCAM-1-Fc DNA construct,
ICAM-1-Fc DNA construct, TS2/16 antibody, and KIM185 antibody,
respectively. We are also grateful to M. Leenders from the Department
of Hematology for reimmunophenotyping some of the ALL patients and R. Torensma for his help with the triple fluorescence analyses.
| |
FOOTNOTES |
Submitted June 17, 1998; accepted March 11, 1999.
Supported by the Dutch Cancer Society (NKB; Grant No. 96-1358) and the
Netherlands Organization for Scientific Research (NWO; Grant No.
901-09-244).
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 Yvette van Kooyk, PhD, Tumor
Immunology Laboratory, University Hospital Nijmegen, Philips van
Leydenlaan 25, 6525 EX Nijmegen, The Netherlands; Y. van
Kooyk{at}dent.kun.nl.
| |
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K. Gijzen, P. J. Tacken, A. Zimmerman, B. Joosten, I. J. M. de Vries, C. G. Figdor, and R. Torensma
Relevance of DC-SIGN in DC-induced T cell proliferation
J. Leukoc. Biol.,
March 1, 2007;
81(3):
729 - 740.
[Abstract]
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W. ten Hove, L. A. Houben, J. A. M. Raaijmakers, L. Koenderman, and M. Bracke
Rapid Selective Priming of Fc{alpha}R on Eosinophils by Corticosteroids
J. Immunol.,
November 1, 2006;
177(9):
6108 - 6114.
[Abstract]
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A. Cambi, B. Joosten, M. Koopman, F. de Lange, I. Beeren, R. Torensma, J. A. Fransen, M. Garcia-Parajo, F. N. van Leeuwen, and C. G. Figdor
Organization of the Integrin LFA-1 in Nanoclusters Regulates Its Activity
Mol. Biol. Cell,
October 1, 2006;
17(10):
4270 - 4281.
[Abstract]
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A. W. Zimmerman, B. Joosten, R. Torensma, J. R. Parnes, F. N. van Leeuwen, and C. G. Figdor
Long-term engagement of CD6 and ALCAM is essential for T-cell proliferation induced by dendritic cells
Blood,
April 15, 2006;
107(8):
3212 - 3220.
[Abstract]
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D. Brandsma, L. Ulfman, J. C. Reijneveld, M. Bracke, M. J.B. Taphoorn, J. J. Zwaginga, M. F.B. Gebbink, H. de Boer, L. Koenderman, and E. E. Voest
Constitutive integrin activation on tumor cells contributes to progression of leptomeningeal metastases
Neuro-oncol,
April 1, 2006;
8(2):
127 - 136.
[Abstract]
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N. Dakappagari, T. Maruyama, M. Renshaw, P. Tacken, C. Figdor, R. Torensma, M. A. Wild, D. Wu, K. Bowdish, and A. Kretz-Rommel
Internalizing Antibodies to the C-Type Lectins, L-SIGN and DC-SIGN, Inhibit Viral Glycoprotein Binding and Deliver Antigen to Human Dendritic Cells for the Induction of T Cell Responses
J. Immunol.,
January 1, 2006;
176(1):
426 - 440.
[Abstract]
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S. Verploegen, L. Ulfman, H. W. M. van Deutekom, C. van Aalst, H. Honing, J.-W. J. Lammers, L. Koenderman, and P. J. Coffer
Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function
Blood,
August 1, 2005;
106(3):
1076 - 1083.
[Abstract]
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K. P.J.M. van Gisbergen, C. A. Aarnoudse, G. A. Meijer, T. B.H. Geijtenbeek, and Y. van Kooyk
Dendritic Cells Recognize Tumor-Specific Glycosylation of Carcinoembryonic Antigen on Colorectal Cancer Cells through Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin
Cancer Res.,
July 1, 2005;
65(13):
5935 - 5944.
[Abstract]
[Full Text]
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K. P.J.M. van Gisbergen, M. Sanchez-Hernandez, T. B.H. Geijtenbeek, and Y. van Kooyk
Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN
J. Exp. Med.,
April 18, 2005;
201(8):
1281 - 1292.
[Abstract]
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C. V. Cox, R. S. Evely, A. Oakhill, D. H. Pamphilon, N. J. Goulden, and A. Blair
Characterization of acute lymphoblastic leukemia progenitor cells
Blood,
November 1, 2004;
104(9):
2919 - 2925.
[Abstract]
[Full Text]
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J.-F. Arrighi, M. Pion, M. Wiznerowicz, T. B. Geijtenbeek, E. Garcia, S. Abraham, F. Leuba, V. Dutoit, O. Ducrey-Rundquist, Y. van Kooyk, et al.
Lentivirus-Mediated RNA Interference of DC-SIGN Expression Inhibits Human Immunodeficiency Virus Transmission from Dendritic Cells to T Cells
J. Virol.,
October 15, 2004;
78(20):
10848 - 10855.
[Abstract]
[Full Text]
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V. I. Otto, T. Schurpf, G. Folkers, and R. D. Cummings
Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes
J. Biol. Chem.,
August 20, 2004;
279(34):
35201 - 35209.
[Abstract]
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E. Van Liempt, A. Imberty, C. M. C. Bank, S. J. Van Vliet, Y. Van Kooyk, T. B. H. Geijtenbeek, and I. Van Die
Molecular Basis of the Differences in Binding Properties of the Highly Related C-type Lectins DC-SIGN and L-SIGN to Lewis X Trisaccharide and Schistosoma mansoni Egg Antigens
J. Biol. Chem.,
August 6, 2004;
279(32):
33161 - 33167.
[Abstract]
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I. S. Ludwig, A. N. Lekkerkerker, E. Depla, F. Bosman, R. J. P. Musters, S. Depraetere, Y. van Kooyk, and T. B. H. Geijtenbeek
Hepatitis C Virus Targets DC-SIGN and L-SIGN To Escape Lysosomal Degradation
J. Virol.,
August 1, 2004;
78(15):
8322 - 8332.
[Abstract]
[Full Text]
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A. W. Zimmerman, J. M. D. T. Nelissen, S. E. van Emst-de Vries, P. H. G. M. Willems, F. de Lange, J. G. Collard, F. N. van Leeuwen, and C. G. Figdor
Cytoskeletal restraints regulate homotypic ALCAM-mediated adhesion through PKC{alpha} independently of Rho-like GTPases
J. Cell Sci.,
June 1, 2004;
117(13):
2841 - 2852.
[Abstract]
[Full Text]
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J. W. Lee, H. Y. Chung, L. A. Ehrlich, D. F. Jelinek, N. S. Callander, G. D. Roodman, and S. J. Choi
IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells
Blood,
March 15, 2004;
103(6):
2308 - 2315.
[Abstract]
[Full Text]
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H. Honing, T. K. van den Berg, S. M. A. van der Pol, C. D. Dijkstra, R. A. van der Kammen, J. G. Collard, and H. E. de Vries
RhoA activation promotes transendothelial migration of monocytes via ROCK
J. Leukoc. Biol.,
March 1, 2004;
75(3):
523 - 528.
[Abstract]
[Full Text]
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A. Cambi, F. de Lange, N. M. van Maarseveen, M. Nijhuis, B. Joosten, E. M.H.P. van Dijk, B. I. de Bakker, J. A.M. Fransen, P. H.M. Bovee-Geurts, F. N. van Leeuwen, et al.
Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells
J. Cell Biol.,
January 5, 2004;
164(1):
145 - 155.
[Abstract]
[Full Text]
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I. van Die, S. J. van Vliet, A. K. Nyame, R. D. Cummings, C. M.C. Bank, B. Appelmelk, T. B.H. Geijtenbeek, and Y. van Kooyk
The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x
Glycobiology,
June 1, 2003;
13(6):
471 - 478.
[Abstract]
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T. B.H. Geijtenbeek, S. J. van Vliet, E. A. Koppel, M. Sanchez-Hernandez, C. M.J.E. Vandenbroucke-Grauls, B. Appelmelk, and Y. van Kooyk
Mycobacteria Target DC-SIGN to Suppress Dendritic Cell Function
J. Exp. Med.,
January 6, 2003;
197(1):
7 - 17.
[Abstract]
[Full Text]
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A. A. Bashirova, L. Wu, J. Cheng, T. D. Martin, M. P. Martin, R. E. Benveniste, J. D. Lifson, V. N. KewalRamani, A. Hughes, and M. Carrington
Novel Member of the CD209 (DC-SIGN) Gene Family in Primates
J. Virol.,
December 6, 2002;
77(1):
217 - 227.
[Abstract]
[Full Text]
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L. Wu, T. D. Martin, R. Vazeux, D. Unutmaz, and V. N. KewalRamani
Functional Evaluation of DC-SIGN Monoclonal Antibodies Reveals DC-SIGN Interactions with ICAM-3 Do Not Promote Human Immunodeficiency Virus Type 1 Transmission
J. Virol.,
May 13, 2002;
76(12):
5905 - 5914.
[Abstract]
[Full Text]
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M. Lechmann, D. J.E.B. Krooshoop, D. Dudziak, E. Kremmer, C. Kuhnt, C. G. Figdor, G. Schuler, and A. Steinkasserer
The Extracellular Domain of CD83 Inhibits Dendritic Cell-mediated T Cell Stimulation and Binds to a Ligand on Dendritic Cells
J. Exp. Med.,
December 17, 2001;
194(12):
1813 - 1821.
[Abstract]
[Full Text]
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A. A. Bashirova, T. B.H. Geijtenbeek, G. C.F. van Duijnhoven, S. J. van Vliet, J. B.G. Eilering, M. P. Martin, L. Wu, T. D. Martin, N. Viebig, P. A. Knolle, et al.
A Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Nonintegrin (DC-SIGN)-related Protein Is Highly Expressed on Human Liver Sinusoidal Endothelial Cells and Promotes HIV-1 Infection
J. Exp. Med.,
March 12, 2001;
193(6):
671 - 678.
[Abstract]
[Full Text]
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F. Prosper and C. M. Verfaillie
Regulation of hematopoiesis through adhesion receptors
J. Leukoc. Biol.,
March 1, 2001;
69(3):
307 - 316.
[Abstract]
[Full Text]
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P. G. Corn, B. D. Smith, E. S. Ruckdeschel, D. Douglas, S. B. Baylin, and J. G. Herman
E-Cadherin Expression Is Silenced by 5' CpG Island Methylation in Acute Leukemia
Clin. Cancer Res.,
November 1, 2000;
6(11):
4243 - 4248.
[Abstract]
[Full Text]
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G. J. A. Wanten, T. B. H. Geijtenbeek, R. A. P. Raymakers, Y. van Kooyk, D. Roos, J. B. M. J. Jansen, and A. H. J. Naber
Medium-Chain, Triglyceride-Containing Lipid Emulsions Increase Human Neutrophil {beta} 2 Integrin Expression, Adhesion, and Degranulation
JPEN J Parenter Enteral Nutr,
July 1, 2000;
24(4):
228 - 233.
[Abstract]
[PDF]
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A. Sigal, D. A. Bleijs, V. Grabovsky, S. J. van Vliet, O. Dwir, C. G. Figdor, Y. van Kooyk, and R. Alon
The LFA-1 Integrin Supports Rolling Adhesions on ICAM-1 Under Physiological Shear Flow in a Permissive Cellular Environment
J. Immunol.,
July 1, 2000;
165(1):
442 - 452.
[Abstract]
[Full Text]
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J. M. D. T. Nelissen, I. M. Peters, B. G. de Grooth, Y. van Kooyk, and C. G. Figdor
Dynamic Regulation of Activated Leukocyte Cell Adhesion Molecule-mediated Homotypic Cell Adhesion through the Actin Cytoskeleton
Mol. Biol. Cell,
June 1, 2000;
11(6):
2057 - 2068.
[Abstract]
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A. Puig-Kroger, C. Lopez-Rodriguez, M. Relloso, T. Sanchez-Elsner, A. Nueda, E. Munoz, C. Bernabeu, and A. L. Corbi
Polyomavirus Enhancer-binding Protein 2/Core Binding Factor/Acute Myeloid Leukemia Factors Contribute to the Cell Type-specific Activity of the CD11a Integrin Gene Promoter
J. Biol. Chem.,
September 8, 2000;
275(37):
28507 - 28512.
[Abstract]
[Full Text]
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D. A. Bleijs, G. C. F. van Duijnhoven, S. J. van Vliet, J. P. H. Thijssen, C. G. Figdor, and Y. van Kooyk
A Single Amino Acid in the Cytoplasmic Domain of the beta 2 Integrin Lymphocyte Function-associated Antigen-1 Regulates Avidity-dependent Inside-out Signaling
J. Biol. Chem.,
March 23, 2001;
276(13):
10338 - 10346.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION