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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 754-764
By
From the Departments of Tumor Immunology and Hematology, University
Hospital Nijmegen, Nijmegen, The Netherlands.
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
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 Among the 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 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 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 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- 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 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.
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
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.
VLA-4-mediated adhesion of B-lineage CD10+ ALL
cells.
Similar to
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.
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.
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.
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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TOP
ABSTRACT
INTRODUCTION
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
1-integrin (CD29)
L MoAb NKI-L15 and the anti-
