Expression of integrins and examination of their adhesive function in

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1993 81: 112-121
Expression of integrins and examination of their adhesive function in
normal and leukemic hematopoietic cells
JL Liesveld, JM Winslow, KE Frediani, DH Ryan and CN Abboud
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Expression of Integrins and Examination of Their Adhesive Function
in Normal and Leukemic Hematopoietic Cells
By Jane L. Liesveld, Jill M. Winslow, Karen E. Frediani, Daniel H. Ryan, and Camille N. Abboud
Adhesion of hematopoietic progenitor cells to marrow-derived adherent cells has been noted for erythroid, myeloid,
and lymphoid precursors. In this report, we have characterizedvery late antigen (VIA) integrin expressionon normal
CD34' marrow progenitors, on leukemic cell lines, and on
blasts from patients with acute myelogenousor monocytic
leukemias. CD34+ progenitor cells expressed the integrin
8, chain (CD29). VLA-4a (CD49d). and VIA-5a (CD49e).
The myeloid lines KGI and KGla also expressed CD49d
and CD49e as did the Mo7e megakaryoblastic line. CD29,
CD18, and CDI 1a were also present on each of these cell
lines. Only the Mo7e line expressed the cytoadhesins
GPllbllla or GPlb. Binding of KGla to marrow stroma was
partially inhibited by antibodies t o CD49d and its ligand,
vascular cell adhesion molecule (VCAM-1). The majority of
leukemic blasts studied expressed CD49d and CD49e as
well. Blasts from patients with acute myelomonocytic leukemia consistently bound to stroma at levels greater than
20%. and adhesion t o stroma could in some cases be partly
inhibited by anti-CD49d. No role for glycosylphosphatidylinositol (GPI)-linkedstructures was demonstrated in these
binding assays because the adhesion of leukemic blasts to
stroma was not diminished after treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). These
studies indicate that CD34+ myeloid progenitors, myeloid
leukemic cell lines, and leukemic blasts possess a similar
array of VLA integrins. Their functional importance individually or in combination with other mediators of attachment
in adhesion, transendothelial migration, and differentiation
has yet to be fully elucidated.
0 1993 by The American Society of Hematology.
N
in development. immune response, platelet aggregation, and
tissue
and it is currently the focus of intense study
regarding its potential role in myeloidi7 and lymphoid hematop~iesis.'~,'~
Integrins are heterodimer glycoproteins
consisting of noncovalently linked CY and p chains. They are
classified according to their p chain into the very late antigen
(VLA) integrins (PI), leukocyte integrins (p2),cytoadhesin
integrins (p3),and additional molecules expressing p4, ps,
p6, p7 (&), or be chains. The P,(VLA) subfamily consists
primarily of receptors for cell matrix components and is expressed in both hematopoietic and nonhematopoietic
cells.20.22
The seven members of the VLA subfamily are distinguished by the association of different a-chains with the
common integrin Pl-chain.'9,2'-23Hematopoietic expression
of the VLA integrins includes VLA-1, VLA-2, and VLA-6
(CD49a, b, e) presence on T ceUs,23.25and VLA-2 and VLA-6
expression on platelets.17,25 VLA-4 (CD49d) and VLA-5
(CD49e) are more widely expressed within the hematopoietic
system,12,14,16,I7,25
ORMAL HEMATOPOIESIS in adult humans occurs
within the bone marrow (BM) under influences of
the marrow microenvironment.' These influences, which
include cellular,2 ~ y t o k i n e ,and
~ . ~extracellular matrix int e r a c t i o n ~all
, ~ contribute to an environment that allows
the hematopoietic progenitor cells to proliferate and differentiate normally. In long-term culture of the BM, an in
vitro system simulating the in vivo microenvironment, an
adherent stromal layer consisting of fibroblasts, macrophages, endothelial cells, and extracellular matrix is important for sustaining hematopoiesis.6 Early progenitor
cells are preferentially associated with the stromal layer,7.'
and adhesion of early progenitor cells to marrow-derived
adherent cells has been previously documented for erythroid,' myeloid,10," and l y m p h ~ i d ' ~precursors.
.'~
Cellcell and cell-matrix interactions between the BM microenvironment and hematopoietic progenitors likely play an
important role in the proliferation and maturation required
for normal hematopoiesis.
A number of potential cell adhesion molecules, which may
mediate essential cell-cell or cell-matrix interactions, have
been identified in experimental models of BM.l4-" The integrin superfamily of cell adhesion molecules plays a key role
From the Hematology Unit and the Departments ofMedicine (Hematology Unit) and Pathology and Laboratory Medicine, University
of Rochester Medical Center, Rochester, NY.
Submitted June 12, 1992; accepted September 8, 1992.
Supported in part by USPHS Grants Kl1 HL02385-OIAI. HL18208, CA-32737, and HL-07152; and the James P. Wilmot Foundation (J.L.L.). J.L.L. was a Special Fellow of the Leukemia Society
of America.
Address reprint requests to Jane L. Liesveld, MD, U M C , Box
610, Hematology Unit, 601 Elmwood Ave, Rochester, N Y 14642.
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.
0 1993 by The American Society of Hematology.
0006-4971/93/8101-0036$3.00/0
112
We have previously investigated the interaction of myeloid progenitors with BM stroma in vitro and reported
that a subset of human marrow cells that possesses the
CD34 antigen binds to marrow stromal monolayers" and,
to a lesser extent, to extracellular matrices of marrow and
to fibronectin and laminin." We also reported that both
normal CD34+ myeloid progenitors and myeloblastic cell
lines (KG I and KGla) express the p2 integrin (CDI 8), but
the adhesive interactions between these myeloid progenitors and marrow stromal layers were not inhibited by antiCDI 8 monoclonal antibodies (MoAbs) with functional
blocking properties." In this report, we examine further
the expression of integrins on normal CD34+ myeloid progenitors as well as on the leukemic cell lines KG1, KGla,
and Moi'e and myeloblasts obtained from leukemic patients. We also investigate the possible involvement of the
VLA integrins in adhesive interactions between myeloid
precursors and the marrow microenvironment through
studies of binding inhibition.
Blood, Vol81, No 1 (January 1). 1993: pp 112-121
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113
INTEGRINS OF HEMATOPOIETIC CELLS
MATERIALS AND METHODS
Cell Separation and Culture
Marrow CD34+ myeloid precursors. A sterile two-step flow cytometric technique used to isolate CD34+ marrow cells has been
described previously.loBM aspirates were obtained from normal volunteer donors in accordance with institutional guidelines of the Research Subjects Review Board of the University of Rochester. 5 X
IO’-light-density marrow cells per sample were stained with a 1:20
dilution of fluorescein isothiocyanate (FITC)-conjugated HPCA- 1,
an antibody that recognizes the CD34 antigen (Becton Dickinson,
Mountain View, CA), and with phycoerythrin (PE)-conjugated antiCDlO (CALLA antigen; Coulter, Hialeah, FL). One to 5% of cells
were positive for CD34 and negative for CDIO. Cells were then sorted
aseptically using an EPICS C flow cytometer (Coulter). An initial
sort performed at high speed resulted in a population that was 60%
to 70% CD34+. A second slow-speed sort resulted in a 96% to 99%
pure CD34’CDIO- population. These cells, which were 95% viable
at the conclusion of the second sort, constituted the purified CD34+
myeloid precursors subsequently used in binding assays and indirect
immunofluorescencephenotyping studies. At the completion of the
sort, centrifugation was performed to remove sorting sheath fluid,
and the cells were resuspended in RPMI medium with 20% fetal
bovine serum (FBS) for overnight storage at 37°C if further studies
could not be conducted soon after sorting conclusion.
In certain experiments, CD34’ cells were isolated by an avidinbiotin column adsorption technique (CellPro, Inc, Bothell, WA)?6
Cell lines. The leukemic cell lines KGI, KGla, and Mo7e were
obtained from Dr D.W. Golde (Memorial Sloan Kettering, Rye, NY).
KGI and KGla were maintained in RPMI-1640 (GIBCO, Grand
Island, NY) culture medium with 10% FBS (Hyclone, Logan, UT).
Mo7e is an interleukin-3 (IL-3)dependent cell line and was maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (GIBCO)
with 20% FBS and 50 pmol/L IL-3 (kindly provided by Dr S. Clark,
Genetics Institute, Cambridge, MA).
Leukemic patient samples. Marrow or blood obtained with informed consent was layered over Ficoll-Hypaque (specific gravity
1.077;Pharmacia, Piscataway, NJ). Light-density interface cells were
washed twice and resuspended in RPMI with 10% FBS. Samples
were used in binding assays only if they had greater than 80% blasts.
Stromal cell layers. Marrow stromal cell layers were established
from BM aspirates from normal volunteers and were cultured and
passaged as previously described.” Briefly, lightdensity BM cells were
cultured in McCoy’s SA culture medium (GIBCO) supplemented
with 12.5%FBS, 12.5% horse serum (Hyclone), and I pmol/L hydrocortisone (Sigma Chemical Co, St. Louis, MO). These adherent
cell layers were used in binding assays only after passage of more
than three times. At final passage, the adherent layers were seeded
into either Falcon 24-well plates (Becton Dickinson, Lincoln Park,
NJ) for cell line chromiumbinding studies or into 35-mm tissue
culture plates (Coming, Coming, NY) for CD34’ progenitor cell
adhesion assays. The adherent layers were allowed 5 to 7 days of
incubation after the final passage to ensure adequate confluent matrix
formation for cell-binding assays.
CA; (CD49b [VLA-2 a chain, IgGI], CD49c [VLA-3 a chain, IgGI],
and CD49e [VLA-5 a chain, IgG3]), AMAC, Westbrook, M E
(CD49d [VLA-4 CY chain, IgGI], CD49f [VLAd a chain, IgG2a1,
CD6 1 [GPIIIa or b3,IgGI], and CD34 F’ITC conjugate), Tago, Burlingame, CA; (RTC-conjugated F(ab)’ goat anti-mouse Ig, and PEconjugated F(ab’)’ goat anti-mouse Ig). Antibodies L1 (anti-LFA-1
a chain, CD1 la), 44 alpha (anti-Mola-chain, CDl lb), L29 (antip150,95 a-chain, CDI IC), and 10F12 (anti-&chain, CD18) were
gifts of Dr A. Amaout (MassachusettsGeneral Hospital, Boston, MA).
Antibody 12.8, an IgM molecule recognizing CD34, was a gift of Dr
R. Berenson, Seattle, WA. Anti-VCAM (4B9, IgG1) was a gift from
Dr J. Harlan (Harborview Medical Center, Seattle, WA). For diagnostic immunophenotyping of leukemic blasts, anti-CD14, antiCD19, anti-CD33, and anti-CD34 were obtained from Coulter. Antibodies API (anti-GPIb), AP2 (anti-GPIIbIIIa), AP4 (anti-GPIIb),
and AP5 (anti-GPIIa) were gifts of Dr T.J. Kunicki (The Blood Center
of Southeastem Wisconsin, Milwaukee).Phosphatidylinositol-specific
phospholipase C (PI-PLC) was purchased from ICN Biomedicals
(Costa Mesa, CA). Granulocyte-macrophage colony-stimulating
factor (GM-CSF) and IL-3 were gifts of Dr S. Clark.
Adhesion Protein Expression
Cell lines were stained for indirect immunofluorescence by incubating with antibody to the adhesion protein at 4°C for 30 minutes
followed by three washes in PBS. Cells were then incubated at 4°C
for 30 minutes with RTC-conjugated goat anti-mouse Ig. After the
final wash, cells were resuspended in I% paraformaldehyde in PBS
at 4°C for flow cytometry analysis. Stained cells were analyzed on
an EPICS C flow cytometer. The mean log green fluorescence channel
of the cell lines was determined directly from a single parameter
histogram. The mean log fluorescence channel was converted to linear
equivalents as previously described?’ In order to estimate the fluorescence intensity specifically due to the test adhesion protein antibody, the mean fluorescence intensity of cells stained with isotypespecific control antibody was subtracted from that of cells stained
with the adhesion protein antibody to obtain the final mean fluorescence intensity measurement. To ensure that only specific receptor
expression was evaluated, only fluorescence measurements 2 5 were
defined as positive after subtraction of background staining.
For phenotyping of adhesion proteins on normal CD34’ myeloid
progenitors, two-color immunofluorescence was used with light-density marrow cells stained with an adhesion marker versus CD34. 2
X 106-light-density bone marrow cells were incubated with 12.8 antiCD34 and unconjugated test antibody for 30 minutes at 4°C in PBS
plus 20% human AB serum and then washed twice with cold PBS.
The cells were then incubated with FITC-conjugated goat anti-mouse
IgG specific for the isotype of the unconjugated “test” antibody used
and with goat antimouse IgM PE. After staining, cells were resuspended in 1% paraformaldehyde in PBS and kept at 4°C for flow
cytometry analysis. Stained cells were analyzed on an EPICS Profile
flow cytometer (Coulter) using standard light scatter gates for mononuclear cells to obtain a two-parameter histogram of log green fluorescence (FITC) versus log red fluorescence (PE).
Binding Assays
Antibodies and Reagents
Antibodies and additional reagents were obtained from Becton
Dickinson Immunocytometry Systems, Mountain View, CA; (HPCA1 [CD34, IgGI] and FITC-conjugatedgoat antimouse isotype control
for IgGl), Coulter Immunology, Hialeah, n,
(CD29 [VLA 6, chain,
IgGl], CDIOPE, and goat antimouse isotype controls for IgG I, IgG2a,
and IgG3), Fisher Scientific,Orangeburg, NJ; (RTC-conjugated isotype specific goat antimouse IgG 1, PE-conjugated isotype specific
goat antimouse IgG2b and IgG3), Telios Pharmaceuticals, San Diego,
Chromium binding assay. Binding experiments with cell lines
were performed as previously described” with minor modifications.
KGI, KGla, or M07e cells ( 5 X IO6) were labeled with 100 pCi 51Cr
(Amersham, Arlington Heights, IL) for 30 minutes at room temperature. Cells were then washed three times in more than 10 vol of
PBS. Cells were then resuspended in RPMI- I640 medium with 10%
FBS at a concentration of 1.0 X IO6 cells/mL. One-half milliliter ( 5
X IO5 cells) were plated over the stromal layer or plastic-coated 24well tissue culture plate for 2 hours at 37°C. The medium and non-
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LIESVELD ET AL
114
VLA-6 N chains (CD49e and CD49f). Expression of the leukocyte integrin subfamily molecules (CAMS) was also similar
in the threecell lines(Tab1e I). LFA-I (CDI la)and thecommon p2 chain (CD18) were expressed in all three cell lines
with no significant expression of Mol (CDl I b) and ~ 1 5 0 . 9 5
(CDI IC) on KG I or KGla. Mo7e expressed a small amount
of CDI IC. Expression of GPlb and GPIIbIlIa of the cytoadhesin integrin subfamily was seen on Mo'le, a megakaryoblastic cell line. KG 1 and KG la cell lines expressed none of
the cytoadhesin molecules, with the exception of low-intensity
GPlb on K G l a (Table I).
120
100
80
60
40
20
Expression qf V I A Inregrins by Normal Human Myeloid
Progenitors and by Myeloblasts From Leukemic Patient
Samples
0
CD29
CD49b
CD49c
CD49d
CD49e
CD491
Adhesion Receptors (VLA-2-6)
Fig 1. The V I A integrin expression of the KG1 (0). KG1a (m),
and Mo7e (B)
leukemia cell lines is shown. Shown is the mean 2
SEM of fluorescence intensity expressed in arbitrary linear units as
described in the text. n
=-
5 for each set of antibody data.
adherent cells were aspirated and wells washcd twice with PBS. The
initial aspiiated medium and washes were pooled. The adherent layers
or plastic wells with hound cells were then treated with 0.05% trypsin0.5 mmol/L EDTA, (GIBCO) for 20 minutes. The entire adherent
contents were then aspirated and counted in a gamma scintillation
counter (Packard. Meriden, CT).The entire adherent layer cell counts
were calculated as a percentage of total recovered counts and reg
resented the percentage of plated cells that hound to the surface
(stromal layer or plastic) under study.
Co/ony/iwningitnit (CFL') assay. Binding experiments using
purified CD34' cells from normal marrow were performed as previously described" with minor modification. Briefly. CD34' cells ( 5
X IO') in I mL RPMI-I640 medium with 10% FRS were plated over
stromal cell-coated or plasticcoated X-mm tissue culture plates.
ARer a 2-hour incubation at 37°C. the medium and nonadherent
cells were aspirated and the plates washed twice with PBS. The plates
were then overlaid with I mL of a 0.5% agar mixture containing
Iscove's medium (GIBCO). 10%bovine Serum albumin (fraction V;
Sigma Chemical Co). 30% FRS. and 10% Mo cellconditioned medium (CM) and 5% GCT-CM as sources of hematopoietic growth
factors. Granulocyte-macrophagecolony-forming unit (CFU-GM)
colonies (>20 cells) were scored after incuhation for 14 days at 37°C
in 570 C 0 2 .
RESULTS
Expression c?f V U Inregrin Moleciclrles by the Myeloblastic
Cell Lines KGI/KGla and the Megakaryohlasric Cell Line
Mo 7e
The expression of integrins on myeloblastic cell lines was
examined using immunofluorescence and flow cytometric
analysis. The fluorescence intensity of integrin expression on
the cell lines KG I , KG la, and Mo7e is shown in Fig I . Qualitatively, the expression of the VLA integrins was similar in
all three.cell lines examined. The common VLA B1 chain
(CD29) and the VLA-4 a chain (CD49d) were expressed in
all three cell lines, as were to a lesser degree the VLA-5 and
To further investigate whether differences of integrin
expression between normal human myeloid progenitors and
malignant myeloid lineage precursors might be evident, the
expression of the VLA integrins was examined on CD34+
progenitors from normal BM as well as on myeloblasts obtained from leukemic patient BM. Using two-color immunofluorescence, CD34' cells derived from normal BM were
found to express the common VLA B1chain (CD29) and the
VLA-4 and VLA-5 N chains (Fig 2). No significant VLA-6
a chain o r CD61 (integrin BS)was expressed (Fig 2).
Myeloblasts from leukemic patient samples showed a pattern of VLA integrin expression qualitatively similar to that
found on normal myeloid progenitors. Eleven patient samples
were examined for VLA integrin expression (Table 2): 7 of
7 patients tested expressed the common
chain, IO of I I
expressed VLA-4 a,and IO of I 1 expressed V L A J a. The
intensity of expression of VLA-4a and VLA-Sa was quite
variable (Table 2). VLA-2, VLA-3, and VLA-6 a expression
was minimal in the myeloblasts, similar to the expression of
these a chains seen in normal myeloid precursors. Qualitatively, the only difference apparent in VLA integrin expression
between normal myeloid precursors and leukemic blasts was
a relatively higher expression of the VLA-5 a chain in some
patient leukemic samples as compared to normal myeloid
precursors. This degree of VLA-5 a expression was also rel-
Table 1. LEU-CAM and Cytoadhesion Expression of Cell Lines
Cell tines
KG 1
KGla
M07e
17.1 24.2
3.0 2 1.7
15.0 2 3.2
2.0 2 1.7
1.02 1.7
15.2 2 4.7
18.7 2 4.2
~~
LEU-CAM
CD1 l a (LFA-la)
CD1 l b (Mola)
C D l l c (~150.95)
CD18 (82)
Cytoadhesin
GPlb
~
0.0 2 0.0
10.3 2 1.7
1.6 2 0.6
8.02
GPllbllla
GPllb
0.3 2 0.3
1.7 2
GPllla (83)
0.7 2
1.8
0.3
4.7 2 1.7
8.323.2
14.3 2 4.5
18.027.6
25.0 2 5.7
8.5 2 0.5
6.2 2 3.6
Values shown represent mean 2 SEM fluorescence intensity obtained
0.3 2 0.3
0.3
0.3 2 0.3
0.7 2 0.3
~~~
by flow cytometryand staining with a particular antibody (n = 3).Intensity
is expressed in arbitrary linear units as described.
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INTEGRINS OF HEMATOPOIETIC CELLS
115
lgG3 Control
P
P
Table 2. VLA lnteorin Exmession of Patient Leukemic Blasts
VLA-gl
VLA-2a
VLA-3a
VLA-4a
VLA-5a
VLA-6a
48
94
81
62
80
9
0
2
0
0
7
2
1
5
0
2
2
7
18
65
55
43
32
7
28
5
1
42
18
ND
ND
ND
1
8
74
49
6
15
3
27
12
8
36
10
ND
37
CJ
0
VLA-5
Using a 5'Cr-labeling adhesion assay like that used previously to demonstrate the ability of normal CD34+ myeloid
progenitors and myeloid leukemic cell lines to bind to marrow
stromal layers," the adhesion of myeloblasts from 16 freshly
obtained pretreatment marrow samples from leukemic patients was similarly examined (Table 3). In all 16 cases, 220%
(range 20% to 65%) of blasts bound to stromal layers, and in
ND
ND
ND
3
B
0
Adhesion of Freshly Isolated Leukemia Myeloblasts to
Marrow Stromal Layers
1
4
3
2
6
2
0
5
1
VLA-4
VLA-2
atively higher than that seen in the myeloblastic cell lines
examined.
1
VLA-3
0
Fig 2. Two-parameter flow cytometry histograms of light-density B M cells gated for forward
angle light scatter and 90" light scatter; 50,000 cells
were counted. Cells were stained with CD34 (or
irrelevant IgM) plus the test antibody (or irrelevant
isotype-specific control antibody), followed by PEconjugated goat anti-mouse I g M (y-axis; log red
fluorescence) and FITC-conjugated goat anti-mouse
l g G l ( V U - 2 [CD49b], VLA-3 [ C D ~ ~ C V
] ,U - 4
[CD49d]. CD61 US]). lgG3 ( V U - 5 [CD49e]), or
lgG2a (VLA-6 [CD49f] control not shown) (x-axis;
log green fluorescence). Only panels f(VLA-4) and
g(VLA-5) show a significant population of dually labeled cells.
2
3
4
5
6
7
8
9
10
11
lgGl Control
2
B
0
Patient
lgG3 Control
2
1
1
3
0
0
5
2
Shown is the mean linear fluorescence intensity noted with FACS analysis with the indicated antibodies.
Abbreviations: ND = not done; VLA-01 = CD29; VLA-2a through
VLA-6a are CD49a through CD49f. respectively.
cD61
VLA-6
all cases, binding to stromal layers was greater than to tissue
culture-grade plastic (mean 29% o 6% of cells bound to stroma
2) plastic). All cases studied had features of acute
myelo(mono)cytic leukemia with the exception of case no.
14, a case of acute promyelocytic leukemia. The degree of
CD34 or CD33 positivity of the myeloblasts did not correlate
Table 3. Adhesion of Leukemic Blasts t o Marrow Stromal Layers
Percent Binding To
Percent Blasts Positive For
Patient
CD34
CD33
CD14
CD19
1
2
3
4
5
6
7
8
9
10
ND
ND
ND
89
8
14
67
19
24
81
5
67
3
53
68
0
67
88
2
0
74
5
89
16
88
89
12
5
ND
0
11
12
13
14
15
16
ND
0
79
48
98
3
20
5
51
23
11
6
63
11
4
0
1
ND
ND
ND
5
4
22
5
1
67
0
1
2
0
1
1
2
ND
Stroma
Plastic
23 + 4
20 f 2
58 + 7
6 0 + 16
36 f 7
40 f 4
28 k 3
60 + 3
44 f 3
39 IT 1
53 + 8
64 + 2.4
35 f 5
65 + 5.4
29 f 5
30 + 2
7+1
3+0
25 + 8
621
8+3
3+0
322
4+2
18 + 7
3f 1
14 t 2
47 f 5.3
8.3 f 1.4
17 f 7.6
10 + 2
26 f 3
Shown is a partial blast antigenic phenotype for individual leukemia
cases and the percentage of each which adhered to stromal layers or to
plastic. Blasts from case 16 had Auer rods and this was therefore considered a case of myelogenous leukemia.
Abbreviation: ND = not done.
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116
LIESVELD ET AL
with the degree of binding to stroma nor did possession of
CD 14, a monocytic marker, correlate with degree of binding
to tissue culture plastic. In five cases, the effect of PI-PLC on
leukemic blast binding to stroma was investigated. Cells were
treated with 120 mU PI-PLC for 30 minutes at 37"C, washed
once, and plated in the previously described adherence assay.
As shown in Fig 3, in only two cases did mean binding decrease in presence of PI-PLC; by 21% in sample 3 and by
27% in sample 5.
Inhibition of Adhesion by Blocking Antibodies
To study which of the expressed adhesion proteins might
be involved with adhesion of myeloid progenitors to the BM
stroma, we used an in vitro model of stromal adhesion to
conduct inhibition studies with blocking antibodies to the
integrins of interest.
Each of the adhesion receptor antibodies used in the inhibition experiments blocks adhesion at the concentration
used. CD29 and CD49d antibodies inhibited adhesion of a
B-cell line, NALM-6, to BM fibroblasts by 57% and 52%,
respectively.l 2 The CD49e and anti-VCAM antibodies were
used at a concentration previously demonstrated to show
blocking of VLA-5-dependent and VCAM-dependent adhesion, respectively.
CD34+ cell lines and leukemic myeloblasts. The effect
of various blocking antibodies on adhesion of the myeloblastic
cell lines KG 1, KG la, and Mo7e to bone marrow stroma is
shown in Fig 4. Binding of the KGla, KGl, and Mo7e cells
to normal bone marrow stroma was not significantly decreased by anti-CD49e (anti-VLA-Sa) or CD29 (anti-0, ).
Anti-CD49d (anti-VLA-4a), and anti-VCAM had no effect
on the adhesion of KG 1 or Moi'e to marrow stromal layers.
As shown in the case of Mo7e, the combination of antiVLA4-a and anti-CD29 did not block adhesion either. In
contrast, KGla binding to marrow stroma was partially inhibited by both anti-VLA-4a and anti-VCAM (P< .05 by
paired t-test) (Fig 4A). The combination of these two anti-
bodies did not result in greater inhibition than either used
alone. No difference in the degree of inhibition was observed
between addition of the test antibody at the time of target
cell incubation over stromal layers versus preincubation of
antibody with ligand-containing cells for 30 minutes at 4°C.
In three cases of myeloid leukemia where sufficient blasts
were obtained to study effects of antibodies to VLA integrins
on stromal adhesion, anti-VLA-4a inhibited blast binding
by a mean of 18% f 4.9% (range 10%to 27%). In two cases
studied, anti-VLA-Sa did not result in significant inhibition
of blast binding.
Normal CD34 progenitors. We have previously shown
that normal CD34+ progenitors attach to stromal layers using
a binding assay in which the readout is numbers of CFUGM colonies." Using this same assay, inhibition ofadhesion
of normal CD34' progenitors to BM stroma was examined.
When the binding assay was performed in the presence of
anti-CD29 (0')
(1: 100 dilution of 4B4 antibody), inhibition
of adhesion of normal progenitors to marrow stroma was
53% (+4.2%, n = 3). Data from these three experiments are
shown in Fig 5A. Addition of anti-VLA-4a chain antibody
did not enhance the inhibition seen with anti-0, alone.
In addition, to assess effects of these blocking antibodies
upon the entire population of CD34+ cells, "Cr labeling of
column-adsorbed CD34+ cells was performed followed by
the standard adhesion assay. As shown in Fig 5B, anti-CD29
(VLA-0, chain) gave significant binding inhibition in this
population as well (P < .O 1 by paired t-testing), whereas antiVLA-4a, anti-VCAM, and anti-VLA-Sa did not.
Effects of cytokines on binding of leukemic cell lines. To
assess whether incubation of progenitor or leukemic cells with
growth factors would enhance adhesion as has been previously
reported for transplanted marrow in murine models,2sor to
examine whether exposure of the stromal layers to inflammatory/immune modulators would influence adhesion,
adhesion assays were performed after incubation with appropriate cytokines. As shown in Table 4, neither IL-3 nor
+
T
70
60
50
40
30
20
10
0
1
2
3
4
ACUTE LEUKEMIA SAMPLE #
5
Fig 3. Shown is the percentage of myeloblasts
bound to stromal or plastic layers in the presence or
absence of PI-PLC treatment in five cases of acute
myeloblastic leukemia (AML). Similar treatment of
blood monocytes with PI-PLC decreased CD14 antigen density by greater than 50%. N = 3 except for
sample No. 5 performed in duplicate. (W), Stroma
1, stroma + PLC; (El), plastic PLC; (0).
plastic + PLC.
-
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INTEGRINS OF HEMATOPOIETIC CELLS
117
Ami-IgGla
Anti-IgGla
Anti-VCAM
Ami-VCAM
.U
r
v
AMCVLA-4
.O Anti-VU4
p-y
Anti-IgG1b
C
L
23
AntCVLAOl
Anti-VLAO1
0
C
Anti-lgG3
, t-p
Antl-VLA-5
AMCVLA-5
0
10
A
20 30 4 0
50
10
0
60 70 80 90 100
20 30 4 0
B
KG1-a Adhesion (%)
5
50
60 70 80 90 100
KG1 Adhesion (%)
c
0
I
Fig 4. Shown is the mean 2 SEM percentage of cells adherent to
marrow stroma in the presence of the indicated antibodies for (A) KG1a
(n = 3). ( 6 )KG1 (n 3). and (C) Mo7e (n 3-5). Antibodies were used
at dilutions indicated in Materials and Methods. In (A), anti-IgGla represents paired control experiments for anti-VCAM. anti-lgG1 b paired
controls for anti-VIA-4 and 484, and anti-lgG3 for anti-VIA-5. In ( E ) .
anti-lgG1a represents paired control experiments for anti-VCAM, antilgGl b represents paired control experiments for VIA-4 and 4 8 4 and antilgG3 for anti-VIA-5. In (C), anti-lgG1a represents controlled paired experiments for anti-VCAM, anti-lgGl bfor 484. anti-VIA-4, and the combination of anti-VIA-4 and 4B4, and anti-lgG3 for anti-VIA-5. VIA-4
CD49d. VIA-5 CD49e. and V W l CD29.
~
~
GM-CSF at concentrations of SO U/mL and 100 U/mL. respectively, affected binding of KGla or KGI to marrow
stromal layers. Also. pretreatment ofstromal layers with IO-"
mol/L PMA (phorbol myristate acetate) or IO U/mL IL-In
did not alter the degree of adhesion of the KGla or Mo7e
cell lines nor of normal CD34' myeloid progenitors to such
layers (Table 5).
DISCUSSION
The 8, (VLA) integrins are generally associated with adhesion to extracellular matrix components. In this study. it has
been documented that early myeloid cells, including normal
CD34' progenitors, early leukemic cell lines. and acute leukemia myeloblastsall possess a similar array of VLA integrins.
Most prominently expressed are CD29 (the common S I )
chain: CD49e (VLA-Sa), a receptor for the RGDS cell-bindingsite of fibronectin: and CD49d (VLA-4n). which can bind
to fibronectin domains29.was well as to a cell-associated ligand, VCAM-I.3' CD49b and CD49c were minimally expressed on all of these cell types. VLAd. a receptor for laminin. was present only on established leukemic cell lines, and
most prominently on Mo7e. a line with some megakaryoblastic properties. We have shown previously that normal
CD34' myeloid progenitors posms CD18." and here it is
shown that immature cell lines (CD34') also possess CD18
and CDI la. N o significant expression of cytoadhesins was
found on the three cell lines tested except on Mo7e. which
C
0
AmCbQGl8
0
An(CVCAY
-m
AmClgGlB
2
AICVLAfJl
5
AnICVLA-4
C
0
L
n
V U 4 + An(CVLAfJ1
An(CtG3
An1bvLA-5
0
C
10
20 30 40
50
60 70 80 90 100
Mo7E Adhesion (%)
has megakaryocytic properties. Normal CD34' progenitors
did not possess the Pz chain (CD61. vitronectin B receptor/
gpIIIa).s2
The spectrum of 8-integrin antigen expression documented
here is in keeping with that noted by others using different
assay methods. Saeland et al-" have reported a similar array
of integrin antigen expression on CD34+ cells from marrow
and cord blood, and a repertoire of integrin expression during
erythroid maturation has also been described.34Soligo et aI,l7
using an immunohistochemical detection method. also found
VLA-4 presence on immature hematopoietic cells and restriction of /& cytoadhesin molecules to megakaryocytes and
platelets when unfractionated RM was studied. They also
noted only a minor role for fi2 integrins in early hematopoiesis
and found variability of & integrin expression on acute myelogenous leukemia specimens. whereas VLA-2, VLA-3. and
VLA-6 were absent on nonlymphoid pr~genitors.'~
In the study presented here, VLA-5n (CD49e) was seen
on all early myeloid cell types (normal CD34' progenitors,
leukemic cell lines. and acute leukemia myeloblasts) with a
relative increase seen in some cases of acute myelocytic leukemia (AML), probably reflecting the cell-maturation spectrum involved in leukemogcnesis." In addition to VLA-Sa
and the common PI chain (CD29). only VLA-4n (CD49d)
was prominently expressed on normal early myeloid cells.
Within the acute leukemia samples studied. VLA-4a expression did not seem to correlate with maturation as even an
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LIESVELD ET AL
Anti-lgG3
.-0
E Anti-VLA-5 1
-1
1
c
0
u
C
.-0
-
Anti-lgG1
m
5 Anti-VCAM
0
C
- Antl-VLA4
~
Anti-CD29
,
I
Control
lgGl
Anti-CD29
0
B
Incubation Condition
10
o/o
20
30
40
CD34+ Cells Bound
Fig 5. (A) Shown are the mean ? SEM percentage of CFU-GM bound to marrow stroma in the presence of anti-CD29 [anti-&) or in the
presence of an lgGl control antibody as compared to binding to stroma without antibody present (100%);n = 3 for all conditions. ( 6 )The
percentageof CD34’ cells bound to marrow stroma in the presence of the indicated antibodies is shown with all data obtained in a parallel
fashion with the appropriate isotype specific control antibody; n = 4 for all lgGl -paired conditions and n = 3 for VIA-5/lgG3. V I A 4 =
CD49d and VIA-5 r CD49e.
M3 specimen with no CD34’ blasts expressed significant levels of VLA-40. VLA-4 has been found on cultured T lymphocytes to function as a receptor for the heparin-I1 and IllCS
domains of fibronectin.’’ VLA-4a has also been found to be
a ligand for VCAM-I, an adhesion molecule of the immunoglobulin gene superfamily found on endothelial cells stimulated with IL-la, lipopolysaccharide, or tumor necrosis factor a. It is also constitutively expressed on dendritic cells,
renal tubular epithelial cells, and tissue macrophages..”
VCAM has also been found on marrow stromal cell layers
such as those used here.”
To what extent CD49d ( V L A l a ) or CD49e (VLA-Sa)
antigens participate in homing phenomena or in attachment
of myeloid progenitors to marrow microenvironmental
components remains unknown. The data presented here show
no role for VLA-Sa in adhesion of CD34’ cells to stroma as
measured in a specific adhesion assay. This is in keeping with
previous studies that show no role for RGD sequences in
In contrast. RGD sequences have
such adhesion proce~ses.~..”
been found to have a role in adhesion oferythroid progenitors
to marrow.33
Antibody inhibition data shown here suggest a role for
CD29 (8,integrin) in adherence ofCD34’ CFU-GM-forming
cells to marrow stroma and participation of VLA-40 (CD49d)
in adhesion of some myeloid progenitors (KG la cell line and
three samples from leukemia patients) to stroma. Data shown
here with the KGla cell line would suggest that VCAM-I on
human marrow stromal cells may participate in leukemic
blast adhesion as the ligand for VLA-4. Such an interaction
has also been noted for binding of lymphocytes or lymphocyte
progenitors to endothelial cell^,'^^^^ to murine marrow stromal
or to human marrow fibroblasts expressing V-CAMI ,Izand, more recently, for adhesion of myeloid and erythroid
progenitors to marrow stromal cells.35
In no instance did antibodies to VCAM-I, VLA-4a
(CD49d). B1(CD29). or other VLA integrins completely inhibit myeloid progenitor adhesion to marrow stroma. and,
in most cases, inhibition of adhesion noted was minimal.
This might suggest a role for other classes of integrins or
other types of adhesion receptors in the interaction of leu-
Table 4. Effect of GM-CSF and IL-3 on GKla and KG1 Adhesion
KGla
Stroma
Plastic
KGla
t GM-CSF
KGla
KGla + IL-3
6 5 + 3.0
1.3 fO.6
63 + 6.1
2.0+ 1.0
63 f 11
3.8 + 1.2
68 9.0
4.0+0.7
+
KG 1
KG1
+ GM-CSF
KG1
68 f 2.9
2.7 f 0.6
74 f 3.5
3.7 f 1.2
18+5
ND
+ IL-3
- - - Stroma
Plastic
KG1
19f6
ND
Data represent the mean ? standard deviation percentage of the indicated cell line which adhered to marrow stromal layers or plastic in the
presence or absence of GM-CSF (100 U/mL) or IL-3 (50 U/mL). Experiments were performed in a paired fashion with n = 3 for all conditions
except for KG1a + IL-3 experiments and their controls for which n = 5.
Abbreviation: ND = not done.
Table 5. Effect of IL-la and Phorbol Myristate Acetate (PMA)
on Cell Adhesion
Mo7e
CD34’ Progenitors
KGla
Stroma
Stroma
+ IL-la
14 ? 3.2 (n = 3)
23 f 9.5 (n = 4)
43
17 f 3.5 (n = 3)
26 + 8.6 (n = 4)
49 f 12 (n = 3)
Stroma
Stroma
+ PMA
38 f .89 (n = 3)
41 ? 4.7 (n = 3)
48 (n = 2)
41 f 2.3 (n = 3)
42
+ 4.7 (n = 3)
53 (n = 2)
+9
(n = 3)
Data shown are mean + SEM percentage of the indicated cell type
bound to stromal layers grown in the presence or absence of 10 U/mL
mol/L PMA for 24 hours. Experiments were performed in
IL-la or
a paired fashion, n = number of experiments.
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119
INTEGRINS OF HEMATOPOIETIC CELLS
kemia cells with marrow. These might include heparin-binding receptors. proteoglycans, L-selectin and its mucin-like
ligand,33.41 CD44-hyaluronate
hemonectin?’
thrombospondin.” other collagen receptors,” or other fibroA rolc for thc 33/66-Kd carboxy-terminal
nectin
heparin-binding cell adhesion domains of fibronectin in interactions between primitive marrow progenitors and intact
irradiated marrow stroma has been found.46This interaction
appearcd to involve the FN-C/H I I site. Also, other anchored
growth factors such as membrane-bound basic fibroblast
growth factor?’ macrophage colony-stimulating factor (MCSF. CSF-I)?* or c-kit ligand (stem cell factor)49may participate in this adhesion process. Figure 6 illustrates schematically some of the possible receptor-ligand interactions
that might contribute to precursor (CD34’) attachment to
marrow stroma.
The lack of inhibition seen with anti-CD29 in the case of
myeloid/megakaryoblastic cell lines does not necessarily rule
out a role for /3, integrins in their adhesion to stroma. Changes
in conformation of some integrin receptors after antibody
binding can “activate” or alter the affinity of ligand recognition. This has been reported for another monoclonal antibody to CD29 (8A2) that stimulated the binding of U937
cells to CHO cells transfected with VCAM-I complementary
DNA ( c D N A ) and
~ for glycoprotein IIbIIIa.aII&3.51Whether
such a phenomenon might be operational in the adhesion of
myeloid leukemic progenitors to stroma can only be speculated on. It is also possible that to detect the role of the
CD49d(VLA4a)/VCAM complex in mediating progenitor
adhesion to the microenvironment, one may have to maximize the stromal expression of VCAM-I by cytokine combinations. as recently shown by Simmons et aL3’
In this work, the presence of a similar array of VLA integrins on freshly isolated leukemia blasts as on tissue cultureadapted cell lines and normal CD34’ progenitors was identified. Leukemic marrow blasts demonstrated, on average, a
comparable degree of binding to marrow stromal layers as
did these other cell types. Although the number of samples
reported upon here is small, the presence of CD49d and
CD49e was consistently noted, and the degree of blast adhesion to stromal layers was independent of subtype; ie, myelogenous versus monocytic versus promyelocytic. In those
cases where adhesion inhibition was studied with antibody
to CD49d, a small degree of inhibition was noted.
This ability of leukemic blasts to adhere to marrow stromal
layers is surprisinggiven the common presence of blood blasts
in these cases even when marrow cellularity does not suggest
a crowding effect. Because VLA integrin expression appears
similar to that of normal progenitors, it is possible that blast
egress occurs because of altered function or affinity of these
receptors for their respective ligands. It is also possible that
these blasts lack adhesive receptors of other uncharacterized
classes as has been described for progenitors in chronic myelogenous leukemia (CML).5ZUnlike the situation in CML,
the acute myelo(monocytic) blast adhesion to stroma was
not consistently PI-PLC linked.53Another explanation for
blast exit from the marrow would be altered receptor affinity
or changes in integrin phenotype on egress. Such possibilities
could be explored by comparing the adhesive properties of
circulating blood blasts to marrow blasts. Hematopoietic
growth factors such as GM-CSF and IL-3 have been reported
to influence adhesion or aggregation of mature granulocytes
and monocyte^,'^.'^ but their presence did not influence
binding of early myeloid cell lines to marrow stromal layers
in our hands.
As cells of the granulocyte lineage mature, they lose VLA4a. whereas monocytes and eosinophil^'^ retain this protein.
Changes in integrin phenotype with blast differentiation may
partially explain the propensity of monoblasts for tissue invasion. Other factors regulating acute myelogenous leukemia
blast traffic may relate to their responses to chemotactic factors or their ability to elaborate collagenases* or enzymes
rlcD18,
1182)
-
ICAM.1, ICAM.2, ICAM-3
CD58 (LFA-3)
Fig 6. This diagram indicates
many of the membrane-bound
antigen receptors present on
CD34‘ precursor cells and their
associated ligands. These receptors singly or in various
combinations promote the
adhesion of these early progenitors to their respective ligands
within the marrow microenvironment. b-FGF, basic fibroblast
growth factor; LFA, leukocyte
function antigen; SCF, stem cell
factor; CSF-1, colony-stimulating factor 1; and M-CSF, macrophage CSF; ICAM, intercellular adhesion molecule; VIA,
very late antigen; VCAM, vascular cell adhesion molecule.
of flbronectln
,
c02
CD34
n
eklt
Stem Cell Factor/
KR Ligand
CSF-11 M-CSF
(CD49dlCD29)
Laminin
Fibronectln
(ass11
(CD49elCD29)
Basic Flbrablast Growth Factor
WGF)
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120
LIESVELD ET AL
such as the human heparin-binding elastase homologue
(CAP37, azurocidin) that mediates reversible fibroblast and
endothelial cell ~ontractility,’~
thereby facilitating cell egress
into extravascular spaces. Further studies will be required to
assess the role of the VLA integrins in leukemia blast egress
from the marrow and any role that they might play in stem
cell homing during transplantation.”
ACKNOWLEDGMENT
We thank Abigail Wilson Harbol for excellent technical assistance
and Michelle Abdo for assistance in manuscript preparation.
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`