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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3909-3919
Constitutive Chemokine Production Results in Activation of Leukocyte
Function-Associated Antigen-1 on Adult T-Cell Leukemia Cells
By
Yoshiya Tanaka,
Shinichiro Mine,
Carl G. Figdor,
Atsushi Wake,
Hideyasu Hirano,
Junichi Tsukada,
Megumi Aso,
Koichi Fujii,
Kazuyoshi Saito,
Yvette van Kooyk, and
Sumiya Eto
From The First Department of Internal Medicine and the Department of
Biochemistry, University of Occupational and Environmental Health,
Japan, School of Medicine, Kitakyushu, Japan; the Department of Tumor
Immunology, University Hospital Nijmegen, Nijmegen, The Netherlands;
and the Department of Internal Medicine, Kokura Memorial Hospital,
Kitakyushu, Japan.
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ABSTRACT |
Adult T-cell leukemia (ATL) is characterized by massive infiltration
of circulating ATL cells into a variety of tissues, a finding often
associated with poor prognosis. Leukocyte migration from circulation
into tissue depends on integrin-mediated adhesion to endothelium, and
integrins are tightly regulated by several stimuli, such as
inflammatory chemokines. However, the exact mechanisms that enhance
adherence of leukemic cells to the endothelium and infiltration into
tissues remain to be fully understood. We investigated the mechanisms
of extravasation of leukemic cells using ATL cells and report the
following novel features of endogenous chemokine-induced adhesion of
ATL cells to the endothelium. ATL cells spontaneously adhered to
endothelial cells without exogenous stimulation. Integrin leukocyte
function-associated antigen-1 (LFA-1) on ATL cells was spontaneously
activated. ATL cells produced high amounts of chemokines, macrophage
inflammatory protein-1 (MIP-1), and MIP-1 . Adhesion of ATL
cells to endothelial cells and the expression of activated form of
LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or
anti-MIP-1 and MIP-1 antibodies or transfection with antisense
of MIP-1 or MIP-1. Spontaneous polymerization of cytoskeletal
F-actin was observed in ATL cells, which was also inhibited by
pertussis toxin and wortmannin. We propose that ATL cells adhere to
endothelial cells through an adhesion cascade similar to normal
leukocytes and that the chemokines produced by ATL cells are involved
in triggering integrin LFA-1 through cytoskeletal rearrangement induced
by G-protein-dependent activation of phosphoinositide 3-kinases in an
autocrine manner. These events result in a strong adhesion of ATL cells
to the endothelium and spontaneous transendothelial migration.
| |
INTRODUCTION |
ADULT T-CELL LEUKEMIA (ATL) is a
peripheral CD4+ T-cell malignancy caused by infection with
human T-lymphotropic virus-1 (HTLV-1) and is associated
with a marked increase of peripheral ATL cells with monoclonal growth
during the acute phase. ATL is characterized by a rapid infiltration of
circulating ATL cells into several tissues and secondary lymphoid
organs, a process often associated with poor prognosis.1,2
Extravasation and infiltration of hematologic malignant cells into
tissues probably reflect the biologic properties of these cells,
expression and function of relevant adhesion molecules on these cells,
and their adhesive interaction with endothelial
cells.3 In a series of studies from our
laboratories, we have reported that one integrin, the leukocyte
function-associated antigen-1 (LFA-1), plays a central role in ATL cell
adhesion to its calcium-dependent ligand, intercellular adhesion
molecule-1 (ICAM-1), a process involving heparan sulfate proteoglycan
on the cell by binding heparin-binding chemokines.4-6
Leukocyte integrin LFA-1 and very late antigen-4 (VLA-4) mediate
adhesion of circulating leukocytes to endothelial ligands.7 The adhesive capacity of integrins expressed on peripheral leukocytes is tightly regulated. Although integrins expressed on resting cells do
not mediate firm adhesion to endothelial ligands, stimulation of these
cells results in a rapid increase in integrin function.8-10 Thus, activation of integrin is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the
functionally inactive integrin to an active adhesive configuration. In
this regard, we have previously reported that the chemokine macrophage
inflammatory protein-1 (MIP-1) triggers integrin and induces
adhesion of T-cell subsets to endothelial
integrin-ligands.11-13 Several recent studies have
supported the potential importance of chemokines in inflammatory
responses, specifically that various chemokines, including MIP-1,
produced in large amounts at the site of inflammation activate
integrins on leukocytes and result in their accumulation in the
tissues. The mechanisms causing activation of integrin are
thought to involve conformational changes of ectodomain of integrins
and/or clustering of integrins on the cell membrane that may
induce active, adhesive configuration of integrins, resulting from
cytoskeletal actin-polymerization associated with endodomain of
integrins.14-16
However, the exact mechanisms that enhance the adherence of circulating
leukemic cells to the endothelium and their subsequent infiltration
into the tissues or those regulating integrin adhesiveness to
endothelium are not very clear at present. We investigated in the
present study the mechanisms of integrin-mediated adhesion of leukemic
cells to endothelial ligands and examined the signaling and
cytoskeletal interaction of integrins using ATL cells. ATL is a unique
model for the following reasons. (1) It is characterized by a malignant
expansion of peripheral mature CD4+ T cells. (2) ATL cells
result from monoclonal proliferation and form in each patient a
phenotypically and functionally homogeneous population of cells. (3)
There is a high tendency for malignant cells to infiltrate multiple
organs. The present study was performed to investigate the relevance of
integrin expression to its adhesive function and regulatory mechanisms,
with special emphasis on signaling and rearrangement of the
cytoskeleton upon adhesion to the endothelium and subsequent tissue
infiltration of ATL cells.
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MATERIALS AND METHODS |
ATL cells and ATL cell lines.
We examined 20 patients with ATL, 1 control patient with chronic T-cell
leukemia (T-CLL) which was not caused by infection with HTLV-1, 4 established HTLV-1-infected T-cell lines (MT-1, MT-2, HUT-102, and
SALT-3; a kind gift from Prof K. Sagawa, Kurume University Medical
School, Kurume, Japan), and 10 normal healthy volunteers. ATL was
diagnosed based on clinical features, hematologic findings, serum
antibodies against HTLV-1, and monoclonal integration of HTLV-1
proviral genome.1,17 Highly purified CD4+ T and
ATL cells were prepared by exhaustive negative selection10 from peripheral blood mononuclear cells of normal donors and ATL patients using magnetic beads (Dynal, Oslo, Norway) and a cocktail of a
variety of antibodies, including CD19 monoclonal antibody (MoAb) FMC63,
CD16 MoAb 3G8, CD11b MoAb NIH11b-1, CD14 MoAb 63D3, and CD8 MoAb
B9.8.4.
Antibodies and reagents.
The following MoAbs were used as purified Ig in the preparation of T
and ATL cells, staining and analysis of cell surface molecules, and
blocking of cellular adhesion: activated LFA-1 MoAb
NKI-L16,18 CD19 MoAb FMC63 (H. Zola, Bedford Park,
Australia), CD8 MoAb B9.8.4 (B. Malissen, Marseille, France), CD11b
MoAb NIH11b-1, CD49d (VLA-4) MoAb NIH49d-1, CD54 (ICAM-1) MoAb 84H10
(S. Shaw, Bethesda, MD), CD49d MoAb HP2/1 (F. Sanchez-Madrid, Madrid,
Spain),19,20 CD16 MoAb 3G8 (D. Siegel, Bethesda, MD), CD69
MoAb FN50, phycoerythrin (PE)-conjugated CD45RO MoAb UCHL-1,
CyChrom-conjugated CD4 MoAb Leu-3a (Fujisawa, Osaka, Japan), CD14 MoAb
63D3, CD11a (LFA-1) MoAb TS1/22, major histocompatibility complex
(MHC) class I MoAb W6/32, and control MoAb Thy1.2 (ATCC, Rockville,
MD). ICAM-1 was purified by affinity column chromatography from the
Reed-Sternberg cell line L428 as previously described.10,21
Multiple inhibitors of intracytoplasmic signaling were applied to each
assay system and all reagents were used at the indicated
concentrations. At these concentrations, none of these inhibitors
produced cytotoxic effects on ATL cells, as confirmed by trypan blue
staining. We used wortmannin (Wako Pure Chemical, Osaka, Japan), a
phosphoinositide 3 (PI 3)-kinase inhibitor; pertussis toxin, uncoupler
of certain G-proteins from their complex; H88 and H89, A-kinase
inhibitors; H7 and staurosporine (Seikagaku, Tokyo, Japan), C-kinase
inhibitors; herbimycin A (Sigma, St Louis, MO) and genestein
(Carbiochem, San Diego, CA), tyrosine kinase inhibitors; and
cytochalasin B (Sigma) and cytoskeleton-disrupting reagent.
Preparation of sense and antisense oligonucleotide of
MIP-1 and MIP-1 and their transfection
into ATL cells.
Sense and antisense oligonucleotide sequences of MIP-1 and MIP-1
were 5 -CACCTGCTCAGAATCA, 5-TGATTCTGAGCAGGTG,
5-ATGAAGCTCTGCGTG, and 5-CACGCAGAGCTTCAT, respectively.
We synthesized 15-base deoxyribonucleotides on an automated solid-phase
synthesizer (Sawady Technology, Tokyo, Japan). The oligomers were
purified by affinity-gel chromatography embedded ether-toyopearl
(Tosoh, Tokyo, Japan) carrying hydrophobic affinity and gel filtration
effect (DNA stec-1000; ASTEC, Fukuoka, Japan), precipitated with
ethanol, lyophilized to dryness, and dissolved in the culture medium.
Oligonucleotides were introduced into ATL cells using a cationic
liposome-mediated transfection method.22 Oligonucleotides
dissolved in 100 µL of serum-free medium (OPTI-MEM; Life
Technologies, Gaithersburg, MD) were mixed with 5 µL of Lipofectin
reagent (LipofectAMINE; Life Technologies) in the same volume of
OPTI-MEM and incubated for 10 minutes at room temperature. The
oligonucleotide/liposome complex was added to ATL cells plated in a
6-well culture dish (3 × 105 cells/well), incubated
for 6 hours in OPTI-MEM, and then replaced with 10% fetal calf serum
(FCS) containing RPMI1640 (Nissui, Tokyo, Japan) for 24 hours. The
concentration of oligonucleotides in the conditioned medium was 2.2 mmol/L.
Adhesion assay.
Adhesion assay of ATL cells, cell lines to human umbilical vein-derived
endothelial cells (HUVECs), or purified ICAM-1 glycoproteins was
performed as previously described.5,10 HUVECs were placed onto 48-well culture plates (Costar, Cambridge, MA) coated with 2%
gelatine and cultured to confluence in Dulbecco's modified Eagle's
medium (D-MEM; Nissui) containing 100 U/mL penicillin G,
100 U/mL streptomycin, 20% heat-inactivated FCS, endothelial mitogen
20 µg/mL (Biomedical Technologies, Stoughton, MA), and Heparin (10 U/mL). After washing with phosphate-buffered saline (PBS), HUVECs were
stimulated with 1 ng/mL interleukin-1 (IL-1; Otsuka, Tokyo,
Japan) for 4 hours at 37°C. Purified ICAM-1 (50 ng/well) was
applied to 48-well plates in Ca/Mg-free PBS at 4°C overnight.
Binding sites were subsequently blocked with Ca/Mg-free PBS/3% human
serum albumin (HSA; Green-Cross, Osaka, Japan) for 2 hours at 37°C
to reduce nonspecific attachment. The plates were washed three times
with PBS before the addition of T cells. A total of 2 × 105 T cells, ATL cells, and ATL cell lines were labeled
with 51Cr (Dupont NEN, Wilmington, DE) in RPMI1640 with 1%
HSA and were added to the culture with or without the relevant adhesion
blocking MoAb in the presence or absence of phorbol myristate acetate
(PMA; 10 ng/mL; Sigma), which is a pharmacologically
relevant trigger of integrin adhesiveness. All MoAbs were used at a
saturating concentration of 10 µg/mL, which was shown in previous
studies to produce a maximum inhibition of the relevant adhesive
interaction.10 After a settling phase of 30 minutes at
4°C, which also allowed MoAb binding, the plates were rapidly
warmed to 37°C for 30 minutes and then gently washed twice with
RPMI-1640 at room temperature to completely remove nonadherent T cells
and ATL cells. The contents of each well containing adherent T cells or
ATL cells were lysed with 250 µL of 1% Triton X-100 (Sigma), and the
emission of the contents of each well was measured using a  -counter.
Data were expressed as the mean percentage of the binding of indicated
cells from a representative experiment.
Flow microfluorometry.
Staining and flow cytometric analyses of freshly obtained ATL or normal
T cells were performed using a FACScan (Becton Dickinson, Mountain
View, CA) and standard procedures as described
previously.6,10 Briefly, 2 × 105 cells
were incubated with negative control MoAb thy1.2, LFA-1 (CD11a) MoAb
TS1/22, VLA-4 (CD49d) MoAb NIH49d-1, activated LFA-1 MoAb NKI-L16, CD69
MoAb FN50 in fluorescence-activated cell sorting (FACS) media
consisting of Hanks' balanced salt solution (HBSS; Nissui), 0.5% HSA,
and 0.2% NaN3 (Sigma) for 30 minutes at 4°C. After
washing the cells three times with FACS media, they were further
incubated with fluorescein isothiocyanate
(FITC)-conjugated goat antimouse IgG Ab for 30 minutes at
4°C. After washing and incubation with irrelevant MoAbs, the cells
were further incubated with a mixture of PE-conjugated CD45RO MoAb
UCHL-1 and CyChrom-conjugated CD4 MoAb Leu-3a for 30 minutes at
4°C. The staining of cells with MoAbs was detected by gating on CD4
and CD45RO double-positive cells using FACScan. Amplification of the
MoAb-binding was provided by a three-decade logarithmic amplifier.
Quantification of cell surface antigens on single cells was calculated
using standard beads (QIFKIT; DAKO Japan, Kyoto, Japan).
Enzyme-linked immunosorbent assay (ELISA) of MIP-1
and MIP-1 in supernatant or cytosol of ATL cells.
Freshly isolated normal T cells and ATL cells (1 × 106) were washed in PBS and lysed with 250 µL of PBS
containing 2% N-octyl--D-glucopyraide (OGP; Sigma). The culture
supernatants were collected from normal T cells (1 × 106) and ATL cells (1 × 106) after 24 hours of incubation in RPMI1640 with 5% FCS at 37°C without any
stimulation. The level of MIP-1 protein in each sample was measured
by MIP-1 and MIP-1 ELISA system (R&D Systems, Minneapolis, MN).
The sensitivity of the assay was 4.0 pg/mL of MIP-1 and MIP-1 and
was not affected by the presence of OPG at the concentration used to
lyse cells. Results were expressed in nanograms per milliliter per 1 × 105 cells.
F-actin polymerization assay.
For microscopic analysis, T cells, ATL cells, and cell lines were
allowed to settle for 30 minutes at 4°C on fibronectin-coated slides. After incubation for 1 minute at 37°C, the cells were fixed
with 1% formaldehyde. F-actin was stained with rhodamine-phalloidin (1 U/slide; Molecular Probes, Inc, Eugene, OR) and analyzed later with a
confocal laser microscope system (model LSM 410UV, LD Achroplan 20 objective lens; Carl Zeiss, Juna, Germany).
Transmigration assay.
We assessed the transmigration of ATL cells in 3-mm pore, 12-well
microchemotaxis chambers (Transwell; Costar) precoated with IL-1-activated HUVECs for 48 hours at 37°C. ATL cells and ATL cell lines were labeled with 51Cr (1 × 106/well in RPMI-1640 with 10% FCS) and placed in the
insert wells. After incubation for 2 hours, cells that migrated into
each of the lower wells were retrieved and dissolved in 250 µL of 1%
Triton X-100, and -emission of well contents was determined. Data
were expressed as the mean percentage of transmigrated cells from a representative experiment.
| |
RESULTS |
ATL cells and cell lines spontaneously adhered to HUVECs.
One of the most characteristic features of ATL cells is their marked
infiltration into tissues during the acute phase.1,17,23 Leukocyte migration from circulation into the tissue is mediated by
adhesion of leukocyte integrins to their ligands, including ICAM-1, on
endothelial cells. However, leukocyte integrins are normally inactive;
hence, the regulation of integrin-dependent adhesion is critical to the
migration of virtually all hematopoietic cells.8,10 As
shown in Fig 1, ATL cells obtained from 6 representative ATL patients and 4 ATL cell lines spontaneously adhered
to IL-1-activated HUVECs after 30 minutes of incubation, without any
exogenous stimulus. In contrast, resting peripheral T cells did not
adhere to HUVECs and T cells bound only after they were activated with
PMA, a potent stimulator of integrins. CD4+ leukemic cells
from a patient with HTLV-1-negative T-cell leukemia also did not bind
to HUVECs.
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| Fig 1.
Spontaneous adhesion of ATL cells to IL-1-activated
HUVECs. Adhesion of resting or PMA-activated peripheral normal
CD4+ T cells, ATL cells freshly obtained from 6 representative ATL patients, CD4+ leukemic cells from a
patient with HTLV-1-negative T-CLL, and 4 ATL cell lines (MT1, MT2,
SALT-3, and HUT-102) that were labeled with 51Cr to
IL-1-activated HUVECs was assessed after 30 minutes of incubation
at 37°C. -Emission of the lysate of only adherent cells was
determined. Data are expressed as the mean percentage of binding of
indicated cells from a representative experiment.
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ATL cells expressed an activated form of LFA-1.
Using flow cytometry, we next examined whether the augmented adhesion
of ATL cells to endothelial cells depends on increased expression of
adhesion molecules or the expression of activated molecules. The
expression of LFA-1 on ATL cells was significantly lower than on normal
T cells (Fig 2a). In contrast, the
expression of VLA-4 on ATL cells was similar to that on normal T cells
(Fig 2b). LFA-1 requires an active configuration to bind to its ligand, a process that can be induced by a variety of stimuli,24-27
and NKI-L16 MoAb reacts with a Ca2+-dependent activation
epitope located on the ectodomain of -chain of
LFA-1.18,28 The expression of the activated form of LFA-1 as recognized by NKI-L16 MoAb was significantly higher on most ATL
cells than on normal resting CD4+CD45RO+ T
cells (Fig 2c). The majority of ATL cells also expressed CD69 (Fig 2d)
as well as MHC class II antigens and CD25 (data not shown), both
regarded as early activation markers. These results suggest that
spontaneous adhesion of ATL cells to the endothelium depends on
activated LFA-1 rather than on the amount of adhesion molecules expressed by ATL cells.
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| Fig 2.
Phenotypic analysis of fresh ATL cells by flow cytometry.
Staining and flow cytometric analyses of resting peripheral blood T
cells from 10 normal volunteers ( ) and ATL cells freshly obtained from peripheral blood of 20 ATL patients ( ) were performed with (a)
LFA-1 (CD11a) MoAb TS1/22, (b) VLA-4 (CD49d) MoAb NIH49d-1, (c)
an anti-activated form of LFA-1 MoAb NKI-L16, and (d)
CD69 MoAb by gating on CD4 and CD45RO double-positive cells using
FACScan. Each point represents the number of molecules expressed per
cell, calculated using standard QIFKIT beads. Bars represent the mean ± SD of each group and statistical significance was determined by the
Student's t-test.
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ATL cells spontaneously secreted chemokines MIP-1 and
MIP-1.
We and others have proposed that chemokines such as MIP-1 and
MIP-1 functionally trigger T-lymphocyte
integrins.11,29,30 ATL cells produced significant amounts
of MIP-1 and MIP-1 protein in the culture supernatant as well as
in the cytosol without any stimulation, whereas normal resting T cells
did not produce any of these chemokines
(Fig 3). These results suggest that
MIP-1 and MIP-1 are spontaneously produced by ATL cells and might
trigger integrin-mediated adhesion of ATL cells to endothelial cells in an autocrine manner.
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| Fig 3.
Spontaneous MIP-1 and MIP-1 production from ATL
cells. The cytokine levels in culture supernatants (a and c) collected
from normal CD4+ T cells (group A) and the ATL cells
(group B) after 24 hours of incubation at 37°C without any
stimulation or cytosol (b and d) of normal CD4+ T cells
(group A) and ATL cells freshly obtained from peripheral blood of ATL
patients (group B) were determined by MIP-1 (a and b) and MIP-1
(c and d) ELISA system. Each point represents the concentration of
MIP-1 and MIP-1 in the lysate or supernatant derived from 1 × 105 cells of individual subjects. Bars represent the mean ± SD of each group (Student's t-test).
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Signaling pathways involved in increased integrin-dependent adhesion
of ATL cells to ICAM-1 and HUVECs.
ATL cells spontaneously adhered to purified ICAM-1
(Fig 4a) as well as IL-1-activated
HUVEC (Fig 4b). MoAb blocking studies, in which ATL cell adhesion to
activated HUVECs or ICAM-1 were inhibited by anti-LFA-1/VLA-4 MoAbs or
by anti-LFA-1 MoAb alone, respectively, indicated that the adhesion
was mediated by ATL cell integrins and their endothelial ligands.
Chemokine receptors belong to the serpentine family of seven
transmembrane G-protein-coupled receptors.31 To determine
if G-proteins are involved in the induction of ATL cell adhesion to the
endothelium, we also analyzed the ability of several signaling
inhibitors to block adhesion. Pretreatment of cells with pertussis
toxin (which uncouples certain G-proteins from their complex) reduced
integrin-dependent increased adhesion of ATL cells to purified ICAM-1
and activated HUVECs in a concentration-dependent manner
(Fig 5a and b). Adhesion also diminished by
10 to 100 nmol/L of wortmannin, a PI 3-kinase inhibitor. Two A-kinase
inhibitors, H89 and H88, also slightly decreased the adhesion. However,
H7 and staurosporin (C-kinase inhibitors) and herbimycin A and
genestein (tyrosine kinase inhibitors) did not influence
adhesion. Furthermore, the data in Fig 4 show that increased adhesion was also inhibited by pretreatment of ATL cells with
a mixture of anti-MIP-1 and anti-MIP-1 Abs and was reduced by
transfection of antisense oligonucleotides, but not sense
oligonucleotides, of either MIP-1 or MIP-1. Thus, these data
indicate that, although the increased and spontaneous integrin-mediated
adhesion of ATL cells to ICAM-1 and HUVECs is mediated by a number of
signaling pathways, it mainly depends on activation of
G-protein-sensitive PI 3-kinase that is stimulated by endogenous
chemokines MIP-1 and MIP-1.
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| Fig 4.
Adhesion of peripheral ATL cells to IL-1-activated
HUVECs and purified ICAM-1. A proportion of ATL cells were transfected with sense or antisense oligonucleotides of MIP-1 and MIP-1 and
preincubated for 24 hours at 37°C. Another group of ATL cells were
pretreated with a mixture of anti-MIP-1 and anti-MIP-1 Abs for
4 hours at 37°C. Adhesion assay of ATL cells to purified ICAM-1 (a)
or IL-1-activated HUVECs (b) was performed in the presence or absence of indicated adhesion-blocking MoAbs (10 µg/mL). Data are
expressed as the mean percentage of bound cells from a representative experiment of 4 patients.
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| Fig 5.
Effects of multiple signaling inhibitors on adhesion of
peripheral ATL cells to IL-1-activated HUVECs and purified ICAM-1. ATL
cells were pretreated with or without indicated concentrations of
multiple inhibitors of intracytoplasmic signaling to purified ICAM-1
(a) and IL-1-activated HUVECs (b). Adhesion assay was performed as
described in the Materials and Methods. Data are expressed as the mean
percentage of bound cells from a representative experiment of 4 patients.
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Signaling pathways involved in expression of activated form LFA-1 on
ATL cells.
Figure 6 shows histograms of conventional
LFA-1 chain (CD11a) and the activated form of LFA-1 recognized by
NKI-L16 MoAb on normal resting T cells and ATL cells from a
representative normal donor and from an ATL patient. The
resting T cells (Fig 6a) and ATL cells (Fig 6b) expressed LFA-1
equally. However, the activated form of LFA-1 was highly expressed on
ATL cells (Fig 6b), but not on resting T cells (Fig 6a). The
spontaneous expression of activated LFA-1 on ATL cells diminished by
pretreatment of these cells with pertussis toxin (Fig 6c) or wortmannin
(Fig 6d), but not with herbimycin A (Fig 6e). In contrast, the
expression of conventional LFA-1 did not change by these inhibitors
(Fig 6c, d, and e). Furthermore, pretreatment of cells with a mixture of anti-MIP-1 and anti-MIP-1 Abs resulted in a decrease of the activated form of LFA-1 (Fig 6f), but not LFA-1 molecules (Fig 6f) on
ATL cells (mean fluorescence intensity for NKI-L16 MoAb-staining decreased from 370 to 260; the number of molecules/cell bound with
NKI-L16 MoAb decreased from 4,200 to 1,000; calculated by QIFKIT). These results suggest that the spontaneous
expression of activated form of LFA-1 on ATL cells may depend on
cytoskeletal rearrangement induced through G-protein-dependent
activation of PI 3-kinase stimulated by endogenous chemokines.
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| Fig 6.
Staining for the activated form of LFA-1 (CD11a) on
peripheral ATL cells using NKI-L16 MoAb. Peripheral normal T cells (a) and ATL cells (b through f) were pretreated with or without 1 µg/mL
pertussis toxin for 1 hour at 37°C (c), 100 nmol/L wortmannin (d),
10 µmol/L herbimycin A (e), or a mixture of anti-MIP-1 and anti-MIP-1 Abs (f) for 2 hours at 37°C. The cells were
subsequently stained with CD11a MoAb TS1/22 or NKI-L16 MoAb. Cells in
the CD4 and CD45RO gate (triple fluorescence) were analyzed by using a flow cytometer. Representative histograms from each one of the volunteers or patients are shown.
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Actin polymerization in ATL cells was inhibited by pertussis toxin
and wortmannin.
Actin polymerization is a dynamic process critical to cellular adhesion
and functional LFA-1 is associated with polymerized F-actin.25,29 Resting T cells seeded on fibronectin did not spread and their F-actin content and distribution remained constant as
observed by confocal microscopy (Fig 7A).
In contrast, freshly obtained ATL cells (Fig 7B and C) showed increased
expression of F-actin in the cell cortex, and marked spreading and
polymerization of F-actin was observed without any stimulation.
However, when ATL cells were pretreated with pertussis toxin or
wortmannin, F-actin-polymerization was markedly reduced (Fig 7D and E).
These results suggest that the spontaneous polymerization of F-actin in
ATL cells may be induced by signaling through G-protein-dependent activation of PI 3-kinase.
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| Fig 7.
Confocal microscopical analysis of polymerized F-actin on
ATL cells. Resting normal T cells (A), ATL cells obtained from 2 representative ATL patients (B and C), and ATL cells pretreated with 1 µg/mL pertussis toxin (D) or 100 nmol/L wortmannin (E) for 2 hours at
37°C were incubated on fibronectin-coated slides for 1 minute, and
the F-actin in these cells was stained with rhodamine-phalloidin and
was observed by confocal microscopy (original magnification
×1,000).
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Endogenous MIP-1 and MIP-1 were
involved in spontaneous transmigration of ATL cells.
Finally, we assessed the effects of endogenous MIP-1 and MIP-1 on
transendothelial migration. ATL cells and MT1 cell lines spontaneously
transmigrated through IL-1-activated HUVECs without any exogenous
stimuli after 2 hours of incubation (Fig 8a
and b). The increased transendothelial migration was reduced by
pretreatment of ATL cells with a mixture of anti-MIP-1 and
anti-MIP-1 Abs as well as by transfection of antisense
oligonucleotides, but not by sense oligonucleotides, of either MIP-1
or MIP-1. Thus, spontaneous transendothelial migration of ATL cells
as well as increased integrin-mediated adhesion of ATL cells to
endothelial ligands is mediated by a number of signaling pathways that
are likely to be mediated by endogenous chemokines MIP-1 and
MIP-1.
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| Fig 8.
Transmigration of ATL cells through IL-1-activated
HUVECs. Transmigration of ATL cells was assessed in 3-µm pore,
12-well microchemotaxis chambers precoated by IL-1-activated HUVECs
for 48 hours at 37°C. An ATL cell line MT1 (a) and ATL cells from patients (b), both labeled with 51Cr, were placed in the
insert wells. After incubation for 2 hours at 37°C, cells that have
migrated into each of the lower wells were retrieved and dissolved and
-emissions of the well contents were determined. Data are expressed
as the mean percentage of transmigrated cells from a representative
experiment.
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| |
DISCUSSION |
The process of leukocyte transmigration from circulation into the
tissue involves a series of processes, including tethering, triggering
of integrins, firm integrin-mediated adhesion, and diapedesis.8,15,32 Leukocyte integrins adhere firmly to
their endothelial ligands that belong to the Ig superfamily, including ICAM-1 and VCAM-1. However, integrins on resting circulating leukocytes do not mediate such adhesion until activated by certain
stimuli.7,9,10 In contrast, the present results showed that
ATL cells of ATL patients spontaneously adhered to endothelial cells as
well as purified ICAM-1 and subsequently transmigrated through
endothelial cells without any exogenous stimulation. In addition, ATL
cells spontaneously expressed the activated form of integrin LFA-1 as recognized by NKI-L16 MoAb that reacts with a
Ca2+-dependent activation epitope located on the ectodomain
of -chain of LFA-1.18,28,33 Active configuration of
LFA-1 is thought to be induced by a conformational change of LFA-1
and/or clustering of the LFA-1 molecules.24-27
Several studies have suggested that F-actin polymerization and
association with integrins are involved in activation of
integrins.18,25,29,34,35 ATL cells showed a clear increase
of F-actin in the cell cortex and marked spreading, polymerization, and
rearrangement of F-actin without the addition of any stimulus, whereas
the distribution of F-actin on resting T cells remained stable.
Furthermore, the cytoskeleton-disrupting agent cytochalasin B reduced
spontaneous adhesion of ATL cells to endothelial cells and ICAM-1.
Based on these results, we suggest that cytoskeletal rearrangement may
be involved in activation of integrin and subsequent induction of
adhesion of ATL cells to endothelial cells in vitro.
The adhesive capacity of integrins is tightly regulated through
intracellular signals, a process referred to as inside-out signaling.34 Recent studies suggest that activation of
integrin can be induced by multiple signaling pathways that may depend on different integrin regulators, including G-proteins, tyrosine kinases, protein kinase C, cAMP pathway, and PI 3-kinases. This can
result in actin polymerization and association of LFA-1 with cytoskeletal proteins.15,16,24,29,30,36-39 In the present study, pretreatment of ATL cells with either pertussis toxin or wortmannin reduced polymerization of F-actin, diminished the expression of activated LFA-1, and inhibited integrin-mediated adhesion of the
cells to endothelial ligands. However, the effect of these signaling
inhibitors was incomplete, indicating that other pathways might be
involved as well. For instance, two A-kinase inhibitors, H89 and H88,
also slightly decreased integrin-mediated adhesion of ATL cells to
endothelial ligands. However, herbimycin A and genestein (tyrosine
kinase inhibitors) and H7 and staurosporine (protein kinase
C-inhibitors) did not change the spontaneous adhesion of ATL cells.
G-protein-coupled receptors are known to activate PI 3-kinases and
integrin adhesiveness through ligation of the receptor with fMLP and
certain chemokines such as RANTES and monocyte chemotactic protein-1
(MCP-1).36 Furthermore, it is thought that PI 3-kinase is
controlled by G-protein-coupled chemoattractant receptors and is
involved in cytoskeletal changes associated with localized
polymerization of actin filaments and highly cross-linked membrane-associated fibers.16 Based on these early
findings, our results suggest that G-protein-dependent activation of
PI 3-kinases is likely to be involved in the induction of active LFA-1
through cytoskeletal reorganization in ATL cytoplasm and subsequent
amplification of adhesion of ATL cells, although the pathways
downstream of PI 3-kinase as well as their role in these cytoskeletal
changes are unknown at present.
We and others have previously proposed that chemokine MIP-1 and IL-8
induce integrin-mediated adhesion of T cells and neutrophils, respectively.11,40 ATL cells produced high quantities of
both MIP-1 and MIP-1 in the culture supernatant as well as in the cytoplasmic fraction of ATL cells. We have reported that ATL cells produce a variety of cytokines, including IL-1, IL-6, and tumor necrosis factor- (TNF-).4,41 Among these cytokines,
the spontaneous production of MIP-1 and MIP-1 might be a
characteristic feature of ATL cells, because the HTLV-1-viral product
tax induces both MIP-1 and MIP-1.42,43 Furthermore,
pretreatment of ATL cells with anti-MIP-1 and anti-MIP-1 Abs as
well as transfection of antisense oligonucleotide of either MIP-1 or
MIP-1 into ATL cells partially reduced the expression of activated
LFA-1 and integrin-mediated adhesion of ATL cells to endothelial
ligands and, subsequently, their transendothelial migration. Several
chemokines, including fMLP, platelet activation factor (PAF), and
leukotrien B4, are known to induce leukocyte adhesion through integrin
activation and all these signals are via serpentine G-protein-coupled
receptors on leukocytes.37,44,45 Our finding that pertussis
toxin inhibited both the expression of the active form of LFA-1 and
integrin-mediated adhesion of ATL cells suggests that MIP-1 and
MIP-1 produced by ATL cells could transduce signaling through
G-protein by binding to its serpentine receptor on the cells and induce
continuous triggering of integrin-mediated adhesion of ATL cells by an
autocrine mechanism. We have also reported that ATL cells
characteristically express large amounts of heparan sulfate
proteoglycan on their surface and heparan sulfate binds chemokines
MIP-1 and MIP-1, which are heparin-binding
cytokines.5,11 Heparan sulfate proteoglycan is synthesized
and binds any heparin-binding cytokine in cytoplasmic organelles such
as Golgi bodies, which are then transferred to the cell surface holding
the cytokines46,47 and chemokines. Thus, endogenous
cytokines and chemokines accumulate on the cell surface and are
presented to chemokine receptors in an autocrine manner.
Extravasation and infiltration of circulating hematologic malignant
cells into tissues is likely to be a consequence of their biologic
properties, expression and function of relevant adhesion molecules on
these cells, and their adhesive interaction with endothelial
cells.3 Circulating ATL cells first bind to the endothelium
through a loose tethering of selectin to its ligand such as sialyl
LewisX, because ATL cells express high number of sialyl
LewisX.48 In the present study, we observed
that polymerization of F-actin and effective triggering of LFA-1 on
circulating ATL cells is essential for the binding of ATL cells to the
endothelium. Our MoAb-blocking studies of ATL cell adhesion to the
endothelium indicated that the firm adhesion was integrin-dependent.
Based on these results, we propose that the concept of adhesion cascade involved in leukocyte adhesion to endothelium can be expanded to
include leukemic cell migration and metastasis. Especially for ATL
cells, spontaneous triggering of integrin on ATL cells induced by
endogenous chemokines could explain the strong binding of ATL cells to
the endothelium and their spontaneous transendothelial migration. It is
noteworthy that not only resting T cells, but also other leukemic T
cells such as CEM and Jurkat do not express the activated form of
LFA-1, despite significant differences in LFA-1
expression.18 These results indicate that
chemokine-dependent triggering of LFA-1 through G-protein-dependent
activation of PI 3-kinases and subsequent induction of
integrin-mediated adhesion may result in the aberrant behavior of ATL
cells and clinical features marking the infiltration of cells into
multiple organs during the acute phase. Furthermore, we have also
demonstrated that ATL cells show marked clustering through the
LFA-1/ICAM-1 pathway in vitro,4,18 suggesting that the high
adhesiveness of ATL cells could be involved not only in their
infiltration into tissues, but also in nodular proliferation in
multiple tissues in vivo, both of which lead to poor prognosis. The
findings using ATL cells in this study warrant further studies to
unravel the mechanisms of integrin activation in other types of cells
and the pathogenesis of leukemic cell infiltration and proliferation, which could lead to new pharmacologic approaches to control these diseases.
| |
FOOTNOTES |
Submitted August 6, 1997;
accepted January 5, 1998.
Supported in part by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of Japan.
Address reprint requests to Yoshiya Tanaka, MD, The First Department of
Internal Medicine, University of Occupational and Environmental Health,
Japan, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu
807, Japan.
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.
| |
ACKNOWLEDGMENT |
The authors thank T. Adachi for the excellent technical assistance. We
also thank the following investigators for providing MoAbs and cell
lines: Dr B. Malissen for B9.8.4 MoAb; Dr K. Sagawa for MT1, MT2,
HUT-102, and SALT-3 cell lines; Dr F. Sanchez-Madrid for HP2/1 MoAb; Dr
S. Shaw for 84H10, NIH11-1, and NIH49d-1 MoAbs and L428 cell line;
Dr U. Siebenlist for anti-MIP-1 Ab and anti-MIP-1 Ab; Dr D. Siegel for 3G8 MoAb; and H. Zola for FMC63 MoAb.
| |
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H-Ras Signals to Cytoskeletal Machinery in Induction of Integrin-Mediated Adhesion of T Cells
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December 1, 1999;
163(11):
6209 - 6216.
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T. Fujisaki, Y. Tanaka, K. Fujii, S. Mine, K. Saito, S. Yamada, U. Yamashita, T. Irimura, and S. Eto
CD44 Stimulation Induces Integrin-mediated Adhesion of Colon Cancer Cell Lines to Endothelial Cells by Up-Regulation of Integrins and c-Met and Activation of Integrins
Cancer Res.,
September 1, 1999;
59(17):
4427 - 4434.
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Abstract
Introduction
Abstract