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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 30-38
CHEMOKINES
Increased chemokine receptor CCR7/EBI1 expression enhances the
infiltration of lymphoid organs by adult T-cell leukemia cells
Hitoshi Hasegawa,
Tetsuhiko Nomura,
Masashi Kohno,
Norihiko Tateishi,
Yoji Suzuki,
Nobuji Maeda,
Ryuichi Fujisawa,
Osamu Yoshie, and
Shigeru Fujita
From the First Department of Internal Medicine, Ehime University
School of Medicine, Shigenobu, Ehime 791-0295, Japan; the Department of
Physiology, Ehime University School of Medicine, Shigenobu, Ehime
791-0295, Japan; and the Department of Bacteriology, Kinki University
School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
 |
Abstract |
Adult T-cell leukemia (ATL) is characterized by infiltration of
various tissues by circulating ATL cells, a finding often associated
with a poor prognosis. Leukocyte migration from the circulation into
tissues depends on integrin-mediated adhesion to the endothelium, and
integrins are tightly regulated by several factors, such as chemokines.
In this study, we focused on the interaction between chemokines and
chemokine receptors on ATL cells to understand factors involved in ATL
cell infiltration of lymphoid organs. We compared freshly isolated ATL
cells from patients with and without lymphoid organ involvement for the
expression of the chemokine receptor CCR7/EBI1, the functional
receptor for secondary lymphoid-tissue chemokine
(SLC), which is expressed at high levels by high endothelial venules of
lymph nodes and Peyer's patches. Reverse transcriptase-polymerase
chain reaction and flow cytometric analysis, using anti-CCR7 monoclonal
antibody (CCR7.6B3), revealed that ATL cells from patients with
lymphoid organ involvement expressed significantly more CCR7/EBI1 than control CD4+CD45RO+ T cells and ATL cells
from patients without lymphoid organ involvement. Consequently,
significantly more ATL cells from patients with lymphoid organ
involvement than control
CD4+CD45RO+ T cells and ATL cells from
patients without lymphoid organ involvement adhered to
surfaces coated with ICAM-1 and SLC or EBI1-ligand chemokine (ELC), another ligand for CCR7/EBI1, under static and flow
conditions and migrated toward SLC or ELC at a low concentration (30 ng/ml). These findings suggest that increased CCR7/EBI1 expression plays a role in lymphoid organ infiltration of ATL cells. (Blood. 2000;
30-38)
© 2000 by The American Society of Hematology.
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Introduction |
Adult T-cell leukemia (ATL) is a peripheral
CD4+ T-cell malignancy caused by human T-cell leukemia
virus type 1 (HTLV-1). A frequent manifestation of ATL is infiltration
of various organs, such as the lymph nodes, liver, spleen, lungs, skin,
and intestinal tract, by leukemic cells. ATL cell infiltration often
poses serious clinical problems for patients, affecting the disease
profile and prognosis. Tissue infiltration by ATL cells probably
reflects the cells' biological properties, such as the expression
and function of relevant adhesion molecules and the adhesive
interactions with endothelial cells.
Lymphocytes migrate into lymph nodes, Peyer's patches, and other
secondary lymphoid organs by interacting with organ-specific adhesion
molecules expressed by high endothelial venules (HEVs).1,2 A multistep model involving cell adhesion and activation of leukocyte integrins has been proposed for selective leukocyte trafficking, including the process of lymphocyte homing. According to this model,
the sequential involvement of several receptor-ligand pairs selected
from many potential combinations results in lymphocyte subset-specific
homing. The process involves an initial tethering and rolling step
mediated by L-selectin on the surfaces of lymphocytes and a specific
complex of glycoproteins, known as peripheral lymph node addressin,
expressed on the endothelium.3,4 The rolling interaction is
followed by firm arrest of cells,5,6 which is mediated by
 2 integrin,  L2 (LFA-1) binding to ICAM-1 or ICAM-2 on the
endothelium. Then, lymphocytes transmigrate across the endothelium into
the tissues. Treatment of lymphocytes with pertussis toxin (PTX)
inhibits the L2 (LFA-1)-mediated adhesion to HEVs, indicating
that a G-protein-linked signal-transduction mechanism is a component
of these processes. Chemokines that bind to G-protein-coupled
receptors have emerged as candidates for these integrin activation
signals. However, although in vitro static adhesion assays have shown
that some chemokines increase integrin-mediated lymphocyte
adhesion,7-11 this effect does not appear to be rapid or
robust enough to account for the intravascular firm arrest of
lymphocytes in HEVs. Furthermore, these chemokines are not normally
produced by HEV cells of lymph nodes or Peyer's patches.
Recently, a lymphocyte-specific chemokine termed secondary
lymphoid-tissue chemokine (SLC), also known as 6Ckine, Exodus-2, and
thymus-derived chemotactic agent 4, was reported to be expressed at
high levels in HEV cells and areas of T-cell accumulation in lymph
nodes, Peyer's patches, and spleen.12-19 SLC is a highly efficacious chemoattractant for lymphocytes, including naive T cells,
but does not attract monocytes or neutrophils. Furthermore, SLC induced
firm adhesion of lymphocytes via 2 integrin binding to ICAM-1 under
static and flow conditions, suggesting that SLC plays a role in
lymphocyte homing.16,20,21 SLC is a high-affinity functional ligand for CCR7/EBI1, which was originally identified as a
receptor induced by Epstein-Barr virus infection in B
cells.22 CCR7/EBI1 is expressed on T, B, and mature
dendritic cells and is up-regulated in CD4+ T cells in
response to infection with human herpesvirus (HHV)-6 and
HHV-7.23-29 A recent study demonstrated that each of the 3 chemokines expressed in lymph nodes, SLC, EBI1-ligand chemokine (ELC),
and stromal cell-derived factor (SDF)-1, could promote integrin-mediated arrest of human lymphocytes under flow
conditions.20 ELC is another high-affinity functional
ligand for CCR7/EBI1.30 SDF-1 is the only ligand for
CXCR4 to be identified so far.31-33 In this study, we
compared ATL cells from patients with and without lymphoid organ
infiltration for CCR7/EBI1 and CXCR4 expression levels and have
demonstrated that increased CCR7/EBI1 expression correlates with
infiltration of lymphoid organs by ATL cells.
| |
Materials and methods |
Cells
The human T-cell line HUT78 was obtained from the
American Type Culture Collection (Rockville, MD) and maintained in
RPMI 1640 medium supplemented with 10% heat-inactivated
fetal calf serum (FCS) (Life Technologies, Gaithersburg, MD). Human
umbilical vein-derived endothelial cells (HUVECs) were purchased from
Takara Shuzo (Kyoto, Japan).
ATL patients
Mononuclear cells were isolated by Ficoll-Conray density gradient
centrifugation from lymph nodes and peripheral blood samples from 16 patients with involvement of lymphoid organs, such as the lymph nodes
or spleen, and from 12 patients without lymphoid organ involvement.
Control mononuclear cells prepared from peripheral blood (PBMCs) were
obtained from 8 healthy volunteers. The control lymph node samples were
prepared from the reactive lymph nodes of HTLV-1-seronegative
individuals who had undergone abdominal surgery. ATL was diagnosed
according to the following clinical criteria: serum antibodies against
HTLV-1-associated antigens were present; mononuclear cells showed the
morphologic characteristic of highly convoluted nuclei; the results of
phenotypic analysis of ATL cells with anti-CD2, anti-CD4, and anti-CD25
monoclonal antibodies (mAbs); and the HTLV-1 proviral genome were
integrated monoclonally in the cells. Using Shimoyama's
criteria,34 the 16 ATL patients with lymphoid organ
involvement were diagnosed as having acute ATL. Of these, 6 had
involvement of the skin, gut, or bone marrow. The infiltration of the
lymph organs, skin, gut, or bone marrow by ATL cells was confirmed
radiographically and histochemically by examining biopsy samples or
specimens obtained during autopsy. Of the 12 patients with
noninfiltrating ATL, 8 and 4 had acute and chronic ATL, respectively.
The relevant clinical data, profiles, and extent of organ involvement
of each of the subjects are summarized in the
Table.
Highly purified CD4+ T cells were enriched by negative
immunoselection from mononuclear cells, using a multiple mAb mixture and immunomagnetic beads, as described previously.35
Briefly, PBMCs of ATL patients and control samples were incubated for
30 minutes at 4°C with a cocktail of mAbs against CD8, CD11b, CD14, CD16, and CD20. After washing, Dynabeads (Japan Dynal, Tokyo, Japan)
were added, incubated for 1 hour at 4°C, and then removed with a
Dynal magnetic particle concentrator. The proportion of ATL cells
(CD4+ and CD25+ cells) in the negatively
selected cells of each ATL patient was greater than 90%, and the ATL
cells in all the samples expressed CD45RO.
CD4+CD45RO+ T cells from control samples were
purified by the same negative immunoselection technique, using
additional monoclonal antibodies (mAbs) against HLA-DR and CD45RA, and
the proportion of CD4+CD45RO+ T cells in each
control sample was approximately 90%.
Antibodies and reagents
Mouse mAbs to CD8 (OKT8), CD11b (OKM1), and HLA-DR (OKIa1) were
obtained from the American Type Culture Collection (Rockville, MD).
Mouse mAbs, anti-CD14 (322A-1), anti-CD18 (7E4), anti-CD20 (B1),
anti-CD45RA (2H4), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (OKT4), phycoerythrin (PE)-conjugated anti-CD25 (B1.49.9), and
PE-anti-CD45RO (UCHL1) were purchased from Coulter Co
(Hialeah, FL). Mouse mAbs to CD16 (3G8) were purchased
from Chemicon International Inc (Temecula, CA).
Anti-human CXCR4 mAb (12G5), recombinant human ELC/macrophage
inflammatory protein (MIP)-3, SLC/6Ckine, and ICAM-1 were purchased
from R&D Systems Inc (Minneapolis, MN).
The anti-CCR7 mAb, designated CCR7.6B3 (IgG1,  ), was
produced by fusing SP2/0-Ag14 cells with spleen cells from BALB/c mice immunized with CCR7-peptide, YIGDNTTVDYTLFESLC (H.H. et al, unpublished data). CCR7.6B3 antibody was found to be specific for
CCR7 by flow cytometric analysis, using other chemokine
receptor-transfected cells. This antibody is useful for flow cytometric
analysis but not for immunohistochemistry or immunoblotting, because it
cross-reacts with intracellular proteins.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
The total cellular RNA was extracted from control
CD4+CD45RO+ T cells and isolated ATL cells by
the acid phenol-guanidinium isothiocyanate extraction method, as
described previously.36 The expression of CCR7/EBI1 in ATL
cells and CD4+CD45RO+ T cells from control
samples was analyzed by RT-PCR, using an RNA PCR kit (Takara Shuzo) and
the GeneAmp PCR system 2400 (Perkin Elmer, Foster, CA), as described
previously.37 First-strand cDNA was synthesized from 1 µg
total cellular RNA and subjected to 28 PCR cycles (94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute) with specific
primers for CCR7/EBI1 (forward,
5'-TCCTTCTCATCAGCAAGCTGTC-3' and reverse,
5'-GAGGCAGCCCAGGTCCTTGAAG-3'), as described by Yanagihara et al.28 As a control, specific primers for actin
purchased from Clontech (Palo Alto, CA) were used. After staining the
primers with ethidium bromide, the density of each band
was analyzed with FluorImager SI (Molecular Dynamics, Sunnyvale, CA).
In vitro static adhesion assay
Adhesion of ATL cells and control
CD4+CD45RO+ T cells to HUVECs and ICAM-1 was
assayed as described by Tanaka et al.11 Briefly, HUVECs
were placed on 96-well culture plates, cultured to confluence in EBM-2
medium (Takara Shuzo), washed with phosphate-buffered saline (PBS), and
then stimulated with 1 ng/ml interleukin-1 (IL-1) (Otsuka, Tokyo,
Japan) for 4 hours at 37°C. ICAM-1 protein (25 ng/well) in PBS was
applied to 96-well culture plates, which were incubated at 4°C
overnight. The binding sites were then blocked with PBS/1% bovine
serum albumin (BSA) for 2 hours at 37°C to reduce nonspecific
attachment. Cells, which had been labeled with 51Cr (Dupont
NEN, Wilmington, DE) in RPMI 1640 medium supplemented with 1% BSA,
were untreated or preincubated with anti-CD18 mAb (7E4, 20 µg/ml) for
20 minutes at room temperature or with 100 ng/ml PTX (Sigma, St. Louis,
MO) at 37°C for 1.5 hours. Untreated or treated cells
(2 × 105) were incubated with SLC or ELC at a
concentration of 30 ng/ml at 37°C for 30 minutes, and then were
added to each well. The plates were incubated at 37°C for 30 minutes, then gently washed twice with RPMI 1640 medium at room
temperature to remove all the nonadherent cells. The
adherent cells in each well were lysed with 250 µl of 1% Triton
X-100 (Sigma), and the radioactivity of the contents of each well was
measured using a  -counter. The data are expressed as the mean
percentage of the binding of indicated cells from triplicate experiments.
Flow adhesion assay
Adhesion substrates were prepared as described by Tangemann et
al.21 Briefly, adhesion substrates were generated by
coating 10 µg/ml ICAM-1 in Tris-buffered saline (TBS), pH 9.0, onto
petri dishes (Corning, San Mateo, CA) for 2 hours at room temperature. After washing the substrates with PBS, chemokine was
coated at a concentration that induced maximal activation effects (10 µg/ml) in TBS, pH 9.0, for 1 hour. The substrates were washed and
blocked with 3% BSA for 2 hours at room temperature.
A rheoscope combining a transparent 0.8° cone and substrate-coated
dishes with an inverted microscope (Olympus Optics Co, IMT-2, Tokyo,
Japan) was used as described previously.38 Images were
captured using a video camera (Sony Corp, DXC-101, Tokyo, Japan), a
videotape recorder (Sony, SLO-420), and an image processor (Nippon
Avionics Co, TVIP-2000, Tokyo, Japan). Cells (106/ml in
Hank's balanced salt solution supplemented with 0.2% BSA) were
perfused onto each substrate-coated dish at 1 dyne/cm2, and
a single field of view (4 × objective) was recorded for the first 4 minutes. Arrested cells were defined as those that remained stationary
during the 15-second interval by overlaying the succession of captured
images in the computer analysis. The number of arrested cells is shown
by the total number accumulated as arrested cells for the first 4 minutes. Experiments were performed in triplicate.
Chemotactic assay
Chemotactic assays for control CD4+CD45RO+ T
cells and ATL cells were performed in polycarbonate membrane, 6.5-mm
diameter, 5-µm pore size transwell cell culture chambers (Costar
Corp, Cambridge, MA). Aliquots (100 µl) of cells
(5 × 106/ml) suspended in RPMI 1640/0.5% BSA were
added to the upper chambers, and either SLC or ELC, to produce a final
concentration of 30 ng/ml, was added to the lower wells. The cells were
allowed to migrate for 2 hours at 37°C in a 5% CO2
incubator, after which the filters were fixed with 1% glutaraldehyde
in PBS for 30 minutes and stained with 0.5% toluidine blue overnight.
Cell migration was quantified by counting cells in each lower chamber
and cells adhering to the bottom part of the polycarbonate filter. Each assay was performed in triplicate.
Flow cytometric analysis
Flow cytometric analysis of freshly isolated ATL cells and control
CD4+CD45RO+ T cells was conducted as described
previously.35 The cells (2 × 105) were
washed once with 3% FCS-PBS and incubated with anti-CCR7, anti-CXCR4,
IgG1, or IgG2a mouse mAbs (negative control),
at a saturating concentration (10 µg/ml), on ice for 30 minutes.
After washing with 3% FCS-PBS, the cells were stained with
FITC-conjugated goat anti-mouse IgG (Cappel, Westchester, PA) on ice
for 30 minutes, washed, and resuspended in 3% FCS-PBS. The
fluorescence intensity was analyzed using a profile flow cytometer
(Epics; Coulter). The mean fluorescence intensity (MFI) for CCR7 or
CXCR4 on positive cells was corrected by subtracting MFI obtained with
the nonbinding control IgG1 or IgG2a mouse mAb, respectively.
| |
Results |
ATL cells from patients with lymphoid organ involvement expressed
more CCR7/EBI1 than those without involvement of lymphoid organs
One of the major features of ATL cells is their marked infiltration
into tissues. Leukocyte migration from the circulation into tissues is
mediated by adhesion of leukocyte integrins to their ligands, including
ICAM-1, on endothelial cells. Leukocyte integrins are normally
inactive, and chemokines are capable of inducing integrin activation.
SLC is a unique chemokine: It is the first chemokine found to be
expressed at strikingly high levels in HEV cells of lymph nodes and
Peyer's patches and to have the characteristics required to mediate
homing of lymphocytes. Therefore, to investigate ATL infiltration of
lymphoid organs, we focused on the expression of CCR7/EBI1, the
functional receptor for SLC and ELC, on ATL cells. We used RT-PCR to
analyze CCR7/EBI1 expression. The optimal conditions for detection and
quantitation of CCR7/EBI1 expression were established by determining
the relative yields of the PCR products of the
CD4+CD45RO+ T cells and HUT 78 cells after
various numbers of PCR cycles. After 35 cycles, the yield of the
CCR7/EBI1-specific product of each of these cells reached a plateau,
whereas after 25 to 33 cycles, it was in the exponential range,
enabling the initial amounts of mRNA template in the samples to be
compared (data not shown). The actin-specific product was amplified
exponentially by 20 to 30 PCR cycles (data not shown). Therefore, the
CCR7/EBI1 and actin-specific products of
CD4+CD45RO+ T cells and ATL cells were placed
in the same tube and subjected to 28 PCR amplification cycles.
The CCR7/EBI1 expression level was evaluated with the ratio of CCR7/
actin-specific PCR product. As shown in Figure
1A and 1B, the freshly isolated ATL cells
expressed significantly more CCR7/EBI1 than the
CD4+CD45RO+ T cells. Furthermore, the CCR7/EBI1
expression level of ATL cells from patients with lymphoid organ
involvement was significantly higher than that of ATL cells from
patients without involvement of lymphoid organs. Recently, we generated
an anti-CCR7 mAb, designated CCR7.6B3. From flow cytometric analysis
using this mAb, CCR7/EBI1-positive cells were 55% ± 14% of
CD4+CD45RO+ T cells from 8 healthy volunteers,
but more than 90% of ATL cells (data not shown). As shown in Figure
1C, the data obtained from flow cytometric analysis were consistent
with those from RT-PCR analysis. We also examined the expression level
of CXCR4, the receptor for SDF-1, by calculating MFI after staining
with anti-CXCR4 mAb (Figure 1D). CXCR4-positive cells were 76% ± 18% of CD4+CD45RO+ T cells from 8 healthy
volunteers (data not shown). The CXCR4 expression levels of ATL cells
were much higher than those of normal CXCR4-positive
CD4+CD45RO+ T cells, but there were no
significant differences between ATL cells from patients with
and without lymphoid organ involvement.
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| Fig 1.
Analyses for CCR7/EBI1 expression.
(A) Reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis for CCR7/EBI1 expression. PCR amplification of the
CCR7/EBI1 and -actin-specific products in control
CD4+CD45RO+ T cells (lane 1), freshly isolated
adult T-cell leukemia (ATL) cells without (lanes 2, 3, and 4; patients
7, 10, and 6, respectively) and with lymphoid organ involvement (lanes
5, 6, 7, and 8; patients 27, 26, 24, and 23, respectively) was
performed for 28 PCR cycles. (B) RT-PCR analysis for CCR7/EBI1
expression in freshly isolated ATL cells from patients with and without
lymphoid organ involvement. CCR7/EBI1 expression was evaluated with the
ratio of CCR7/-actin-specific product for control
CD4+CD45RO+ T cells obtained from the
peripheral blood of 8 healthy volunteers (A, closed squares), ATL cells
from 12 patients without lymphoid organ involvement (B, open circles;
patients 1 through 12), and ATL cells from 16 patients with lymphoid
organ involvement (C, open circles; patients 13 through 28). RT-PCR
analyses were performed independently in triplicate. (C) Flow
cytometric analysis for CCR7/EBI1 expression on freshly isolated ATL
cells from patients with and without lymphoid organ involvement. Flow
cytometric analysis of control CD4+CD45RO+ T
cells and freshly isolated ATL cells was performed with anti-CCR7 mAb,
CCR7.6B3. Each point shows the mean fluorescence intensity (MFI) for
CCR7/EBI1 staining, which was corrected by substrating MFI obtained
with the nonbinding control IgG1 mouse mAb. Each point of
group A indicates the MFI for CCR7/EBI1 staining of CCR7/EBI1-positive
CD4+CD45RO+ T cells because CCR7/EBI1-positive
cells were 55% ± 14% of control
CD4+CD45RO+ T cells isolated from 8 healthy
volunteers. The bars indicate the mean value of each group; the
statistical analysis was performed using Student t test. (D)
Flow cytometric analysis for CXCR4 expression on freshly isolated ATL
cells from patients with and without lymphoid organ involvement. Flow
cytometric analysis of control CD4+CD45RO+ T
cells and freshly isolated ATL cells was performed with anti-CXCR4 mAb.
Each point shows the MFI for CXCR4 staining, which was corrected by
substrating MFI obtained with the nonbinding control IgG2a
mouse mAb. Each point of group A indicates the MFI for CXCR4 staining
of CXCR4-positive CD4+CD45RO+ T cells because
CXCR4-positive cells were 76% ± 18% of control
CD4+CD45RO+ T cells isolated from 8 healthy
volunteers. The bars indicate the mean value of each group; the
statistical analysis was performed using Student t test.
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Enhancement by SLC and ELC of ATL cell adhesion to HUVECs
and ICAM-1 under static conditions
ATL cells from patients with lymphoid organ involvement adhered
spontaneously and efficiently to IL-1-activated HUVECs and immobilized recombinant ICAM-1 protein, whereas normal
CD4+CD45RO+ T cells and ATL cells from patients
without lymphoid organ involvement adhered poorly to IL-1-activated
HUVECs or ICAM-1 (Figure 2A and 2B). The
addition of SLC or ELC to the adhesion assay buffer increased adhesion
of ATL cells to IL-1-activated HUVECs and ICAM-1. Among ATL cells
from 16 patients with lymphoid organ involvement, ATL cells from
patients with higher CCR7/EBI1 expression, such as
patients 21 through 28, showed higher increases (more than 2-fold) in
the rates of adhesion evoked by adding SLC or ELC than those from other
patients, such as patients 13 through 19 (less than 2-fold). These
increased adhesions were inhibited by anti-CD18 mAb or PTX, indicating
that SLC- and ELC-induced adhesion to both IL-1-activated HUVECs and
ICAM-1 was PTX-sensitive and mediated by integrin 2.
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| Fig 2.
Comparison of secondary lymphoid-tissue chemokine (SLC)-
and EBI1-ligand chemokine (ELC)-induced adhesion of adult T-cell
leukemia (ATL) cells under static conditions.
(A) Comparison of SLC- and ELC-induced adhesion of ATL
cells from patients with and without lymphoid organ involvement to
human umbilical vein-derived endothelial cells (HUVECs) under static
conditions. Cells (2 × 105/well), which had been
labeled with 51Cr, were incubated with SLC or ELC at a
concentration of 30 ng/ml at 37°C for 30 minutes and were added to
the well containing IL-1-activated HUVECs. The plates
were incubated at 37°C for 30 minutes and then gently washed, and
radioactivity of the adherent cells in each well was counted. The cells
are as follows: control CD4+CD45RO+ T cells (A,
closed squares) and ATL cells from patients without (B, patients 1 through 12) and with lymphoid organ involvement (C, patients 13 through
28). ATL cells from patients with lymphoid organ involvement were
blocked with 100 ng/ml pertussis toxin (PTX) for 1.5 hours at 37°C
(C+PT) or with anti-CD18 mAb, 7E4 (20 µg/ml) for 20 minutes at room
temperature (C+CD18 Ab) before adding of chemokines. The data are
expressed as the mean percentage of the binding of indicated cells from
triplicate experiments. The statistical analysis was performed using
Student t test. (B) Comparison of SLC- and ELC-induced adhesion
of ATL cells from patients with and without lymphoid organ involvement
to ICAM-1 under static conditions. The 96-well culture plates were
coated with ICAM-1 protein (25 ng/well).
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|
Comparison of SLC- and ELC-induced adhesion of ATL cells from
patients with and without lymphoid organ involvement to ICAM-1
under flow conditions
Next, we analyzed SLC-induced adhesion of
CD4+CD45RO+ T cells and ATL cells from patients
with and without lymphoid organ involvement to ICAM-1 under flow
conditions. Adhesion was measured by counting the arrested cells for
the first 4 minutes after perfusion. As shown in Figure
3, CD4+CD45RO+ T
cells and ATL cells adhered poorly to surfaces coated with ICAM-1
alone, whereas they adhered efficiently to surfaces coated with ICAM-1
and SLC. Furthermore, ATL cells isolated from patients with lymphoid
organ involvement, such as patients 21 through 28, which had greater
CCR7/EBI1 expression, increased adhesion significantly to surfaces
coated with ICAM-1 and SLC, as compared to
CD4+CD45RO+ T cells and ATL cells from patients
without lymphoid organ involvement. These increased adhesions were also
inhibited by PTX or anti-CD18 mAb. Similar results were obtained when
the adhesion assay was done using surfaces coated with ICAM-1 and ELC.
These findings indicate that the increased expression of CCR7/EBI1 by
ATL cells enhanced their ability to adhere to surfaces coated with
ICAM-1 and SLC or ELC.
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| Fig 3.
Comparison of secondary lymphoid-tissue chemokines (SLC)-
and EBI1-ligand chemokine (ELC)-induced adhesion of adult T-cell
leukemia (ATL) cells from patients with and without lymphoid organ
involvement to ICAM-1 under flow conditions.
Cells (106/ml) were perfused onto the coated dish with
substrates consisting of ICAM-1 (10 µg/ml) alone or also with SLC (10 µg/ml) or ELC (10 µg/ml) at 1 dyne/cm2 for the first 4 minutes. The number of arrested cells is shown by the total number
accumulated as arrested cells for the first 4 minutes in a single field
of view (4X objective). The cells are as follows: control
CD4+CD45RO+ T cells (A, closed squares) and ATL
cells from patients without (B, patients 1 through 12) and with
lymphoid organ involvement (C, patients 13 through 28). ATL cells from
patients with lymphoid organ involvement were blocked with 100 ng/ml
pertussis toxin (PTX) for 1.5 hours at 37°C (C+PT) or with
anti-CD18 mAb, 7E4 (20 µg/ml) for 20 minutes at room temperature
(C+CD18 Ab) before binding to ICAM-1 with SLC or ELC. Experiments were
performed in triplicate. The bars indicate the mean value of each
group; the statistical analysis was performed using Student t
test.
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Comparison of SLC- and ELC-evoked chemotaxis of ATL cells
from patients with and without lymphoid organ involvement
We compared the chemotactic response to SLC and ELC of ATL cells
from patients with and without lymphoid organ involvement. There was no
significant difference between these ATL cells in the chemotactic
responses to more than 100 ng/ml of these chemokines, whereas different
chemotactic responses were observed at 20 to 50 ng/ml (data not shown).
Therefore, the chemotactic assays of ATL cells were performed at a
concentration of 30 ng/ml of SLC and ELC. As shown in Figure
4, ATL cells isolated from
patients with lymphoid organ involvement migrated toward SLC and ELC,
whereas normal CD4+CD45RO+ T cells and ATL
cells from patients without lymphoid organ involvement showed no
significant migration toward SLC and ELC at a concentration of 30 ng/ml.
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| Fig 4.
Comparison of secondary lymphoid-tissue chemokine (SLC)-
and EBI1-ligand chemokine (ELC)-evoked chemotaxis of adult T-cell
leukemia (ATL) cells from patients with and without lymphoid organ
involvement.
Chemotactic assays were performed in the presence of SLC or ELC at a
low concentration (30 ng/ml) as described in Materials and Methods. The
cells are as follows: control CD4+CD45RO+ T
cells (A, closed squares) and ATL cells from patients without (B,
patients 1 through 12) and with lymphoid organ involvement (C, patients
13 through 28). ATL cells from patients with lymphoid organ involvement
were blocked with 100 ng/ml pertussis toxin (PTX) for 1.5 hours at
37°C (C+PT) before chemotactic assays. The data are expressed as
the mean percentage of migrated cells toward SLC or ELC from triplicate
experiments. The bars indicate the mean value of each group; the
statistical analysis was performed using Student t test.
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Flow cytometric analysis of the infiltrating ATL cells
in the lymph nodes
To see whether infiltrating ATL cells in lymph nodes express high
levels of CCR7/EBI1, we examined by flow cytometric analysis, using
CCR7.6B3, the CCR7/EBI1 expression of infiltrating ATL cells prepared
from lymph nodes of 2 representative patients, patients 26 and 27. We also examined, by the same method, the CCR7/EBI1 expression of control CD4+CD45RO+ T cells from
reactive lymph nodes of HTLV-1-seronegative individuals who had
undergone abdominal surgery. Infiltrating ATL cells and control
CD4+CD45RO+ T cells were purified by the
negative immunoselection technique. As shown in Figure
5, infiltrating ATL cells of patients 26 and 27 expressed CCR7/EBI1 at higher levels than control
CCR7/EBI1-positive CD4+CD45RO+ T cells. This
further supports that increased CCR7/EBI1 expression enhances lymphoid
infiltration by ATL cells.
View larger version (15K):
[in this window]
[in a new window]
| Fig 5.
Flow cytometric analysis of the infiltrating adult T-cell
leukemia (ATL) cells in lymph nodes.
CCR7/EBI1 expression levels of infiltrating ATL cells prepared from
lymph nodes of 2 representative patients, patients 26 and 27, and
control CD4+CD45RO+ T cells from reactive lymph
nodes of HTLV-1-seronegative individuals were compared by flow
cytometric analysis using an anti-CCR7 mAb, CCR7.6B3. The infiltrating
ATL cells and control CD4+CD45RO+ T cells were
purified by the negative immunoselection technique as described in
Materials and Methods.
|
|
| |
Discussion |
This is the first report to demonstrate a specific association
between expression of the chemokine receptor CCR7/EBI1 and infiltration
of lymphoid organs by ATL cells and to support that increased CCR7/EBI1
expression enhances the infiltration of lymphoid organs by ATL cells.
Our findings are as follows: 1) RT-PCR and flow cytometric analyses
showed that CCR7/EBI1 expression by ATL cells from patients with
lymphoid organ involvement was significantly higher than that by ATL
cells from patients without lymphoid organ involvement; 2)
significantly more ATL cells from patients with lymphoid organ
involvement adhered to surfaces coated with ICAM-1 and SLC or ELC under
static and flow conditions than did those from patients without
lymphoid organ involvement; and 3) significantly more ATL cells from
patients with lymphoid organ involvement migrated toward SLC or ELC,
even at a low chemokine concentration, than did those from patients
without lymphoid organ involvement.
Lymphocyte homing into lymphoid organs involves a coordinated multistep
process: an initial tethering and rolling step mediated by L-selectin;
chemokine-mediated signaling; and firm arrest mediated by LFA-1 binding
to ICAM-1 or ICAM-2 on HEV cells. To investigate the infiltration of
lymphoid organs by ATL cells, we focused on the interaction between
chemokines and chemokine receptors on ATL cells during this process.
The physiologically relevant chemokines on HEV cells that mediate
integrin activation have not been definitively elucidated, but recent
studies demonstrated that SLC, ELC, and SDF-1 could each promote
integrin-mediated arrest of human lymphocytes under flow
conditions.16,20,21,39 The highest SLC mRNA expression levels were detected in HEV cells of lymph nodes and Peyer's patches, and lower levels were detected in stromal cells in the T-cell zones of
the spleen, lymph nodes, and Peyer's patches.16
Furthermore, dendritic cells in the parafollicular and inner cortex
regions, the so-called T-cell zones of lymph nodes, demonstrated strong ELC mRNA expression.40,41 The high-level expression of SLC, together with the lack of detectable ELC expression by HEV cells, suggests that SLC plays a more prominent role in triggering lymphocyte adhesion to HEV cells. Because ELC is expressed at high levels by the
dendritic cells of the T-cell zone, ELC may instead play an important
role in attracting T cells away from HEVs into the T-cell zone and
promoting T-cell-dendritic-cell encounters. The chemotactic profiles
of SLC and ELC are strikingly similar: both chemokines efficiently
attract naive T cells, memory T cells, and B
cells.16-18,40-43 However, SDF-1 exerts chemotactic
effects on T and B cells, monocytes,44 CD34+
progenitor cells,45 and pre-B and pro-B cell
lines.46 SDF-1 is expressed in stromal cells in lymph
nodes and in a layer of cells surrounding the tonsiller germinal
centers, but whether it is expressed by HEV cells has not been
demonstrated definitively.47
The chemokine receptor CCR7/EBI1, the functional receptor for SLC and
ELC, is expressed in CD4+ and CD8+ T cells as
well as in B cells, but not in natural killer cells, monocytes, or neutrophils in peripheral blood.23,24,41
CCR7/EBI1 expression in CD34+ cells,
colony-forming unit granulocyte macrophage, and mature dendritic cells also has been reported.26-28,48,49
CCR7/EBI1 is expressed strongly in the tissues of various lymphoid
organs, such as the lymph nodes, spleen, thymus, and appendix, and
weakly in the small intestine, colon, placenta, bone marrow, and fetal liver.23,24,41 In lymph nodes, CCR7/EBI1 is expressed in
the parafollicular and inner cortical regions in which mature dendritic cells reside and to which antigen-loaded dendritic cells home. The
expression of CCR7/EBI1 has been reported to be up-regulated in B cells
infected by EB virus, CD4+ T cells infected by HHV-6 or
HHV-7, and T cells treated with IL-2 alone or with
phytohemmaglutinin,23,25,41 whereas CCR7/EBI1 expression
was not up-regulated by Tax using JP×9 cells50
(unpublished data). Although the mechanism responsible for increasing
CCR7/EBI1 expression on ATL cells is unknown, the expression of
CCR7/EBI1 on ATL cells was significantly higher than that on resting
CD4+CD45RO+ T cells. Furthermore, ATL cells
from patients with lymphoid organ involvement expressed CCR7/EBI1 at
significantly higher levels and adhered more efficiently to surfaces
coated with ICAM-1 and SLC under flow conditions than normal
CD4+CD45RO+ T cells or ATL cells from patients
without lymphoid organ involvement. Our findings thus suggest that the
up-regulation of CCR7/EBI1 in ATL cells promotes their responses to SLC
expressed by HEV cells in induction of integrin-mediated adhesion to
ICAM-1, thereby promoting the infiltration of lymphoid organs by ATL
cells. SDF-1 is the only ligand for CXCR4 to be identified so
far,31-33 and CXCR4 expression on cells has been reported
to be down-regulated in the presence of SDF-1.51,52 In
the present study, we showed no correlation between levels of CXCR4
expression and infiltration of lymphoid organs by ATL cells.
Several studies on the localization and infiltration of ATL cells have
been published.11,35,53-57 Other workers and we showed that
increased expression of CD151, 41 integrin,
and 51 integrin on ATL cells contributed to the lymphoma
formation through adhesion of the extracellular matrix and abnormal
proliferation and localization.35,53,54 E-selectin,
41/VCAM-1, and 0×40/gp34 pathways have been shown to be
involved in cell adhesion to endothelial cells and eventually in organ
infiltration by ATL cells.55,56 Three pairs of adhesion molecules, L-selectin/peripheral lymph node addressin, cutaneous lymphocyte-associated antigen/E-selectin, and
47 integrin/mucosal VCAM-1 also have been reported to be
associated with preferential recirculation of ATL cells to the
peripheral lymph nodes, skin, and gastrointestinal mucosa,
respectively.57 The circulating ATL cells from patients
with lymph node, skin, and gut involvement showed higher expression
levels of L-selectin, cutaneous lymphocyte-associated antigen, and
47 integrin, respectively, than those from patients without
involvement of these organs. During chemokine-mediated activation,
MIP-1 and MIP-1 produced by ATL cells activated LFA-1, resulting
in increased adhesion of ATL cells to HUVEC and ICAM-1 under static
conditions, although whether these chemokines exist and function
efficiently on HEV cells is unknown.11 These reports
and the fact that ATL cells adhere spontaneously to
endothelial cells and ICAM-1 suggest that multiple mechanisms are
involved in ATL cell infiltration of lymphoid organs. The results of
our study support that increased CCR7/EBI1 expression on ATL cells plays a role in the infiltration of lymphoid organs by these cells.
| |
Acknowledgment |
We are grateful to Kenji Kameda for his excellent technical assistance.
| |
Footnotes |
Submitted April 19, 1999; accepted August 17, 1999.
Supported in part by a grant-in-aid from the Ministry of Education,
Science, and Culture of Japan.
Reprints: Hitoshi Hasegawa, First Department of Internal
Medicine, Ehime University, School of Medicine, Shigenobu, Ehime
791-0295, Japan; e-mail: hitoshih{at}m.ehime-u.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction